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Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2013 Article ID 286524 24 pageshttpdxdoiorg1011552013286524
Review ArticleGenomic Structure and Variation of Nuclear Factor(Erythroid-Derived 2)-Like 2
Hye-Youn Cho
Laboratory of Respiratory Biology National Institute of Environmental Health Sciences National Institutes of Health111 TW Alexander Dr Building 101 MD D-201 Research Triangle Park NC 27709 USA
Correspondence should be addressed to Hye-Youn Cho cho2niehsnihgov
Received 11 February 2013 Accepted 22 April 2013
Academic Editor Mi-Kyoung Kwak
Copyright copy 2013 Hye-Youn Cho This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
High-density mapping of mammalian genomes has enabled a wide range of genetic investigations including the mapping ofpolygenic traits determination of quantitative trait loci and phylogenetic comparison Genome sequencing analysis of inbredmouse strains has identified high-density single nucleotide polymorphisms (SNPs) for investigation of complex traits which hasbecome a useful tool for biomedical research of human disease to alleviate ethical and practical problems of experimentation inhumans Nuclear factor (erythroid-derived 2)-like 2 (NRF2) encodes a key host defense transcription factor This review describesgenetic characteristics of human NRF2 and its homologs in other vertebrate species NRF2 is evolutionally conserved and sharessequence homology among species Compilation of publically available SNPs and other genetic mutations shows that humanNRF2 is highly polymorphic with a mutagenic frequency of 1 per every 72 bp Functional at-risk alleles and haplotypes have beendemonstrated in various human disorders In addition other pathogenic alterations including somatic mutations andmisregulatedepigenetic processes in NRF2 have led to oncogenic cell survival Comprehensive information from the current review addressesassociation of NRF2 variation and disease phenotypes and supports the new insights into therapeutic strategies
1 Overview
The gene nuclear factor (erythroid-derived 2)-like 2(NFE2L2) or more commonly the used synonym nuclearfactor erythroid 2- (NF-E2-) related factor 2 (NRF2) andits mouse homolog (Nfe2l2 Nrf2) encode a ubiquitoustranscription factor belonging to the basic leucine zipper(bZIP) protein family [1 2] NRF2 modulates downstreamgenes by binding to their cis-regulatory module antioxidantresponse elements (AREs) NRF2 targets include ARE-bearing effector genes such as reactive oxygen species (ROS)scavenging enzymes (eg superoxide dismutases SODs)phase-2 defense enzymes (eg glutathione-S-transferaseGST heme oxygenase-1 HO-1) drug efflux pumps(eg multidrug resistance proteins MRPs) and variousinteracting and indirectly modulated proteins [3ndash6] TheNRF2-ARE pathway has emerged in mechanisms of humandiseases in which oxidative stress is implicated Importantly
three lines of gene-targeted (knockout) mice weregenerated by Drs M Yamamoto (Nfe2l21199051198981119872119910119898) Y W Kan(Nfe2l21199051198981119884119908119896) and P A Ney (Nfe2l21199051198981119873119890119910) [7ndash9] and 124gene-trapped or gene-targeted cell lines have been established(httpwwwinformatics jaxorgsearchesallele reportcgimarkerID=MGI108420) During the last decade or morewide application of the knockout mice to human diseasemodels has led to new insights into disease pathogenesis andtherapeutic potential (Figure 1)
Kelch-like ECH-activating protein 1 (KEAP1 for humansKeap1 for mice or iNrf2 for rats) is a cytoplasmic suppressorof NRF2 and is critical in NRF2 homeostasis and activity [10]Substantial efforts have led to the discovery of the molec-ular mechanisms of KEAP1-mediated NRF2 regulation Inunstressed conditions the NRF2-bound KEAP1 homodimeris complexed to a ubiquitin ligase (Cullin 3-based E3 ligase)which polyubiquitinates NRF2 for proteasomal degradationand maintains NRF2 homeostasis (20min half-life of cellular
2 Oxidative Medicine and Cellular Longevity
Carcinogenesis
Mammary carcinoma (+)Urinary bladder carcinogenesis (+)
Skin tumor (+)
Kidney and bladder
Diabetic nephropathy (+)As toxicity (+)
Brain and neuromuscular systemIschemic brain injury (+)
Cerebral ischemia and stroke (+)Hearing loss and ototoxicity (+)
AirwaysAcute lung injury (+)COPDemphysema (+)Allergy and asthma (+)Pulmonary fibrosis (+)
Steroid resistance (+)
Inflammationimmunity and developmentSepsis (+)Autoimmune encephalomyelitis (+)Rheumatoid arthritis and cartilage destruction (+)
Heart and circulation
Hemolytic anemia (+)
MetabolismLipid homeostasis (+)
Glucose homeostasis (+)
Skin and eyeSkin healing (+)
Uveitis (+)
GastrointestineAlcoholic liver injury (+)
Acetaminophen hepatotoxicity (+)Bile acid homeostasis (+)
Colitis (+)
Various xenobiotic toxicity (+)Cholestatic liver injury (+)
Steatohepatitis (+)Gastroparesis (+)Liver fibrosis (+)
Acute intestinal mucosal injury (+)
Traumatic brain injury (+)
Spinal cord injury complications (+)
Brain leukoencephalopathy with astrogliosis (+)Gliosis and dopaminergic nigrostriatal degeneration (+)Intracerebral hemorrhage-induced brain injury (+)
Cardiac hypertrophy (+)
Ischemia-induced neovascularization (+)Atherosclerosis (minus)
Lupus-like autoimmune nephritis (+)Spermatogenesis (+)
Gastric neoplasia (+)Colorectal cancer (+)
Lung adenomas (minus)
Pseudomonas aeruginosa infection (+)
Bronchopulmonary displasia (+)Respiratory syncytial virus disease (+)
High-fat-diet induced obesity (+)
UV-induced skin aging (+)
Retinal ischemia-reperfusion (+)Retinopathy of prematurity (+)
Ischemic nephrotoxicity (+)Cisplatin nephrotoxicity (+)
Pollutant (diesel exhaust O3) toxicity (+)
Parkinsonrsquos disease (+)
Nrf 2
Cardiomyocyte damage (+)
Figure 1 Role of Nrf2 in human pathogenesis learned from model studies using mice genetically deficient in Nrf2 (+) indicates protectiveor beneficial effects and (minus) indicates aberrant roles
NRF2 [11]) However modifications of KEAP1 (eg cysteineresidues) and NRF2 (eg serine residues) under stressedconditions activate NRF2 by liberating it from a ldquohinge andlatchrdquo NRF2-KEAP1 affinity binding allowing its nucleartranslocation [12]
In the current review I address genetic aspects of humanNRF2 and its homologs in other vertebrate species Sequencevariations in humanNRF2 andmurineNrf2 including single-nucleotide polymorphisms (SNPs)were collected frompublicdatabases and compiled Mutations that have been associatedwith disease risks are defined Nongenetic variations includ-ing somatic mutations and epigenetic modifications are alsodescribed Although the current review does not deal withmutations in other species recent characterization of nrf2mutant zebrafishwhichwere hypersensitive to environmentaltoxicants [13] also provides a useful investigational tool
2 Sequence of NF-E2-Related Factor 2and Cross-Species Homology
Homology scores of gene (coding DNA sequences cds) andprotein across 10 species were compared with human NRF2The highest sequence homology (98-99) was with chim-panzee and rhesus monkey while the lowest similarity wasfound with zebrafish (Table 1) While there is approximately
about 83 homology in cds and protein sequences of humansand rodents 51015840-untranslated regions (51015840-UTR UTR-5) ofthese strains extend differentially (114 bp in human 233 bp inmouse and 82 bp in rat) and the human 51015840-UTR does notshare significant sequence homology with either rat ormouse(the rat is 94 homologous with the 31015840 portion of mouse 51015840-UTR)
Human NRF2 is located in the cytogenetic band 2q312of chromosome 2 spanning 178095031ndash178129859 bp as acomplementary sequence (gene ID 4780 Table 1) MurineNrf2 maps as a complementary sequence to chromosome2 C3 (4475 centimorgan) and spans 75675519ndash75704641(gene ID 18024 Table 1) The complete cds of NRF2 is2859 bp and there are 14 transcript variants reported(httpuseastensemblorgHomo sapiens) Mouse Nrf2mRNA spans 2469 bp and another variant has been re-ported (httpuseastensemblorgMus musculus) The hu-man NRF2 protein (ID NP 006155) contains 605 amino acid(aa) residues with molecular weight of 677 kDa (isoform1) and total 12 isoforms are published (the National Centerfor Biotechnology Information NCBI httpwwwncbinlmnihgovrefseqrsg eEnsemble httpuseastensemblorgindexhtml UniProt Consortium httpwwwuniprotorguniprotQ16236 httpwwwuniprotorguniprotQ60795)Mouse Nrf2 protein (ID NP 035032) comprises 597 aa at668 kDa (Table 1) Structurally there are 6 NRF2-ECH
Oxidative Medicine and Cellular Longevity 3
Table1Geneo
rtho
logy
ofNF-E2
-related
factor
2acrossthes
pecies
Species
Hum
anHom
osapiens
Cat
Felis
catus
Chim
panzee
Pantro
glodytes
Dog
Canislupus
familiaris
Cattle
Bostaurus
Officialfullname
nucle
arfactor
(erythroid-derived
2)-like
2Antho
rnam
enu
clear
factor
erythroid2-related
factor
2NF-E2
-related
factor
2NFE
2-related
factor
2Antho
rnam
eHEB
P1mdash
mdashmdash
mdashGenes
ynon
yms
NFE
2L2NRF
2NFE
2L2NRF
2NFE
2L2NRF
2NFE
2L2NRF
2NFE
2L2NRF
2Ch
romosom
emap
2q312
C12B
362
Genom
esequence
(NCB
IBuild)
NC
0000
0211
(GRC
h37p
10)
NC
1087301
(Feliscatus-62)
NC
0064703
(Pan
troglod
ytes-214)
NC
0066183
(CanFam31)
AC00
01591
(Bos
taurus
UMD
31)
Prim
arysource
HGNC
7782
mdashmdash
mdashBO
S1716
GeneID
mapping
(orie
ntationlowast
)
4780
178095031minus178129859(minus)
1010
9881
2165356974minus
165392728(minus)
7426
22181721941minus181756837(minus)
4788
1320989206minus21021893
(minus)
4970
2419659540minus19692048
Ensembleg
eneID
(mapping
)EN
SG00
00011604
4(178092323minus178257425)
mdashEN
SPTR
G00
000012677
(181721949minus181756285)
ENSC
AFG
0000
0013506
(20987569minus
21022230)
ENSB
TAG00
000019255
(19659540minus19692048)
RefSeq
IDm
RNA
size
NM
006164
4(G
I372620346)2859b
p
XM0039908931
(GI410968909)
predicted2394
bp
XM00114
58762
(GI332814815)predicted
2850
bp
XM5359753
(GI345797173)
predicted2570
bp
NM
0010116
782
(GI147904941)240
9bp
ProteinID
(aaMW
sect )NP00
61552
(60567696)
XP0039909421
(60667797)
XP00114
58762
(60567686)
XP5359751
(60167087)
NP0010116
782
(60767813)
UniProt
IDQ16236
mdashA2T
6Y9
mdashQ5N
UA62
Varia
nts
14transcrip
ts12
isoform
s3transcrip
ts3iso
form
s5transcrip
ts5iso
form
s2transcrip
ts2iso
form
smdash
Hom
olog
ygeneprotein
100100
893914
999999
893888
905891
4 Oxidative Medicine and Cellular Longevity
Table1Con
tinued
Species
Rhesus
mon
key
Macacamulatta
Chicken
Gallusgallus
Zebrafish
Daniorerio
Mou
seMus
musculus
Rat
Rattu
snorvegicus
Officialfullname
nucle
arfactor
(erythroid-derived
2)-like
2nu
clear
factorerythroid-derived
2lik
e2Antho
rnam
enu
clear
factor
erythroid2-related
factor
2NF-E2
-related
factor
2NFE
2-related
factor
2Antho
rnam
eHEB
P1mdash
mdashmdash
mdash
Genes
ynon
yms
NFE
2L2NRF
2Mmu966
NFE
2L2NRF
2nfe2l2N
rf2N
rf2a
wufc15g09wufj6
7e03
Nfe2
l2N
rf2A
I194320
Nfe2
l2N
rf2
Chromosom
emap
127
92C
3(4475
cM)
3q23
Genom
esequence
(NCB
IBuild)
NC
0078691
NC
0060
943
NC
0071205
(Zv9)
NC
0000
687
(GRC
m38p1)
NC
0051023
(Rno
r50)
Prim
arySource
mdashCG
NC
4960
4ZF
INZ
DB-GEN
E-030723-2
MGI108420
RGD620360
GeneIDmapping
(orie
ntationlowast
)70
7606
40848940minus40
8846
00(minus)
3960
1415304546minus15320356
3601
491643746minus
1670288
1802
475675519minus757046
41(minus)
8361
969041647minus69069070
(minus)
Ensembleg
eneID
(mapping
)EN
SMMUG00
0000
01861
(40848948minus
40884174)
ENSG
ALG
0000
0009240
(16897956minus
16922637)
ENSD
ARG
0000
0042824
(1643631minus1672715)
ENSM
USG
0000
0015839
(75675513minus
757046
41)
ENSR
NOG00
0000
01548
(58366
693minus
58394118)
RefSeq
IDm
RNA
size
NM
0012576071
(GI383872375)2335b
pNM
2051171
(GI45384113)2555
bpNM
1828891
(GI33504557)2149
bpNM
0109023
(GI76573877)2469
bpNM
0317892
(GI402692377)2305b
pProteinID
(aaMW
sect )NP001244
5361
(606
67703)
NP9904
481
(58265160)
NP8783091
(58665757)
NP0350321
(59766770)
NP1139771
(59766825)
UniProt
IDF7GPD
8Q90834
Q8JIM
1Q60795
O54968
Varia
nts
2transcrip
ts2iso
form
s1transcript1isoform
1transcript1isoform
1transcript
1transcript1isoform
Hom
olog
ygeneprotein
988990
726674
546491
834825
838832
Referto
httpwwwncbinlm
nihgovhom
ologene2412
and
reference
[14]forcross-speciesho
molog
yDetails
ofho
molog
yscores
and
sequ
encescan
bealso
obtained
byblastagainsthu
man
sequ
ence
(http
blastn
cbin
lmnihgov)
Furtherinform
ation
ontranscrip
tvaria
ntsand
protein
isoform
sareview
able
bygene
IDat
NCB
I(http
wwwncbinlm
nihgovrefseqrsg)
and
eEn
semble
(http
useastensembleo
rgin
dexhtml)or
byproteinID
atUniProt(http
wwwun
iproto
rg)
sect MWpredicted
molecularweightfrom
NCB
I(MW
inUniProtvarie
sslightly)Re
fSeqreference
sequ
enceaaam
ino
acidsandlowastgene
incomplem
ento
rientation
Oxidative Medicine and Cellular Longevity 5
Table 2 Protein domains of NF-E2-related factor 2
Domains Amino acid positionslowast Predicted functionsHuman (605 aa) Mouse (597 aa)
Neh216ndash89
DLG motif 17ndash32ETGF motif 77ndash82
16ndash89 KEAP1 repression through DLGETGF motif-DC motif binding fastredox-sensitive proteasomal degradation
Neh4 111ndash134 111ndash134 Translocation and transactivation Phosphorylation or CBP bindingNeh5 182ndash209 172ndash201
Neh6 337ndash394(or 338ndash388) 328ndash385 Degron motif-associated constitutive turnover slow redox-insensitive
(Keap1-independent)
Neh1435ndash568
basic motif 503ndash518leucine zipper 525ndash539
427ndash560basic motif 494ndash509leucine zipper 517ndash531
Dimerization for nuclear translocation DNA binding through basicmotif-leucine zipper
Neh3 569ndash605 561ndash597 CHD6 binding stability or transactivationlowastVaries slightly among publications
homology (Neh) domains configuring the protein sequenceof either species (Table 2) and the potential functions of eachregion particularly the highly conserved KEAP1-bindingNeh2 and DNA- (ARE-) binding Neh1 domains have beenintensively investigated [12 14 15]
3 Genetic Variation of NF-E2-Related Factor 2in Human and Mouse
31 Evolution Genome Sequence and Polymorphism Dis-covery in Human and Mouse While rare and monogenicMendelian diseases are inheritable mutations in a single gene[16] many common diseases are complex traits and thedisease phenotypes are affected by variants inmultiple geneticloci Recent advancements in high-throughput technologyhave enabled sequencing of entire mammalian genomes[17ndash19] and information on DNA sequence and variationhas facilitated the study of complex traits of human dis-orders Genome-wide association studies (GWAS) examinewhether SNPs are associated with important disease traitsand ascertains ldquoat-riskrdquo genotypes that are significantly moreprevalent in the affected group than in the nonaffected groupThe HapMap Project (httphapmapncbinlmnihgov) hasmapped combinations of alleles at specific loci (haplotypes)that is common patterns of sequence variation in severalhuman populations It has supported efficient mapping ofmultiple loci for complex traits in GWAS Candidate geneapproaches based on findings from GWAS of similar dis-orders have also been useful for determining the potentialgenetic mechanisms of diseases
The evolutionary divergence of human and mouse lin-eages occurred for roughly 75million years and their genomesequences have been altered by nearly one substitution (ordeletion or addition) for every twonucleotides [20]Howeverthis slow evolution process resulted in a high degree ofconservation across the two species which allows alignmentof orthologous sequences gt90 of the human and mousegenomes is partitioned into corresponding regions of con-served synteny and at the nucleotide level approximately 40of the human genome is aligned to themouse [20]Due to this
fact biomedical studies of human genes are complemented byexperimental manipulation of corresponding mouse genesand they have aided functional understanding of genesin human health Following the 2003 completion of theHuman Genome Project of approximately 31 giga base pairs(Gbp) the Mouse Genome Project assembled the completegenome sequence of one strain (C57BL6J 2716965481 bp)in 2011 Using this reference strain whole genome sequenc-ing data across 16 additional inbred strains were done(httpwwwsangeracukresourcesmousegenomes [21])Discovery of high-density SNPs in the mouse genome sup-ports evolutionary history of the strain and provides a toolto investigate models of human disease processes that cannotoften be practically achieved through direct human studies
32 GeneticMutations in HumanNRF2 HumanNRF2 codesthree major isoforms of protein (Figure 2) Transcript variant2 (NM 0011454122 2746 bp) has an alternate promoter 51015840-UTR and a downstream start codon compared to variant 1(NM 0061644 2859 bp) It encodes an isoform 2 missing N-terminal 16 aa (NP 001138884 or Q16236-2 589 aa) relativeto isoform 1 (NP 0061552 or Q16236 605 aa) Isoform 3(NP 0011388851 or Q16236-3 582 aa) is encoded by tran-script variant 3 (NM 0011454132 2725 bp) and lacks aninternal segment relative to isoform 2 due to an alternate in-frame splice site in the 31015840 coding region In public databasesmore than 583 sequence mutations are reported in NRF2(34827 bp) and 7000 bp upstream (Table S1 data acquired asof December 2012) (See Table S1 in Supplementary Materialavailable on line at httpdxdoiorg1011552013286524)NRF2 locates on 178130354-178129304 bp of GRCh37p10PrimaryAssembly and Figure 2 shows sequences of proximalpromoter (minus1 to minus500) partial mRNA variant 1 including 51015840-UTR (exon 1 up to TSS) and protein isoform 1 (NP 006155encoded by variant 1 NM 0061644 556ndash2373 bp) Basedon the current assembly and sequence update previouspromoter positions minus686 [22]minus653 [23] are identified asminus214 minus684 [22]minus651 [23] as minus212 and minus650 [22]minus617 [23]asminus178Overall frequency ofNRF2 SNPs and othermutationsis about 1 per 72 bp The genetic mutations include 37 in
6 Oxidative Medicine and Cellular Longevity
Translation start site for variants 2 and 3
1 missed in isoforms 2 and 3pA72T pS99P pQ121H missed in isoform 3
pP406S pE423D pK428R pR437W pR449C
minus400
minus500
minus50minus100
minus200
minus300
minus450
minus350
minus250
minus150
g minus
g
pE152K pV155I pL177V
pV268M
pR517K pH453L
pS365I pS387N pK389R pL309F pI317M pD341V
pK219R pP233S pK237 = fs
51101151201251301351401451501551601
Protein isoform 1 (NP 006155)
g minus66minus65insC
Termination codon (c +2371 +2373)
Translation start site (c +1)
151
101151201251301351401451501551601
2351
frame stop codonc minus156 154 in-old + 1 [23]
old + 1 [22]
g minus1 (178130354)
c minus96delC
c minus438 minus437delTT
minus89 minus87delGCC
transcription start site)+1 (c minus555
p R43Q p R43W
Genomic DNA (reversed contig GRCh37 p10 primary assemblyNM 0061644)minus477
minus214 g minus212gminus188
c 9minus95
g minus178
c minus304
c 8minus13
c 2minus16
TgtA
CgtT
GgtA
GgtA
GgtA
GgtT
GgtCAgtG
AgtCAgtG
Figure 2 DNA (variant 1 partial promoter and exons 1 and 5) and protein (isoform 1) sequence of Human NRF2 SNPs and amino acidresidues for nonsynonymous SNPs are marked
the 51015840 flanking promoter and 59 in exons (Tables 3 and S1)Among exon SNPs 26 are nonsynonymous (Cns) mutationsA triplet repeat variation (rs143406266 GCC
4versus GCC
5
previously published as minus20minus6) in the 51015840-UTR was uniquelyidentified in Asian populations [22 24]
33 SNPs Haplotypes and Association with Disease RiskThe use of gene knockout mice in model systems hasprovided potential insights into the role of NRF2 in thepathogenesis of various human disorders (see Figure 1)Recent epidemiological and association studies have revealedsignificant associations of NRF2 sequence variations withdisease risks which further supportsNRF2 as a susceptibilitygene Most of the phenotype-associated variants are in thepromoter region and presumed to be involved in NRF2 generegulation Table 4summarizes NRF2 SNP andor haplotypealleles that have been associated with oxidant-related diseaserisks Interestingly there is no evidence for exon SNPs as at-risk alleles For convenience and consistency intronic and31015840 distal SNP alleles are presented as chromosome contig(HGVS) alleles while promoter and exon SNPs are presentedas reversed contig alleles throughout the text
Pulmonary Diseases NRF2 SNPs in the promoter and intron1 sequences have been investigated for their potential asso-ciations with risk of pulmonary critical disorders includingacute lung injury (ALI) cigarette smoke-induced chronicobstructive pulmonary disease (COPD) and asthma Aheterozygous CA SNP at minus178 position (rs6721961TgtCor TgtG previously minus617 or minus650) significantly increasedthe risk for developing ALI following major trauma inEuropean and African-American populations (odds ratioOR 644 95 confidence interval CI 134ndash308 allelicfrequency = 119 at 21180) [23] Promoter activity ofthe A allele (AC or AA) determined in vivo and invitro was significantly lower than CC allele at that locus(minus178 in an ARE-like motif) indicating that it is a func-tional SNP for autoregulation [23] The minus178GG werealso nominally associated with ALI-related 28-day mor-tality following systemic inflammatory response syndrome[37] In a Japanese cohort SNP haplotype (rs2001350Trs6726395Ars1962142Ars2364722Ars6721961T) co-tainingthe minus178AA homozygote was associated with an annualdecline of rapid forced expiratory volume in one second(FEV1) in relation to cigarette-smoking status [34] In addi-
tion a promoter and 51015840-UTR SNP haplotype consistingof minus214G allele (52 rs35652124 previously minus686minus653)
Oxidative Medicine and Cellular Longevity 7Ta
ble3Geneticmutations
inprom
oter
andexon
sofh
uman
NRF
2
IDMap
onchromosom
e2lowast
(HGVS
name)
Positionin
Nfe2
l2
(HGVS
name)
Region
s(position
from
mRN
A)
Varia
tioncla
ssand
consequences
(HGVS
name)dagger
Minor
allelefrequ
ency
(MAF)
MAcoun
tsDagger(coh
ort
size)
MAFsources
rs16865105
g178136629AgtC
cminus555minus
6770Tgt
G51015840Flanking
(minus6770)
SNP
C=01928421
1000
Genom
esrs7557529
g178135097CgtT
cminus555minus
5238GgtA
51015840Flanking
(minus5238)
SNP
nars6750320
g178131796CgtT
cminus555minus
1937GgtA
51015840Flanking
(minus1937)
SNP
T=0030266
1000
Genom
esrs18116
2518
g178131774TgtC
cminus555minus
1915AgtG
51015840Flanking
(minus1915)
SNP
C=000
051
1000
Genom
esrs1900
44775
g178131746CgtT
cminus555minus
1887GgtA
51015840Flanking
(minus1887)
SNP
T=000
051
1000
Genom
esrs185117338
g178131704AgtG
cminus555minus
1845Tgt
C51015840Flanking
(minus1845)
SNP
G=000
051
1000
Genom
esrs149947189
g178131697CgtT
cminus555minus
1838GgtA
51015840Flanking
(minus1838)
SNP
T=000
092
1000
Genom
esrs139771244
g178131625AgtG
cminus555minus
1766Tgt
C51015840Flanking
(minus1766)
SNP
G=000
092
1000
Genom
esrs6747203
g17813160
4CgtG
cminus555minus
1745GgtC
51015840Flanking
(minus1745)
SNP
G=000
613
1000
Genom
esrs193101749
g178131504TgtC
cminus555minus
1645AgtG
51015840Flanking
(minus1645)
SNP
C=000
4610
1000
Genom
esrs190630762
g178131495GgtC
cminus555minus
1636Cgt
G51015840Flanking
(minus1636)
SNP
C=000
092
1000
Genom
esrs183764
402
g178131366CgtA
cminus555minus
1507GgtT
51015840Flanking
(minus1507)
SNP
A=000
051
1000
Genom
esrs19122296
4g178131211GgtA
cminus555minus
1352Cgt
T51015840Flanking
(minus1352)
SNP
A=000
092
1000
Genom
esrs187137522
g178131165TgtG
cminus555minus
1306AgtC
51015840Flanking
(minus1306)
SNP
G=000
092
1000
Genom
esrs182620359
g178131158AgtG
cminus555minus
1299Tgt
C51015840Flanking
(minus1299)
SNP
G=000
051
1000
Genom
esrs4893819
(rs61433302)
g178131134CgtT
cminus555minus
1275GgtA
51015840Flanking
(minus1275)
SNP
C=04263931
1000
Genom
es
rs191547130
g178131017CgtT
cminus555minus
1158GgtA
51015840Flanking
(minus1158)
SNP
T=000
051
1000
Genom
esrs188422217
g178131003AgtG
cminus555minus
1144TgtC
51015840Flanking
(minus114
4)SN
PG=000143
1000
Genom
esrs143047764
g178130865AgtG
cminus555minus
1006Tgt
C51015840Flanking
(minus1006)
SNP
G=000
6915
1000
Genom
esrs74432849
g178130766CgtA
cminus555minus
907GgtT
51015840Flanking
(minus907)
SNP
nars116
79252
g178130691CgtG
cminus555minus
832GgtC
51015840Flanking
(minus832)
SNP
nars12993217
g178130516AgtG
cminus555minus
657TgtC
51015840Flanking
(minus657)
SNP
nars115644
826
g17813044
2TgtA
cminus555minus
583AgtT
51015840Flanking
(minus583)
SNP
A=0015133
1000
Genom
esrs140803524
g178130431GgtA
cminus555minus
572CgtT
51015840Flanking
(minus572)
SNP
A=000
4610
1000
Genom
es
rs776844
20g178130427TgtC
cminus555minus
568AgtG
51015840Flanking
(minus568)
SNP
C=0033974
C=0190(84)
1000
Genom
es[24]
rs183651094
g178130336AgtT
cminus555minus
477TgtA
51015840Flanking
(minus477)
SNP
T=000184
1000
Genom
es
rs35652124
sect
(rs57695243)
g178130073TgtC
cminus555minus
214AgtG
51015840Flanking
(minus214)
SNP
C=03512767
T=0429(84)
T=0413181Dagger
C=033827Dagger
C=03517
69
1000
Genom
es[24]
[22]
[23]
[25]
rs6706649
sectg178130071CgtT
cminus555minus
212GgtA
51015840Flanking
(minus212)
SNP
T=0078170
T=004
8(84)
T=004
821Dagger
T=00753Dagger
1000
Genom
es[24]
[22]
[23]
rs1506
48896
g178130047CgtG
cminus555minus
188GgtC
51015840Flanking
(minus188)
SNP
G=000235
G=000
6(84)
1000
Genom
es[24]
rs6721961
sect
(rs117801448)
g178130037TgtC
TgtG
cminus555minus
178AgtC
AgtG
51015840Flanking
(minus178)
SNP
T=0150328
T=0283124Dagger
T=031325Dagger
T=0321(84)
1000
Genom
es[22]
[23]
[24]
8 Oxidative Medicine and Cellular Longevity
Table3Con
tinued
IDMap
onchromosom
e2lowast
(HGVS
name)
Positionin
Nfe2
l2
(HGVS
name)
Region
s(position
from
mRN
A)
Varia
tioncla
ssand
consequences
(HGVS
name)dagger
Minor
allelefrequ
ency
(MAF)
MAcoun
tsDagger(coh
ort
size)
MAFsources
rs20134560
4g178129924178129925insG
cminus555-66minus555-65insC
51015840Flanking
(minus66minus65)
Insertion
G=0017939
1000
Genom
es
rs200432479
g178129741178
129742delAA
cminus438minus437delT
Texon
1UTR
-51015840(118minus119
)Dele
tion
-=000378
-=000
6(84)
1000
Genom
es[24]
rs75485459
g178129608CgtA
cminus304GgtT
Exon
1UTR
-51015840(252)
SNP
nars192086766
g17812946
6CgtT
cminus162GgtA
Exon
1UTR
-51015840(394)
SNP
T=002248
1000
Genom
esmdash
g17812944
2GgtA
cminus138CgtT
Exon
1UTR
-51015840(418)
SNP
A=0012(84)
[24]
rs7166
8246
g17812940
0delG
cminus96delC
Exon
1UTR
-51015840(460)
Dele
tion
nars187291840
g178129399CgtT
cminus95GgtA
Exon
1UTR
-51015840(461)
SNP
T=0054912
01000
Genom
es
rs143406266
g178129391178129393delGGC
cminus89minus87delGCC
Exon
1UTR
-51015840(467minus469)
Dele
tion
-=064
4282Dagger
-=0589(84)
[22]
[24]
rs182428269
g178098918GgtA
c127Cgt
TEx
on2(682)
Cns(pArg43Trp)
A=000
051
1000
Genom
esrs35248500
g178098917CgtT
c128GgtA
Exon
2(683)
Cns(pArg43Gln)
T=000
613
1000
Genom
esrs1135118
g178098831CgtT
c214GgtA
Exon
2(769)
Cns(pAla72Th
r)TDagger
[23]
rs19969166
0g178098829AgtT
c216Tgt
AEx
on2(771)
Cs(pAla72=)
namdash
g178098769GgtA
c276Cgt
TEx
on2(831)
Cs(pIle92=)
TDagger[23]
rs5031039
g178098750AgtG
c295Tgt
CEx
on2(850)
Cns(pSer99P
ro)
G=0Dagger
[23]
rs200239262
g178098017CgtG
c363GgtC
Exon
3(918)
Cns(pGln121H
is)na
rs183034165
g178098008TgtC
c372GgtA
Exon
3(927)
Cs(pAla124=
)T=000
092
T=000
6(84)
1000
Genom
es[24]
rs199970826
g178097996CgtT
c384GgtA
Exon
3(939)
Cs(pPro128=
)T=000
051
1000
Genom
esrs201992337
g178097260CgtT
c454GgtA
Exon
4(100
9)Cn
s(pGlu152Lys)
nars201589693
g178097251CgtT
c463GgtA
Exon
4(1018)
Cns(pV
al155Ile)
T=000
051
1000
Genom
es
rs35577826
g178097185AgtC
c529Tgt
GEx
on4(1084)
Cns(pLeu177Va
l)C=000143
Alt0005Dagger
1000
Genom
es[23]
rs181513314
g178096710CgtT
c621GgtA
Exon
5(1176)
Cn(pLeu207=
)T=000
051
1000
Genom
esrs60132461
g178096675TgtC
c656AgtG
Exon
5(1211)
Cns(pLys219Arg)
C=000184
1000
Genom
es
rs139187151
g178096634GgtA
c697Cgt
TEx
on5(1252)
Cns(pPro233Ser)
A=000
051
A=0012Dagger
(84)
1000
Genom
es[24]
rs35557421
g178096620d
elT
c711delA
Exon
5(1266)
Fram
eshiftdeletio
n(pLys237=fs)
na
rs34154613
g178096529CgtT
c802GgtA
Exon
5(1357)
Cns(pV
al268M
et)
T=000184
1000
Genom
esrs141363120
g178096
406GgtA
c925Cgt
TEx
on5(14
80)
Cns(pLeu309Ph
e)A=000378
1000
Genom
esrs201661476
g178096380AgtC
c951Tgt
GEx
on5(1506)
Cns(pIle
317M
et)
na
Oxidative Medicine and Cellular Longevity 9
Table3Con
tinued
IDMap
onchromosom
e2lowast
(HGVS
name)
Positionin
Nfe2
l2
(HGVS
name)
Region
s(position
from
mRN
A)
Varia
tioncla
ssand
consequences
(HGVS
name)dagger
Minor
allelefrequ
ency
(MAF)
MAcoun
tsDagger(coh
ort
size)
MAFsources
rs199673454
g178096309TgtA
c1022AgtT
Exon
5(1577)
Cns(pAs
p341Va
l)A=000
051
1000
Genom
esrs35007548
g178096299GgtA
c1032CgtT
Exon
5(1587)
Cs(pSer344=
)A=000
092
1000
Genom
esrs200209692
g178096287TgtC
c104
4AgtG
Exon
5(1599)
Cs(pLeu348=
)C=000
051
1000
Genom
es
mdashg178096237CgtA
c1094GgtT
Exon
5(1649)
Cns(pSer365Ile
)A=0125273
A=000
6Dagger(84)
PubM
ed[24]
rs201214197
g178096171CgtT
c1160GgtA
Exon
5(1715)
Cns(pSer387Asn)
T=000
051
1000
Genom
esrs200494292
g178096165TgtC
c1166AgtG
Exon
5(1721)
Cns(pLys389Arg)
nars186171287
g178096115GgtA
c1216CgtT
Exon
5(1771)
Cns(pPro4
06Ser)
A=000
051
1000
Genom
esrs182276775
g178096062CgtA
c1269GgtT
Exon
5(1824)
Cns(pGlu423A
sp)
A=000
051
1000
Genom
esrs201560221
g17809604
8TgtC
c1283AgtG
Exon
5(1838)
Cns(pLys428Arg)
nars189238236
g178096043AgtG
c1288TgtC
Exon
5(1843)
Cs(pLeu430=
)G=000
051
1000
Genom
esrs184287392
g178096022GgtA
c1309CgtT
Exon
5(1864)
Cns(pArg437Trp)
A=000
051
1000
Genom
esrs201871588
g178095986GgtA
c1345CgtT
Exon
5(19
00)
Cns(pAg449Cy
s)na
rs181294188
g178095985TgtC
c1346GgtA
Exon
5(19
01)
Cns(pArg44
9His)
T=000
092
1000
Genom
esrs20169046
6g178095973TgtA
c1358AgtT
Exon
5(19
13)
Cns(pHis4
53Leu)
A=000
051
1000
Genom
esrs105704
4(rs52789869)
g178095781CgtT
c1550GgtA
Exon
5(2105)
Cns(pArg517Lys)
TDagger[23]
rs200750800
g178095603AgtG
c1728TgtC
Exon
5(2283)
Cs(pTyr576=
)na
rs200175942
g178095567AgtG
c1764TgtC
Exon
5(2319)
Cs(pAsp588=
)G=000
051
1000
Genom
esrs77547666
g178095495GgtC
clowast18Cgt
GEx
on5UTR
-31015840(2391)
SNP
C=000
6915
1000
Genom
esrs73031353
g178095425TgtC
clowast88AgtG
Exon
5UTR
-31015840(2461)
SNP
C=000184
1000
Genom
esrs675944
3g178095345TgtC
clowast168AgtG
Exon
5UTR
-31015840(2541)
SNP
C=000
419
1000
Genom
esrs188674558
g178095279CgtA
clowast234GgtT
Exon
5UTR
-31015840(2607)
SNP
A=000
051
1000
Genom
esrs77685897
g178095247AgtG
clowast266TgtC
Exon
5UTR
-31015840(2639)
SNP
G=000
092
1000
Genom
esrs1057092
g178095162TgtG
clowast351AgtC
Exon
5UTR
-31015840(2724)
SNP
nars3197704
g178095162TgtG
clowast351AgtC
Exon
5UTR
-31015840(2724)
SNP
nars184701151
g178095159TgtC
clowast354AgtG
Exon
5UTR
-31015840(2727)
SNP
C=000276
1000
Genom
esrs11543307
g178095153AgtG
clowast360TgtC
Exon
5UTR
-31015840(2733)
SNP
nars111874043
g178095146AgtG
clowast367TgtC
Exon
5UTR
-31015840(2740)
SNP
nars34012004
g178095102AgtC
clowast411TgtG
Exon
5UTR
-31015840(2784)
SNP
C=0071
[24]
10 Oxidative Medicine and Cellular Longevity
Table3Con
tinued
IDMap
onchromosom
e2lowast
(HGVS
name)
Positionin
Nfe2
l2
(HGVS
name)
Region
s(position
from
mRN
A)
Varia
tioncla
ssand
consequences
(HGVS
name)dagger
Minor
allelefrequ
ency
(MAF)
MAcoun
tsDagger(coh
ort
size)
MAFsources
rs201481890
g178095090178095091in
sTclowast
422lowast423insA
Exon
5UTR
-31015840
(2795minus
2796)
Insertion
na
rs3082500
g178095089178095090d
elTT
delTTinsT
clowast423lowast424d
elAAinsA
Exon
5UTR
-31015840
(2796minus
2797)
Dele
tion
insertion
na
rs71792546
(rs71796710)
g178095079d
elT
clowast425delA
Exon
5UTR
-31015840(2798)
Dele
tion
na
rs1057106
g178095078AgtC
AgtT
clowast435TgtA
TgtG
Exon
5UTR
-31015840(2808)
SNP
na
rs34176791
g178095076AgtC
clowast437TgtG
Exon
5UTR
-31015840(2810)
SNP
C=000235
1000
Genom
esrs35911553
g178095045CgtT
clowast46
8GgtA
Exon
5UTR
-31015840(2841)
SNP
T=000
6915
1000
Genom
esSequ
ence
varia
tions
inup
stream
andexon
sofh
uman
NRF
2fro
m655varia
tions
availablein
publicdatabase
asof
Decem
ber2012
(583
activ
esomeSN
Psmergedge14
SNPs
citedin
PubM
ed)lowastNCB
Ireference
sequ
ence
NC
0000
0211
(Hom
osapiens
chromosom
e2G
RCh37p
10prim
arya
ssem
bly)spanning
178095033ndash1781298
59bp
(com
plem
ent34827
bpforexons
andintro
ns)51015840-Flank
ingregions
startat17812
9860
bp(minus1)in
transcrip
tvariant
1HGVS
Hum
anGenom
eVa
riatio
nSo
ciety
Position
sinvaria
nt1(NM
006164
4)2859
bpsectSN
Pscitedin
PubM
edE
xon11781298
59ndash17812
9260
(600
bpT
TS=1781293
04)exon
2178098999ndash
178098733(267
bp)exon
3178098067ndash1780979
78(90b
p)exon41780973
11ndash178097120
(192b
p)and
exon
5178096736ndash
178095033(1704b
p)daggerProteinam
inoacid
(aa)
resid
uesiniso
form
1(N
P00
61552605
aa)Cn
scoding
-non
syno
nymou
sCs
cod
ing-syno
nymou
sDaggerheterozygositydetectednano
tavailable
Oxidative Medicine and Cellular Longevity 11
Table4Hum
anNRF
2SN
Psassociated
with
diseaser
isk
IDMap
onchromosom
e2 (H
GVS
name)
Region
class
Dise
asea
ssociatio
nandreferences
Risk
allelesa
ndstatistics
Ethn
icgrou
p(num
bero
fcase)
rs7557529
g178135097CgtT
minus5238
Ggt
AParkinsonrsquos
disease(2010
[26])
G=0718252Dagger
inhaplotype
OR=090
or040
CI=
060ndash140or
03ndash06
Swedish
Polish
Caucasian
(357)
rs2886162
g178133165AgtG
minus3306
TgtC
Breastcancer
survival(2012[27])
TT=0324
OR=16
87C
I=1105ndash275
FinlandKB
CP(452)
Chronicg
astritisgastric
ulcer(2007
[28])
GDagger
GDaggerin
haplotypefor
ulcer(OR=
252C
I=119ndash
545)
Japanese
(159)
P14methylationin
gastric
cancer
Hpyloriinfectio
n(2008[29])
Gin
haplotype
OR=290C
I=114ndash
736
Japanese
(209)
Gastriccancer
inHpylori-n
egative
cases(2008
[30])
Ain
haplotype
119875=0022
Japanese
(209)
rs35652124
(rs57695243)
g178130073TgtC
minus214Agt
G(previou
slyminus653[23]
orminus686[22])
Ulcerativec
olitis(2008
[31])
AG
(OR=045C
I=022ndash0
93)
G(O
R=257C
I=10
1ndash660)
Japanese
(89)
Lupu
swith
neph
ritisin
female(2010
[25])
GA
OR=18
1CI
=10
4ndash312
Mexican
mestizo(362)
Parkinsonrsquos
disease(2010
[26])
A=0884312Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
COPD
(2010[32])
G=052Dagger
hazard
ratio
=095C
I=091ndash0
99
(Haplotype)
German
(69)
Gastriculcer(2007
[28])
GDaggerin
haplotype
OR=252C
I=119ndash
545
Japanese
(159)
P14methylationin
gastric
cancer
H
pyloriinfection(2008[29])
Gin
haplotype
OR=290C
I=114ndash
736
Japanese
(209)
Gastriccancer
inHpylori-negative
cases(2008
[30])
Gin
haplotype119875=0022
Japanese
(209)
rs6706
649
g178130071CgtT
minus212Ggt
A(previou
slyminus651[23]
orminus684[22])
Ulcerativec
olitis(2008
[31])
AG
(OR=045C
I=022ndash0
93)
G(O
R=257C
I=10
1ndash660)
Japanese
(89)
Maternalacetaminop
henandasthma
(2010[33])
A=02321137Dagger
OR=17
3CI
=12
2245
UKALSPA
C(gt40
00mothersgt
5000
child
ren)
12 Oxidative Medicine and Cellular Longevity
Table4Con
tinued
IDMap
onchromosom
e2 (H
GVS
name)
Region
class
Dise
asea
ssociatio
nandreferences
Risk
allelesa
ndstatistics
Ethn
icgrou
p(num
bero
fcase)
COPD
(2010[32])
G=098Daggerin
haplotype
hazard
ratio
=095C
I=091ndash0
99
German
(69)
Parkinsonrsquos
disease(2010
[26])
G=0972343Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
03ndash06
Swedish
Polish
Caucasian
(357)
Acutelun
ginjury
follo
wingtrauma
(2007[23])
CA=0119Dagger
OR=644
CI=
134ndash
3080
Caucasia
nAfrican-
American
(164
)
Ann
ualF
EV1decline
(2011[34])
A=0082in
haplotype
Japanese
(915)
Vitiligo(2008[35])
CAA
AOR=17
24C
I=13
5ndash221
Chinese(300)
COPD
(2010[32])
G=073Daggerin
haplotype
hazard
ratio
=095C
I=091ndash0
99
German
(69)
rs6721961
(rs117801448)
g178130037TgtC
TgtG
minus178Agt
GAgt
C(previou
slyminus617[23]
orminus650[22])
Parkinsonrsquos
disease(2010
[26])
C=0980346Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
Postm
enop
ausalvenou
sthrombo
embo
lism
(2011[36])
A=033311
OR=25CI
=370ndash8570
Caucasia
n(161)
Breastcancer
survivalandNRF
2proteinexpressio
n(2012[27])
AA
OR=4656CI
=13
5ndash1606
FinlandKB
CP(452)
Acutelun
ginjury-related
mortality
followingsyste
micinflammatory
respon
sesynd
rome(2012
[37])
GG
OR=97
3CI
=12
7ndash7480
Caucasia
n(750)
Infection-indu
cedasthma(
2012
[38])
AC
OR=0437CI
=028ndash0
80
Hun
garia
nCaucasia
nGypsy
(307)
rs143406266
g178129391178
129393delGGC
Exon
1(467ndash4
69G
CC)
COPD
(2010[32])
GCC
4=053Dagger
hazard
ratio
=095C
I=091ndash0
99
Taiwanese(69)
rs2886161
g178127839Tgt
ClowastIntro
n1
Parkinsonrsquos
disease(2010
[26])
T=0878309Dagger
inhaplotype
OR=09or
04CI
=06ndash
14or
03ndash06
Swedish
Polish
Caucasian
(357)
rs2364723
g178126546Ggt
CIntro
n1
Basaland
smoker
FEV
1(200
9[39])
CI=minus6360simminus1780C
=0525Dagger
Also
ashaplotype
Netherla
nd(2542)
rs2364722
g178124787Agt
GIntro
n1
Ann
ualF
EV1decline
(2011[34])
A=0082in
haplotype
Japanese
(915)
rs13001694
g178118990Agt
GIntro
n1
Basaland
smoker
FEV
1(200
9[39])
G=040
11578Dagger
inhaplotype
Netherla
nd(2542)
Oxidative Medicine and Cellular Longevity 13
Table4Con
tinued
IDMap
onchromosom
e2 (H
GVS
name)
Region
class
Dise
asea
ssociatio
nandreferences
Risk
allelesa
ndstatistics
Ethn
icgrou
p(num
bero
fcase)
Breastcancer
[40]
Twith
NQO1N
OS3H
O1risk
allelesOR=15
6CI
=097ndash251
Caucasia
nandothers(505)
rs1806
649
(rs58745895)
g178118152Cgt
TIntro
n1
Basaland
smoker
FEV
1(200
9[39])
T=02631119Daggerin
haplotype
CI=minus8730ndash
(minus17
0)Netherla
nd(2542)
Parkinsonrsquos
disease(2010
[26])
T=0422148Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
Particulatem
attera
ndasthmaCO
PDadmiss
ion(2012[41])
Cwith
lowvitamin
Clevel(OR=
31CI
=15
0ndash630)
UK(209)
rs4243387
(rs6003846
4)g178117765Cgt
TIntro
n1
Basaland
smoker
FEV
1(200
9[39])
T=00914
25Daggerin
haplotype
Netherla
nd(2542)
rs1962142
(rs58448508)
g178113484Agt
GIntro
n1
Ann
ualF
EV1decline
(2011[34])
A=0082in
haplotype
Japanese
(915)
Breastcancer
NRF
2andARE
expressio
n(2012[27])
A(119875=0036)
FinlandKB
CP(452)
rs6726395
(rs57309289)
g178103229Agt
GIntro
n1
Smok
ing-relatedFE
V1decline
and
annu
alFE
V1decline
(2011[34])
G=0884Dagger
A=0082in
haplotype
Japanese
(915)
Basaland
smoker
FEV
1(200
9[39])
G=046
41764Daggerin
haplotype
Netherla
nd(2542)
rs2001350
(rs17515179
rs60883775)
g178100
425Cgt
TIntro
n1
Ann
ualF
EV1decline
(2011[34])
T=0082in
haplotype
Japanese
(915)
Parkinsonrsquos
disease(2010
[26])
T=0986350Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
rs10183914
(rs58731187
rs61374844
)g17809766
6Cgt
TIntro
n3
Parkinsonrsquos
disease(2010
[26])
T=0536188Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
rs2706110
g178092162Tgt
C31015840Flanking
Breastcancer
(2012[27])
TT
OR=2079CI
=118ndash368
FinlandKB
CP(452)
rs2588882
g178087165Ggt
T31015840Flanking
Infection-indu
cedasthma(
2012
[38])
TG
OR=0290CI
=013ndash0
62
Hun
garia
nCaucasia
nGypsy
(307)
minus686in
reference[22]
=minus653in
reference[23]
=currentlyminus214minus684in
reference[22]
=minus651inreference[23]
=currentlyminus212minus651inreference[22]
=minus617in
reference[23]
=currentlyminus178Ch
romosom
econtig(in
tron
31015840flank
ing)
orreversed
(51015840flank
ingprom
oter)a
llelesinbo
ldhave
been
used
inthetextand
TableOR
odds
ratio
CI95confi
denceinterval
14 Oxidative Medicine and Cellular Longevity
minus212GG (98 rs67006649 previously minus684minus651) minus178Callele (73) and GCC
4(53) was predicted to increase
respiratory failure development (hazard ratio = 095 CI091ndash099) in German COPD patients [32] Significant inter-action was also identified between an intronic SNP Gallele (rs6726395 g178103229AgtG 884 frequency) andsmoking status on FEV
1decline relative to the reference
AA allele in the above Japanese cohort [34] Siedlinskiet al [39] reported that the CC genotype of anotherintronic SNP (rs2364723 g178126546GgtC) was associatedwith a lower FEV
1level compared to the wild-type geno-
type (GG) in two Netherland cohorts (CI minus636minus178frequency = 0525 and pooled cohort size = 2542) ThisSNP alone or as a haplotype with 4 more intronic SNPs(rs13001694Grs1806649Trs4243387Trs6726395G)was alsoassociated with high FEV
1levels in individuals that ever
smoked [39] In a Hungarian population of childhoodasthma SNPs at minus178 (CA) and 31015840 flanking (rs2588882TG)loci were inversely associated with infection-induced asthma(OR 0437 CI 028ndash080 OR 0290 CI 013ndash062 resp) andthese SNPs significantly influenced an asthma-environmentalpollution interaction [38]The intronic SNP rs1806649 (CgtT)was associated but not significantly with an increased risk ofhospitalization during high-level particulate matter (PM
10)
periods in asthma or COPD patients (119899 = 209) of the UnitedKingdom (UK) [41] Asthma andCOPDadmission rateswererelated to the increase in environmental PM
10concentration
Importantly effects of interaction between prenatal stressand NRF2 SNPs on descendant pulmonary health wereinvestigated by the Avalon Longitudinal Study in the UKmaternal smoking during pregnancy was not associated withlung function change determined by maximum mild expi-ratory flow (FEF
25ndash75) or with asthma incidence in school-aged children and this relation was not modified by NRF2SNP genotypes [42]However early gestation acetaminophenexposure significantly influenced the risk of asthma andwheezing at the age of 7 years in gt4000 mothers and gt5000children [33] When maternal copies of the minus212A allele werepresent association with asthma (11374891 OR 173 CI122ndash245) and wheezing (11494949 OR 153 95 CI 106ndash220) was significantly increased [33]
Gastrointestinal Disorders While there was no evidence inlung cancer cases studies in Japanese populations suggesteda potential association of NRF2 variations with gastro-intestinal tumorigenesisHelicobacter pylori (H pylori) causesgastritis which can lead to gastric atrophy and cancer Ingastric epithelium from the Japanese cancer cohorts (39gastric cancers 46 controls) H pylori infection was posi-tively correlated with aberrant CpG island methylation oftumor suppressor genes (eg p14) and minus214Gminus212G orminus214Aminus212G NRF2 haplotype was significantly associatedwith increased (OR 290 95 CI 114ndash736) or decreased(OR 033 95 CI 013ndash088) risk of the CpG methyla-tion respectively in the H pylori-infected patients [29]Further study from the same investigators determined thatminus214Aminus212G allele carriers had significantly (119875 = 0022)reduced risk of gastric cancer inH pylori-negative cases [30]The minus214AGminus212AG genotypes were negatively associated
(OR 045 CI 022ndash093) and the minus214Gminus212G genotypeswere positively associated (chronic continuous phenotypeOR 257 CI 101ndash660) with ulcerative colitis (89 patients 141controls) in a Japanese population [31]
Autoimmune Disorders Systemic lupus erythematosus (SLE)is a long-term autoimmune disease more frequently found infemales than in males It affects organs including skin jointskidneys and brain and nephritis is an aggressive character-istic in some patients Genome-wide association studies inhumans identified a suggestive quantitative trait locus nearNRF2 [43] A study of a Mexican Mestizo population (362patients with childhood-onset SLE 379 controls and 212nephritis diagnosed) determined that lupus with nephritiswas significantly (OR 181 CI 104ndash312) associated with theminus214GA SNP in females [25] The same SNPs were notclosely associated with SLE risk in a Japanese cohort [22]Vitiligo is a skin condition in which there is a loss of browncolor (pigment) from areas of skin resulting in irregularwhite patches It is thought to be an autoimmune diseasecaused by loss of cells (melanocytes) that produce brownpigment A study indicated that theminus178A allele increased therisk of vitiligo dose-dependently (OR 1724 95 CI 135ndash221for CA OR 2902 CI 162ndash519 for AA) [35]
Female Disorders It is well known that estrogen metabolites(eg catechols) cause ROS formation suggesting correla-tion of NRF2 and downstream effectors in postmenopausalmammary cancer In a study of a Finish population (KuopioBreast Cancer Project 119899 = 452 patients 370 controls)the minus178AA homozygous genotype (OR 4656 CI = 135ndash1606) and 31015840 flanking rs2706110 (TT OR 2079 CI 118ndash368) genotype were associated with increased risk of breastcancer while the 51015840 flanking minus3306TT homozygous allelewas significantly associated with lower survival (frequency =71219 OR 1687 CI 1105ndash275) [27] suggesting that NRF2genetic polymorphisms affect susceptibility and outcome ofthe patients The minus178A allele carriers together with intronicrs1962142A allele carriers were associated with lowered tissuelevels of NRF2 proteins [27] In postmenopausal women theminus178A allele (OR 179 95CI 370ndash8570) appeared tomodifythe risk of venous thromboembolism caused by oral estrogentherapy (AA or AC frequency = 333) as demonstratedby the French ESTHER study (161 cases 474 controls) [36]An intronic rs1806649CgtT SNP did not associate with breastcancer risk in postmenopausal women [40] However whenthis SNP and other at-risk alleles of ARE-responsive genes(NQO1 HO-1 NOS3) were combined there was a significantgene-dose effect on the breast cancer risk [40] Althoughcoding region SNPs in NRF2 and KEAP1 were identifiedin the Japanese endometrial adenocarcinoma patients noassociation of NRF2 SNPs with the disease was found [44]
Neurodegenerative Diseases Oxidative stress is known to beinvolved in Parkinsonrsquos disease (PD) presumably due toproduction of ROS fromhigh-dopaminemetabolism and lowlevels of antioxidants in the substantia nigra of the brainInvestigators found a protective NRF2 haplotype consistingof four 51015840 flanking SNPs (minus5238Gminus214Aminus212Gminus178C)
Oxidative Medicine and Cellular Longevity 15
and 4 intronic SNPs (rs2886161Ars1806649Ars2001350Ars10183914A) from Swedish (OR 09 CI 060ndash140) and Polish(OR 04 CI 030ndash060) populations (total 165+192 PD cases190+192 controls) [26]The investigators also suggested thatNRF2 haplotype alleles were associated with 2 years earlierage of Alzheimerrsquos disease (AD) onset 4 years earlier age ofposterior subcapsular cataract surgery and 4 years later ageof cortical cataract surgery while they were not significantlyrelated to AD or age-related cataract risk [45]
34 Genetic Mutations in Mouse Nrf2 Tsang et al [46]compiled 673 SNPs in 55 mouse strains and constructedtheir phylogenetic tree to correlate and clarify the originsof strains based on the assembled mouse genome sequenceand SNP data [20 47 48] Recently using the completegenome sequence of C57BL6J (B6) mouse as a referencehigh-density SNP screening in other laboratory strains orin panels of strains has been published (see [17]) Althoughmillions of mouse SNPs (gt10089892 as of December 2012)and haplotype mappings from more than 120 strains havebeen published as valuable references for dissecting thegenetic basis of complex traits [49ndash51] little attention hasbeen paid to polymorphisms of Nrf2 and their correlationwith disease phenotypes
Figure 3 demonstrates the proximal promoter region(minus1 to minus950)51015840-UTR (exon 1 up to TSS) and proteinsequence of mouse Nrf2 based on GRCm38p1 PrimaryAssembly (75704641ndash75675513 bp) mRNA variant1 and protein (NP 035032 encoded by NM 010902234ndash2027 bp) sequences Genetic variations in the Nrf2genome of inbred strains collected from public databasesare listed in Tables S2 and 5 (See Supplementary TableS2) Overall 968 genetic mutations are compiled forNrf2 gene and 5 kb upstream2 kb downstream regions785 SNPs between B6 and another 16 strains wereacquired from the Mouse Phenome Database (MPDhttpphenomejaxorg dbqrtn=snpret1) and additionalSNPs and other mutations were acquired from NCBI dbSNP(httpwwwncbinlmnihgovsnpterm=mus+musculus20nfe2l2) In total 132 mutations are in the promoter (37in proximal 1 kb) 49 in exons (38 in coding region 19 Cns)727 in introns and 60 in the 31015840 flanking region Excludingmutations in the 51015840 and 31015840 flanking sequences murine Nrf2sequences appear to be more highly variable (1 variationper 375 bp) than much of the mouse genome which has anapproximate frequency of one SNP per every 245 bp (httpwwwinformaticsjaxorgmgihomehomepagesstatsallstatsshtmlallstats snp)
Nrf2 was found to be a susceptibility gene from genome-wide linkage analysis in a murine model of hyperoxia-induced ALI [52] A promoter SNP minus103TgtC (previouslypublished as minus336TgtC) in Nrf2 was found and predicted toadd an additional Sp1 binding site in hyperoxia-susceptibleB6 mice but not in resistant C3HHeJ mice [52] Genotypesfrom the SNP and from simple-sequence length polymor-phism markers of the Nrf2 locus (D2Mit248 and D2Mit94)cosegregated in the B6C3F
2mouse cohort [52] and Nrf2
deficient mice were significantly more susceptible to ALI
sub-phenotypes caused by hyperoxia than similarly exposedwild-type mice supporting Nrf2 as a contributor to thephenotypic traits [53] Although no other functional analyseson Nrf2 SNPs or haplotype association studies have beenconducted in inbred mice strains bearing haplotypes suchas multiple Cns in functional domains (eg F71L L451VH543Q and L575M) may be useful to elucidate the role ofNrf2 in differential susceptibility to oxidative diseases
4 Oncogenic Somatic Mutations in HumanNF-E2-Related Factor 2
Somatic mutation is a change in the DNA of somatic cellsthat affects derived cells but is not inherited by offspringEfforts to discover somatic mutations have provided insightinto mutagenesis and cancer development Lung cancerparticularly non-small cell lung cancer (NSCLC) is theleading cause of cancer death worldwide Somatic mutationsof NRF2 and KEAP1 discovered in lung cancer patients havedetermined the oncogenic potential ofNRF2 [54 55] KEAP1somatic mutations were associated with its reduced proteinlevels in lung cancer tissues and cells [56 57] Investigationsof NSCLC in various ethnic populations as well as cancersin gastrointestine breast and prostate have coordinatelydemonstrated that multiple Cns somatic mutations in KEAP1cause dysfunction of the translated protein and in turnconstitutive activation of NRF2 increasing risk of neoplasiaand chemoresistance [12 55 58 59] Somatic mutations ofNRF2 have been detected in various cancer tissues (largelysquamous cell carcinomas) in Asian populations (Table 6)NRF2 mutations were significantly associated with NSCLCcases (squamous cell lung carcinoma adenocarcinoma) ofthe Japanese (107 [54]) the Chinese (23 [60]) andthe Koreans (8 [61]) as well as with lung cancer celllines Smoking history was also correlated with mutationoccurrence in all of the studies [54 60 61] In addition tolung cancers laryngeal squamous carcinoma (13 in [61])esophageal squamous cancer (ESC 22 in [60] 114 in[61]) head and neck cancers (25 in [54]) skin (117 casein [61]) and oral cancer cell lines had somatic changesin NRF2 In contrast to wide-spread KEAP1 mutationsmutations in NRF2 were clustered in DLGETGE motifs ofthe Neh2 domain which are critical in the ldquohinge and latchrdquomodel of KEAP1 binding [12] Similar to KEAP1 somaticmutations it has been postulated that NRF2 mutations incancer cells lead to NRF2 accumulation by suppressing itsubiquitination or KEAP1 binding which eventually confersmalignant potential and resistance to chemotherapy
Most variable sites in NRF2 included aa residues 29 (AspD) 31 (Gly G) 77 (Asp D) and 79 (Glu E) (Table 6)Residue 33 (Ser S) in the Neh2 domain is mutated by eithergenetic or somatic processes (Figure 4) Cns in the EDGFmotif of NRF2 was experimentally determined to impairrecognition of KEAP1 [54] NRF2 mutations were signif-icantly correlated with increased (25-fold) copy number(31 of mutants versus 3 wild types) in Japanese NSCLCcases [63] Aberrant mutation of NRF2 also led to increasedexpression of downstream effectors includingRagD known to
16 Oxidative Medicine and Cellular Longevity
pL575M
pF71L
pH543QpL451V
pA151P pA151V pN159D pQ198delpV105D
pA204T pS224I225S226del pT253S pS278ApD305G
pM409V pR410HpP388L pA389G pT395I
Genomic DNA (reversed contig GRCm38 p1 C57BL6JNM 0109023)
Protein (NP 035032)1
51101151201251301351401451501551
minus400
minus500
minus50
minus100
minus200
minus300
minus450
minus350
minus250
minus150
minus950
minus900
minus850
minus800
minus750
minus700
151
101151201
2001
g minus470gg minusg minus
c minus41c minus
c
c
c c
gg minus 6C
g minus125insTTC g minus118insC
g minus281delC
g minus63delCg minus1 (75704642 c minus234)
Translation start site (c +1)Termination codon (c +2025 +2027)
c minus136 minus134in-frame stop codonc minus8585
g minus41
246
440
minus381
minus11
minus91 minus90
minus332
minus153
minus98
c minus197c minus209 minus202
gminus942 minus899 (cminus1175 minus1132) CpG islands [71]
g 460CgtT
g minus103CCgtT [51]
CgtTCgtG
g minus320CgtT
CgtT
CgtT
CgtTg minus426GgtA
GgtACgtA
CgtA
GgtA
g minus238GgtTTgtC
TgtC
TgtC
TgtCTgtG
g minus229AgtG
CgtG
CgtG
GgtC
gminus826 minus654 (cminus1059 ) CpG islands [71]minus887
Figure 3 DNA (partial promoter and exons 1 and 5) and protein sequence ofmouseNrf2 SNPs and amino acid residues for non-synonymousSNPs are marked Promoter regions bearing 5CpG islands are underlined
151
201251301351401451501551601
Genetic mutation (Table 3 and Figure 2)Somatic mutation (Table 6)
Neh2
Neh3
Neh4Neh5
Neh6
Neh1
Protein isoform 1 (NP 006155)
101151
Figure 4 Genetic and somatic mutation loci in human NRF2 protein
Oxidative Medicine and Cellular Longevity 17
Table5Mou
seup
stream
andexon
varia
tions
ofNfe2
l2locusin17
inbred
strainsR
eference
sequ
ence
isC5
7BL6J
(B6strain
1)geno
me(GI14933824975547698ndash75513576)SNPalleleand
geno
typesareshow
nas
chromosom
econ
tigsequ
enceStrains
2129S1S
vlmJ3AJ
4AKR
J5BA
LBcJ6C3
HH
eJ7C
57BL
6NJ8CA
STEiJ9CB
AJ
10D
BA2J11FVBNJ12LPJ
13N
ODShiLtJ14N
ZOH
ILtJ
15P
WK
PhJ16SPR
ETEiJ
17W
SBEiJ
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs2566
08517
75705565
GA
(CT)
51015840Flankingminus924
NC
GG
GG
GG
GG
GG
GG
GG
AA
Grs47274959
75705562
AT
(TA
)51015840Flankingminus921
NC
AT
TA
TT
AA
TT
AT
AT
TT
Ars216940398
75705554ndash75705555
Tadd(A
add)
51015840Flankingminus913minus914
NC
CGrs239114134
75705540
CT(G
A)
51015840Flankingminus899
NC
CT
TC
TT
CC
TT
CT
CT
CT
Crs46
461765
75705528
TG(A
C)oA
C
51015840Flankingminus887
NC
TG
GT
GG
TT
GG
TG
TG
GG
Trs5144
9853
75705525
CA(G
T)
51015840Flankingminus884
NC
CA
AC
AA
CC
AA
CA
CA
CC
Crs249093111
75705512
CT(G
A)
51015840Flankingminus871
NC
CT
TC
TT
CC
TT
CT
CT
TT
Crs263745200
75705510
CG(G
C)
51015840Flankingminus869
NC
GG
GG
GG
GG
GG
GG
GG
CG
Grs45651867
75705498
TG(A
C)
51015840Flankingminus857
NC
TG
GT
GG
TT
GG
TG
TG
GG
Trs219997531
75705495
GA
(CT)
51015840Flankingminus854
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs251918379
75705451
TA(A
T)
51015840Flankingminus810
NC
TT
TT
TT
TT
TT
TT
TT
TA
Trs27978306
75705449
TA(A
T)
51015840Flankingminus808
NC
TA
AT
AA
TT
AA
TA
TA
TT
Trs216087412
75705429
TG(A
C)lowast
51015840Flankingminus788
NC
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
GTlowast
Tlowast
rs261229914
75705423ndash75705424
AGdel(CT
del)
51015840Flankingminus783minus784
NC
AGrs27978307
75705400
CT(G
A)
51015840Flankingminus759
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs243167395
75705359
GA
(CT)
51015840Flankingminus718
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs27978308
75705307
GA
(CT)
51015840Flankingminus66
6NC
GG
GG
GG
GG
GG
GG
GG
AG
Grs254744
09875705294
AT
(TA
)51015840Flankingminus653
NC
AA
AA
AA
AA
AA
AA
AA
AT
Ars228133419
75705179
GT
(CA
)51015840Flankingminus538
NC
GG
GG
GG
GG
GG
GG
GG
GT
Grs214719520
75705111
GA
(CT)
51015840Flankingminus470
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs2440
87730
75705101
GA
(CT)
51015840Flankingminus46
0NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs227781699
75705081
CG(G
C)
51015840Flankingminus44
0NC
CC
CC
CC
CC
CC
CC
CC
GC
Crs257870353
75705067
CT(G
A)
51015840Flankingminus426
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs24487744
075705022
AG
(TC)
51015840Flankingminus381
NC
AA
AA
AA
AA
AA
AA
AA
AG
Ars234628138
75704973
AC
(TG
)51015840Flankingminus332
NC
AA
AA
AA
AA
AA
AA
AA
AC
A
18 Oxidative Medicine and Cellular Longevity
Table5Con
tinued
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs247275247
75704961
GA
(CT)
51015840Flankingminus320
NC
GG
GG
GG
GG
GG
GG
AG
GG
Grs247519047
75704922
Gdel(Cdel)
51015840Flankingminus281
NC
Grs27978309
(rs51915758)
75704887
GA
C(C
TG
)51015840Flankingminus246
NC
GA
AG
AA
GG
AA
GA
GA
CC
G
rs218139102
75704879
CA(G
T)
51015840Flankingminus238
NC
CC
CC
CC
CC
CC
CC
CC
AC
Crs251990355
75704870
TC(A
G)
51015840Flankingminus229
NC
TT
TT
TT
TT
TT
TT
TT
TC
Trs238746955
75704794
AG
(TC)
51015840Flankingminus153
NC
AA
AA
AA
AA
AA
AA
AA
GA
Ars221664
405
75704786
AG
(TC)
51015840Flankingminus145
NC
AA
AA
AA
AA
AA
AA
AA
AG
Ars217054035
75704766
ndash75704767
GAAadd(TTC
add)
51015840Flankingminus125minus126
NC
GA
rs248182931
75704759ndash75704760
Gadd(C
add)
51015840Flankingminus118
minus119
NC
AGrs217197904
75704744
GA
(CT)
51015840Flankingminus103
(+)S
p1[23]
GA
AG
AA
GG
AA
GA
AA
GG
Ars2644
9364975704704
Gdel(Cdel)
51015840Flankingminus63
NC
Grs2404
81112
757046
82CT(G
A)
51015840Flankingminus41
NC
CC
CC
CC
CC
CC
CC
CC
CT
Crs27978310
75704617
AG
(TC)
UTR
-525
NC
AA
AA
AA
AA
AA
AA
AA
GG
Ars27978311
75704610
CG(G
C)
UTR
-532
NC
GG
GG
GG
GG
GG
GG
GG
GG
Crs27978312
757046
05GA
(CT)
UTR
-537
NC
GA
AG
AA
GG
AA
GA
GA
GG
Grs214784220
757044
99AG
(TC)
UTR
-5143
NC
AA
AA
AA
AA
AA
AA
AA
GG
Ars244146318
757044
98GT
(CA
)UTR
-5144
NC
GG
GG
GG
GG
GG
GG
GG
GT
Grs27978313
757044
97GC
(CG
)UTR
-5145
NC
GC
CG
CC
GC
CC
GC
CC
CC
Crs215431944
757044
93CT(G
A)
UTR
-5149
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs257015697
757044
49GA
(CT)
UTR
-5193
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs252234782
757044
19GT
(CA
)UTR
-5223
NC
Grs1346
0861
75679262
AG
(TC)
Exon
244
6Cn
sF71L
AG
GA
GG
AG
GG
AG
AG
GG
Grs1346
0859
75679202
GA
(CT)lowast
Exon
2506
CsH91=
GA
AG
AA
GG
AA
GA
GA
GG
Grs27978436
75679187
GA
(CT)lowast
Exon
2521
CsT9
6=G
GG
GG
GG
AG
GG
GG
GA
GG
rs226131070
75679178
GA
(CT)
Exon
2530
CsS99=
GG
GG
GG
GG
GG
GG
GG
GA
Grs227683834
75678576
AT
(TA
)Ex
on3
547
CnsV
105D
AA
AA
AA
AA
AA
AA
AA
AT
Ars257321187
75678506
TC(A
G)
Exon
3617
CsP128=
TT
TT
TT
TC
TT
TT
TT
TT
Trs27978444
75678500
TA(A
T)
Exon
3623
CsV130=
TT
TT
TT
TA
TT
TT
TT
AT
Trs227743136
75677686
GA
(CT)
Exon
4662
CsH143=
GG
GG
GG
GG
GG
GG
GG
GA
Grs215327202
75677664
CG(G
C)
Exon
4684
CnsA
151P
CC
CC
CC
CG
CC
CC
CC
CC
Crs247539755
75677663
GA
(CT)
Exon
4685
CnsA
151V
GG
GG
GG
GA
GG
GG
GG
GG
Grs233164
668
75677640
TC(A
G)
Exon
4708
CnsN
159D
TT
TT
TT
TT
TT
TT
TT
TC
T
rs2340
95816
75677162ndash75677160
GCT
del
(AGCdel)
Exon
5826minus
828
Cds-in
delQ
198
GCT
Oxidative Medicine and Cellular Longevity 19
Table5Con
tinued
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs27978452
75677145
CT(G
A)
Exon
5843
CnsA
204T
CC
CC
CC
CC
CC
CC
CC
CC
T
rs225047937
75677086ndash75677078
GAG
ATCG
ATdel
(ATG
CATC
TCdel)
Exon
5902minus
910
Cds-in
del
(S224I225S226)
GAG
ATC
GAT
rs241469868
75676998
TA(G
T)
Exon
5990
CnT2
53S
TT
TT
TT
TT
TT
TT
TT
TA
Trs221602571
75676923
AC
(TG
)Ex
on5
1065
CnsS
278A
AA
AA
AA
AA
AA
AA
AA
AC
Ars252576472
75676876
GA
(CT)
Exon
51112
CsS293=
GG
GG
GG
GG
GG
GG
GG
GA
Grs250802933
75676841
TC(A
G)
Exon
5114
7Cn
sD305G
TT
TT
TT
TT
TT
TT
TT
TC
Trs225425698
75676792
CT(G
A)
Exon
5119
6Cs
P321=
CC
CC
CC
CC
CC
CC
CC
CT
Crs13459064
75676732
CT(G
A)
Exon
51256
CsT3
41=
CT
TC
TT
CT
TT
CT
CT
TC
Crs27978453
75676717
CT(G
A)
Exon
51271
CsA346=
CC
CC
CC
CC
CC
CC
CC
TC
Crs214335034
75676636
AG
(TC)
Exon
51352
CsD373=
AA
AA
AA
AA
AA
AA
AA
AG
Ars24958364
4756766
03AC
(TG
)Ex
on5
1385
CsP3
84=
AA
AA
AA
AA
AA
AA
AA
AC
Ars27978454
75676592
GA
(CT)
Exon
51396
CnsP
388L
GG
GG
GG
GG
GG
GG
GG
AG
Grs215384328
75676589
GC
(CG
)Ex
on5
1399
CnsA
389G
GG
GG
GG
GG
GG
GG
GG
GC
Grs251728286
75676571
GA
(CT)
Exon
51417
CnsT
395I
Grs247602334
75676567
TC(A
G)
Exon
51421
CsV396=
TT
TT
TT
TT
TT
TT
TT
TC
Trs234216231
75676530
TC(A
G)
Exon
51458
CnsM
409V
TT
TT
TT
TT
TT
TT
TT
TC
Trs212904337
75676526
CT(G
A)
Exon
51462
CnsR
410H
CC
CC
CC
CC
CC
CC
CC
CT
Crs252650779
75676522
TC(A
G)
Exon
51466
CsE4
11=
TT
TT
TT
TT
TT
TT
TT
TC
Trs231273560
75676516
TC(A
G)
Exon
51472
CsQ413=
TT
TT
TT
TT
TT
TT
TT
TC
Trs257886949
756764
13GT
(CA
)Ex
on5
1575
CsR4
48=
GG
GG
GG
GG
GG
GG
GG
GT
Grs241537608
756764
04GC
(CG
)Ex
on5
1584
CnsL
451V
GG
GG
GG
GG
GG
GG
GG
GC
Grs227619071
75676312
TC(A
G)
Exon
51676
CsQ481=
TT
TT
TT
TT
TT
TT
TT
TC
Trs258913831
75676290
AG
(TC)
Exon
51698
CsL4
89=
AA
AA
AA
AA
AA
AA
AA
AG
Ars225593319
75676126
AT
(TA
)Ex
on5
1862
CnsH
543Q
Ars4223233
75676105
GA
(CT)
Exon
51883
CsS550=
GG
GG
GG
GA
GG
GG
GG
GG
Grs4223232
75676032
GT
(CA
)Ex
on5
1956
CnsL
575M
Grs4223231
75675816
CG
UTR
-32172
NC
GG
GG
GG
GG
GG
GG
GG
GC
Grs1346
860
75675682
AT
UTR
-32306
NC
ASequ
ence
varia
tions
inmou
seNr
f2wereob
tained
from
Mou
sePh
enom
eDatabase(http
ph
enom
ejaxorgSNP)
andNCB
ISNPdatabase
(http
wwwncbinlm
nihgovSNP)N
CBIreference
sequ
ence
isMus
Musculusstra
inC5
7BL6J
chromosom
e2GRC
m38p1(NC
0000
687
GI37209910875704641ndash756755192
9123
bp)To
tal968
genetic
mutations
arerepo
rted
inNr
f2gene
and5k
bup
stream
(ge75704
642minus
1)and2k
bdo
wnstre
am(le75675512)sequences
asof
Janu
ary2003A
llSN
Psandallelesa
representedas
geno
miccontig
(reversedsequ
ence
indicated)E
xon175704
641ndash75704364(278
bpT
TS=75704
408)
exon
275679431ndash756791
65(267
bp)exon
375678579ndash75678490(90b
p)exon475677713ndash756775
46(168
bp)andexon
57567718
4ndash75678849
(1666
bp)Proteinam
inoacid
(aa)
resid
uesinNP00
61552
(597
aa)Cs
cod
ing-syno
nymou
sCn
scoding
-non
syno
nymou
sAminoacid
A-alanineD-aspartic
acidE
-glutamicacidF-phenylalanineG
-glycineH
-histidineI-iso
leucineL-leu
cineand
M-m
ethion
ineN-
asparagineP-prolin
eQ-glutamineR-arginineS-serineT-threon
ineandV-valin
elowastErrorsin
databasesfi
xedNC
notcon
firmed
20 Oxidative Medicine and Cellular Longevity
Table 6 NRF2 somatic mutations revealed in various human cancers
Domain Amino acid residues DNA mutation Cancer types (cases) ReferencesLocus Wild type Mutant
DLG motif
24 W (Trp) C (Cys) c72GgtCGgtT NSCLC neck ESC [54 62]K (Lys) c72TgtC ESC [62]
26 Q (Gln) E (Glu) c76CgtG NSCLC ESC [54 62]
27 D (Asp) G (Gly)lowast c80GgtA NSCLC [60]Y (Tyr) c79GgtT ESC [61]
28 I (Ile) T (Thr) c83CgtT NSCLC [54]
29 D (Asp) G (Gly) c86AgtG Head and neck ESC [54 62]H (His) c85GgtC NSCLC laynx [60 61]
30 L (Leu) F (Phe) c88CgtT NSCLC ESC [54 62]
31 G (Gly) A (Ala) c92GgtC NSCLC ESC skin [54 60ndash62]
32 V (Val) T (Thr) c95TgtG NSCLC [54]del c93 95delAGT ESC [61]
33ndash36 S-R-E-V S-R-E-V-S-R-E-Vlowast c97 108dupAGTCGAGCCGTA ESC [61]
34 R (Arg) Q (Gln) c101GgtA NCSCL [54 60 61]P (Pro) c101GgtC NSCLC [63]
ETGF motif
75 Q (Gln) H (His) c225AgtC Head and neck ESC [54 62]
77 D (Asp)
V (Val) c230AgtT NSCLC ESC [54 60 62]G (Gly) c230AgtG ESC [62]A (Ala) c230AgtC NSCLC [61]N (Asn) c229GgtA Larynx [61]
78 E (Glu) K (Lys) c232GgtA NSCLC ESC [54 62]
79 E (Glu)
K (Lys) c235GgtA NSCLC ESC [54 61 62]Q (Gln) c235GgtC NSCLC ESC [54 60ndash62]G (Gly) c236AgtG Larynx [61]E-E c234 236dupAGA ESC [61]
80 T (Thr) K (Lys) c239CgtACgtG NSCLC ESC [54 61 62]
80 T (Thr)P (Phe) c238AgtC ESC [62]I (Ile) c239CgtT Head and neck [54]A (Ala) c238AgtG NSCLC [63]
81 G (Gly) V (Val) c242GgtT ESC [61]D (Asp) c242GgtA NSCLC ESC [60ndash62]
82 E (Glu)
D (Asp) c246AgtT ESC oral cancer cell line [54 62]G (Gly) c245AgtG NSCLC [54]Q (Gln)lowast c244GgtC NSCLC ESC [60 61]V (Val)lowast c245AgtT ESC [61]
83 F (Phe) L (Leu)lowast c247TgtC NSCLC [60]NSCLC non-small cell lung cancer ESC esophageal squamous cancer and lowasterrors in reference fixed Number of cases 82 NSCLC and 10 ESC in [62] 125NSCLC 70 ESC 23 larynx and 17 skin in [61] 103 NSCLC and 12 head and neck in [54] 90 NSCLC in [63] 103 NSCLC in [60]
be involved in squamous lung cancer cell proliferation [64]suggesting that the mutation is functional and overcomesKEAP1 inhibition Singh et al [65] determined in vitrothat RNAi-mediated depletion of NRF2 in lung cancer cellsenhanced ROS production and susceptibility to cell deathby ionizing radiation These studies support the concept thatelevated NRF2 and ARE responsiveness provides cancer cellswith proliferative advantage for malignant transformation
and undue protection from anti-cancer therapy Onco-genic epidermal growth factor receptor (EGFR) signaling isrecently found to be critical in NRF2-mediated proliferationof NSCLC cells [66]
Collectively ldquogain of functionrdquo mutations in NRF2 thatreduce KEAP1 recognition are suggested to be predictivemarkers for poor responsiveness to chemotherapy and radi-ation therapy Although NRF2-mediated cellular defense
Oxidative Medicine and Cellular Longevity 21
processes are essential in the initiation stage enhancedNRF2-ARE activity in advanced stages of cancer developmentmay create a favorable intracellular environment for tumorcell growth and survival [67 68] In this context NRF2may be a potential molecular target for the treatment ofradio-resistance cancers especially those that have ldquoloss offunctionrdquo mutations in EGFR KRAS or KEAP1 as well asldquogain of functionrdquo mutations in NRF2
5 Epigenetic Alterations of NF-E2-RelatedFactor 2
Epigenetic modifications are alterations of molecules inter-acting with genes without changes to the primary DNAsequence They include post-translational modification ofhistones DNA methylation events chromatin conforma-tional changes and alterations to noncoding regulatoryRNAs Epigenetic alterations are stable and often inheritablebut are reversible and may affect expression of the geneDysregulation or defects in epigenetic processes particularlyhypermethylation of tumor suppressor gene promoters (egCpG islands) or histone modifications are thought to beassociated with carcinogenesis Investigators have reportedhypermethylation in CpG islands of KEAP1 which wereassociated with reduced KEAP1 expression in human cancersfrom lung prostate colon and so forth [69ndash72] Similar tosomatic mutations epigenetic changes on KEAP1 impairedthe function of its encoded protein leading to constitutiveNRF2 activation
Supporting the role of ldquopathogenic mutationsrdquo in NRF2expression of Nrf2 and downstream Nqo1 was suppressedin prostate tumors of mice (transgenic adenocarcinomaof mouse prostate TRAMP) Among 15 promoter CpGislands located between minus942 and minus654 (cminus1175 cminus1132and cminus1059 cminus887 gap in cminus1131 cminus1060 see Figure 3)hypermethylation of the first 5 CpG islands (minus942 minus899 andcminus1175 cminus1132) was significantly associated with tumorige-nesis [73] Moreover treatment with inhibitors for DNAmethyltransferase and histone deacetylase restored Nrf2expression in these tumor cells [73] A dietary phytochemicalcurcumin known as a DNA hypomethylation agent restoredepigenetically silent Nrf2 expression through CpG demethy-lation in carcinogen-induced mouse tumor cells [74]
The whole genome epigenetic datasets for 5 species arepublicly accessible at NCBI Epigenomics [75 76]The humanNRF2 epigenome of primary cells (breast penis) and H1stem cell line as well as mouse Nrf2 CpG island methylationdata for sperm blood and cerebellum are currently avail-able (httpwwwncbinlmnihgovepigenomics) Althoughno direct evidence of disease-associated epigenetic modu-lation has been identified in human NRF2 various phyto-chemical NRF2 agonists such as sulforaphane and curcuminhave shown their roles in DNA methylation and histonemodification (see reviews by Lee and colleagues eg [77])Taken together epigenetic modifications of theNRF2KEAP1axle are predicted to cause dysregulation of ARE-mediatedcellular defense leading to deleterious health effects and
phytochemical antioxidants as epigenetic modulators forNRF2 are suggested to be useful in cancer prevention
6 Conclusions
NRF2 is evolutionally conserved with high-sequence homol-ogy in many species However it is a highly mutable geneand numerous genetic variants have been discovered inhuman ethnic groups Importantly certain SNPs or haplo-types have been identified in various diseases as ldquoat-riskrdquoalleles and are related to functional alterations In additionto genetic variations multiple somatic mutations identifiedin the KEAP1 recognition domain of NRF2 in cancer cellshave been found to be oncogenic due to dysregulation ofNRF2 homeostasis by its excess ldquogain of functionrdquo Epigeneticalteration of theNRF2 is under investigation and is predictedto have pathogenic influences as learned from mouse andphytochemical agonist studies Continuous updates of Nrf2allelic variants in inbred mouse strains will provide a usefultool for effective experimental designs formodels of oxidativedisorders to provide insight into the disease mechanisms andintervention strategies
Abbreviations
AD Alzheimerrsquos diseaseALI acute lung injuryARE antioxidant response elementbZIP basic leucine zippercds coding DNA sequencesCI confidence intervalCOPD chronic obstructive pulmonary diseaseESC esophageal squamous cancerFEV1 forced expiratory volume in one second
Gbp giga base pairsGST glutathione S-transferaseGWAS genome-wide association studyHGVS Human Genome Variation SocietyHO-1 heme oxygenase-1119867 119901119910119897119900119903119894 119867119890119897119894119888119900119887119886119888119905119890119903 119901119910119897119900119903119894KEAP1 Kelch-like ECH activating protein 1MPD Mouse Phenome DatabaseMRP multidrug resistance proteinNCBI National Center for Biotechnology InformationNeh NRF2-ECH homologyNfe2l2 nuclear factor (erythroid-derived 2)-like 2Nrf2 NF-E2-related factor 2NSCLC nonsmall cell lung cancerNQO1 NAD(P)Hquinone oxidoreductase 1OR odds ratioPD Parkinsonrsquos diseasePM particulate matterROS reactive oxygen speciesSLE systemic lupus erythematosusSNP single nucleotide polymorphismSOD superoxide dismutaseURT untranslated region
22 Oxidative Medicine and Cellular Longevity
Disclosure
Authorrsquos contribution to the Work was done as part of theAuthorrsquos official duties as a NIH employee and is a Work ofthe United States Government Therefore copyright may notbe established in the United States
Conflict of Interests
The author declares that there is no conflict of interests
Acknowledgments
The research related to this paper was supported by theIntramural Research Program of the National Institutes ofHealth of the National Institute of Environmental HealthSciences (NIEHS) Drs Steven Kleeberger and StephanieLondon at the NIEHS provided excellent critical review ofthis paper The author thanks Mrs Jacqui Marzec for herhelpful comments and English editing
References
[1] J Y Chan M C Cheung P Moi K Chan and Y W KanldquoChromosomal localization of the humanNF-E2 family of bZIPtranscription factors by fluorescence in situ hybridizationrdquoHuman Genetics vol 95 no 3 pp 265ndash269 1995
[2] K Itoh K Igarashi N Hayashi M Nishizawa and MYamamoto ldquoCloning and characterization of a novel erythroidcell-derived CNC family transcription factor heterodimerizingwith the small Maf family proteinsrdquo Molecular and CellularBiology vol 15 no 8 pp 4184ndash4193 1995
[3] W O Osburn and T W Kensler ldquoNRF2 signaling an adaptiveresponse pathway for protection against environmental toxicinsultsrdquoMutation Research vol 659 no 1-2 pp 31ndash39 2008
[4] D Papp K Lenti D Modos D Fazekas Z Dul D Tureiet al ldquoThe NRF2-related interactome and regulome containmultifunctional proteins and fine-tuned autoregulatory loopsrdquoFEBS Letters vol 586 no 13 pp 1795ndash1802 2012
[5] X Wang D J Tomso B N Chorley et al ldquoIdentificationof polymorphic antioxidant response elements in the humangenomerdquo Human Molecular Genetics vol 16 no 10 pp 1188ndash1200 2007
[6] D Malhotra E Portales-Casamar A Singh et al ldquoGlobalmapping of binding sites for NRF2 identifies novel targets incell survival response through ChIP-Seq profiling and networkanalysisrdquo Nucleic Acids Research vol 38 no 17 pp 5718ndash57342010
[7] K Chan R Lu J C Chang and Y W Kan ldquoNRF2 a memberof the NFE2 family of transcription factors is not essential formurine erythropoiesis growth and developmentrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 93 no 24 pp 13943ndash13948 1996
[8] K Itoh T Chiba S Takahashi et al ldquoAn NRF2small Mafheterodimer mediates the induction of phase II detoxifyingenzyme genes through antioxidant response elementsrdquo Bio-chemical and Biophysical Research Communications vol 236no 2 pp 313ndash322 1997
[9] FMartin J M vanDeursen R A Shivdasani CW Jackson AG Troutman and P A Ney ldquoErythroid maturation and globin
gene expression inmicewith combined deficiency ofNF-E2 andNrf-2rdquo Blood vol 91 no 9 pp 3459ndash3466 1998
[10] K Itoh N Wakabayashi Y Katoh et al ldquoKEAP1 repressesnuclear activation of antioxidant responsive elements by NRF2through binding to the amino-terminal Neh2 domainrdquo Genesand Development vol 13 no 1 pp 76ndash86 1999
[11] A Kobayashi M I Kang H Okawa et al ldquoOxidative stresssensor KEAP1 functions as an adaptor for Cul3-based E3 ligaseto regulate proteasomal degradation of NRF2rdquo Molecular andCellular Biology vol 24 no 16 pp 7130ndash7139 2004
[12] K Taguchi H Motohashi and M Yamamoto ldquoMolecularmechanisms of the KEAP1-NRF2 pathway in stress responseand cancer evolutionrdquo Genes to Cells vol 16 no 2 pp 123ndash1402011
[13] K Mukaigasa L T Nguyen L Li H Nakajima M Yamamotoand M Kobayashi ldquoGenetic evidence of an evolutionarilyconserved role for NRF2 in the protection against oxidativestressrdquoMolecular and Cellular Biology vol 32 no 21 pp 4455ndash4461 2012
[14] JMaher andMYamamoto ldquoThe rise of antioxidant signalingmdashthe evolution and hormetic actions of NRF2rdquo Toxicology andApplied Pharmacology vol 244 no 1 pp 4ndash15 2010
[15] S C Lo X Li M T Henzl L J Beamer and M HanninkldquoStructure of the KEAP1NRF2 interface provides mechanisticinsight intoNRF2 signalingrdquoThe EMBO Journal vol 25 no 15pp 3605ndash3617 2006
[16] V A McKusick ldquoMendelian inheritance in man and its onlineversion OMIMrdquo American Journal of Human Genetics vol 80no 4 pp 588ndash604 2007
[17] B Yalcin D J Adams J Flint and T M Keane ldquoNext-generation sequencing of experimental mouse strainsrdquo Mam-malian Genome vol 23 no 9-10 pp 490ndash498 2012
[18] D R Bentley S Balasubramanian H P Swerdlow et alldquoAccurate whole human genome sequencing using reversibleterminator chemistryrdquo Nature vol 456 no 7218 pp 53ndash592008
[19] P S G Chain D V Grafham R S Fulton et al ldquoGenomeproject standards in a new era of sequencingrdquo Science vol 326no 5950 pp 236ndash237 2009
[20] RHWaterstonK Lindblad-Toh E Birney J Rogers J F AbrilP Agarwal et al ldquoInitial sequencing and comparative analysis ofthe mouse genomerdquo Nature vol 420 pp 520ndash562 2002
[21] T M Keane L Goodstadt P Danecek M A White K WongB Yalcin et al ldquoMouse genomic variation and its effect onphenotypes and gene regulationrdquoNature vol 477 pp 289ndash2942011
[22] T Yamamoto K Yoh A Kobayashi et al ldquoIdentification ofpolymorphisms in the promoter region of the human NRF2generdquo Biochemical and Biophysical Research Communicationsvol 321 no 1 pp 72ndash79 2004
[23] J M Marzec J D Christie S P Reddy et al ldquoFunctionalpolymorphisms in the transcription factor NRF2 in humansincrease the risk of acute lung injuryrdquo The FASEB Journal vol21 no 9 pp 2237ndash2246 2007
[24] H Fukushima-Uesaka Y Saito K Maekawa et al ldquoGeneticvariations and haplotype structures of transcriptional factorNRF2 and its cytosolic reservoir protein KEAP1 in JapaneserdquoDrug Metabolism and Pharmacokinetics vol 22 no 3 pp 212ndash219 2007
[25] E J Cordova R Velazquez-Cruz F Centeno V Baca and LOrozco ldquoThe NRF2 gene variant -653GA is associated with
Oxidative Medicine and Cellular Longevity 23
nephritis in childhood-onset systemic lupus erythematosusrdquoLupus vol 19 no 10 pp 1237ndash1242 2010
[26] M von Otter S Landgren S Nilsson et al ldquoAssociation ofNRF2-encoding NFE2L2 haplotypes with Parkinsonrsquos diseaserdquoBMCMedical Genetics vol 11 no 1 article 36 2010
[27] JMHartikainenM TengstromVMKosmaV L Kinnula AMannermaa andY Soini ldquoGenetic polymorphisms and proteinexpression of NRF2 and sulfiredoxin predict survival outcomesin breast cancerrdquo Cancer Research vol 72 pp 5537ndash5546 2012
[28] T Arisawa T Tahara T Shibata et al ldquoThe relationshipbetween Helicobacter pylori infection and promoter polymor-phism of the NRF2 gene in chronic gastritisrdquo InternationalJournal of Molecular Medicine vol 19 no 1 pp 143ndash148 2007
[29] T Arisawa T Tahara T Shibata et al ldquoThe influence ofpromoter polymorphism of nuclear factor-erythroid 2-relatedfactor 2 gene on the aberrant DNA methylation in gastricepitheliumrdquo Oncology Reports vol 19 no 1 pp 211ndash216 2008
[30] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene pro-moter polymorphism and gastric carcinogenesisrdquo Hepato-Gastroenterology vol 55 no 82-83 pp 750ndash754 2008
[31] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene promoterpolymorphism is associated with ulcerative colitis in a Japanesepopulationrdquo Hepato-Gastroenterology vol 55 no 82-83 pp394ndash397 2008
[32] C C Hua L C Chang J C Tseng C M Chu Y C Liu andW B Shieh ldquoFunctional haplotypes in the promoter region oftranscription factor NRF2 in chronic obstructive pulmonarydiseaserdquo Disease Markers vol 28 no 3 pp 185ndash193 2010
[33] S O Shaheen R B Newson S M Ring M J Rose-ZerilliJ W Holloway and A J Henderson ldquoPrenatal and infantacetaminophen exposure antioxidant gene polymorphismsand childhood asthmardquo The Journal of Allergy and ClinicalImmunology vol 126 no 6 pp 1141e7ndash1148e7 2010
[34] H Masuko T Sakamoto Y Kaneko et al ldquoLower FEV1 innon-COPD nonasthmatic subjects association with smokingannual decline in FEV1 total IgE levels and TSLP genotypesrdquoInternational Journal of Chronic Obstructive Pulmonary Diseasevol 6 pp 181ndash189 2011
[35] C P Guan M N Zhou A E Xu et al ldquoThe susceptibilityto vitiligo is associated with NF-E2-related factor 2 (NRF2)gene polymorphisms a study on Chinese Han populationrdquoExperimental Dermatology vol 17 no 12 pp 1059ndash1062 2008
[36] J Bouligand O Cabaret M Canonico et al ldquoEffect of NFE2L2genetic polymorphism on the association between oral estrogentherapy and the risk of venous thromboembolism in post-menopausal womenrdquo Clinical Pharmacology amp Therapeuticsvol 89 no 1 pp 60ndash64 2011
[37] D S OrsquoMahony B J Glavan T D Holden C Fong R A BlackG Rona et al ldquoInflammation and immune-related candidategene associations with acute lung injury susceptibility andseverity a validation studyrdquo PLoS ONE vol 7 no 12 Article IDe51104 2012
[38] I Ungvari E Hadadi V Virag et al ldquoRelationship between airpollutionNFE2L2 gene polymorphisms and childhood asthmain aHungarian populationrdquo Journal of CommunityGenetics vol3 no 1 pp 25ndash33 2012
[39] M SiedlinskiD S Postma JMA Boer et al ldquoLevel and courseof FEV1 in relation to polymorphisms inNFE2L2 andKEAP1 inthe general populationrdquo Respiratory Research vol 10 article 732009
[40] C C Hong C B Ambrosone J Ahn et al ldquoGenetic variabilityin iron-related oxidative stress pathways (NRF2 NQ01 NOS3
and HO-1) iron intake and risk of postmenopausal breastcancerrdquo Cancer Epidemiology Biomarkers and Prevention vol16 no 9 pp 1784ndash1794 2007
[41] C Canova C Dunster F J Kelly C Minelli P L Shah CCaneja et al ldquoPM10-induced hospital admissions for asthmaand chronic obstructive pulmonary disease the modifyingeffect of individual characteristicsrdquo Epidemiology vol 23 no 4pp 607ndash615 2012
[42] A J Henderson R B Newson M Rose-Zerilli S M RingJ W Holloway and S O Shaheen ldquoMaternal NRF2 andgluthathione-S-transferase polymorphisms donotmodify asso-ciations of prenatal tobacco smoke exposure with asthma andlung function in school-aged childrenrdquo Thorax vol 65 no 10pp 897ndash902 2010
[43] C Xing A L Sestak J A Kelly et al ldquoLocalization andreplication of the systemic lupus erythematosus linkage signalat 4p16 interaction with 2p11 12q24 and 19q13 in EuropeanAmericansrdquo Human Genetics vol 120 no 5 pp 623ndash631 2007
[44] T F Wong K Yoshinaga Y Monma K Ito H NiikuraS Nagase et al ldquoAssociation of KEAP1 and NRF2 geneticmutations and polymorphisms with endometrioid endometrialadenocarcinoma survivalrdquo International Journal of Gynecologi-cal Cancer vol 21 no 8 pp 1428ndash1435 2011
[45] M von Otter S Landgren S Nilsson et al ldquoNRF2-encodingNFE2L2 haplotypes influence disease progression but not riskin Alzheimerrsquos disease and age-related cataractrdquoMechanisms ofAgeing and Development vol 131 no 2 pp 105ndash110 2010
[46] S Tsang Z Sun B Luke et al ldquoA comprehensive SNP-basedgenetic analysis of inbred mouse strainsrdquoMammalian Genomevol 16 no 7 pp 476ndash480 2005
[47] C M Wade E J Kulbokas III A W Kirby et al ldquoThe mosaicstructure of variation in the laboratory mouse genomerdquoNaturevol 420 no 6915 pp 574ndash578 2002
[48] T Wiltshire M T Pletcher S Batalov et al ldquoGenome-widesingle-nucleotide polymorphism analysis defines haplotypepatterns in mouserdquo Proceedings of the National Academy ofSciences of the United States of America vol 100 no 6 pp 3380ndash3385 2003
[49] K A Frazer E Eskin H M Kang et al ldquoA sequence-basedvariation map of 827 million SNPs in inbred mouse strainsrdquoNature vol 448 no 7157 pp 1050ndash1053 2007
[50] T P Maddatu S C Grubb C J Bult andM A Bogue ldquoMousephenome database (MPD)rdquo Nucleic Acids Research vol 40 ppD887ndashD894 2007
[51] A Kirby H M Kang C M Wade et al ldquoFine mapping in 94inbred mouse strains using a high-density haplotype resourcerdquoGenetics vol 185 no 3 pp 1081ndash1095 2010
[52] H Y Cho A E Jedlicka S P M Reddy L Y Zhang T WKensler and S R Kleeberger ldquoLinkage analysis of susceptibilityto hyperoxia NRF2 is a candidate generdquo American Journal ofRespiratory Cell and Molecular Biology vol 26 no 1 pp 42ndash512002
[53] H Y Cho A E Jedlicka S P M Reddy et al ldquoRole of NRF2in protection against hyperoxic lung injury in micerdquo AmericanJournal of Respiratory Cell and Molecular Biology vol 26 no 2pp 175ndash182 2002
[54] T Shibata T Ohta K I Tong et al ldquoCancer related mutationsin NRF2 impair its recognition by KEAP1-Cul3 E3 ligase andpromote malignancyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 36 pp13568ndash13573 2008
24 Oxidative Medicine and Cellular Longevity
[55] A Singh V Misra R K Thimmulappa et al ldquoDysfunctionalKEAP1-NRF2 interaction in non-small-cell lung cancerrdquo PLoSMedicine vol 3 no 10 article e420 2006
[56] T Ohta K Iijima M Miyamoto et al ldquoLoss of KEAP1 functionactivates NRF2 and provides advantages for lung cancer cellgrowthrdquo Cancer Research vol 68 no 5 pp 1303ndash1309 2008
[57] B Padmanabhan K I Tong T Ohta et al ldquoStructural basisfor defects of KEAP1 activity provoked by its point mutationsin lung cancerrdquoMolecular Cell vol 21 no 5 pp 689ndash700 2006
[58] T Shibata A Kokubu M Gotoh et al ldquoGenetic alteration ofKEAP1 confers constitutive NRF2 activation and resistance tochemotherapy in gallbladder cancerrdquoGastroenterology vol 135no 4 pp 1358e4ndash1368e4 2008
[59] N J Yoo H R Kim Y R Kim C H An and S H LeeldquoSomatic mutations of the KEAP1 gene in common solidcancersrdquo Histopathology vol 60 no 6 pp 943ndash952 2012
[60] Y Hu Y Ju D Lin Z Wang Y Huang S Zhang et alldquoMutation of the NRF2 gene in non-small cell lung cancerrdquoMolecular Biology Reports vol 39 no 4 pp 4743ndash4747 2012
[61] Y R Kim J EOhM S Kim et al ldquoOncogenicNRF2mutationsin squamous cell carcinomas of oesophagus and skinrdquo TheJournal of Pathology vol 220 no 4 pp 446ndash451 2010
[62] T Shibata A Kokubu S Saito et al ldquoNRF2 mutation confersmalignant potential and resistance to chemoradiation therapyin advanced esophageal squamous cancerrdquo Neoplasia vol 13pp 864ndash873 2011
[63] H Sasaki M Shitara K Yokota Y Hikosaka S MoriyamaM Yano et al ldquoIncreased NRF2 gene (NFE2L2) copy numbercorrelates with mutations in lung squamous cell carcinomasrdquoMolecular Medicine Reports vol 6 no 2 pp 391ndash394 2012
[64] H Sasaki M Shitara K Yokota Y Hikosaka S Moriyama MYano et al ldquoRagD gene expression andNRF2mutations in lungsquamous cell carcinomasrdquo Oncology Letters vol 4 pp 1167ndash1170 2012
[65] A Singh M Bodas N Wakabayashi F Bunz and S BiswalldquoGain of NRF2 function in non-small-cell lung cancer cellsconfers radioresistancerdquo Antioxidants and Redox Signaling vol13 no 11 pp 1627ndash1637 2010
[66] T Yamadori Y Ishii SHommaYMorishimaKKurishimaKItoh et al ldquoMolecular mechanisms for the regulation of NRF2-mediated cell proliferation in non-small-cell lung cancersrdquoOncogene vol 31 pp 4768ndash4777 2012
[67] A K Bauer H Y Cho L Miller-Degraff C Walker K HelmsJ Fostel et al ldquoTargeted deletion of NRF2 reduces urethane-induced lung tumor development in micerdquo PLoS ONE vol 6no 10 Article ID e26590 2011
[68] H Y Cho and S R Kleeberger ldquoNRF2 protects against airwaydisordersrdquo Toxicology and Applied Pharmacology vol 244 no1 pp 43ndash56 2010
[69] R Wang J An F Ji H Jiao H Sun and D Zhou ldquoHyperme-thylation of the KEAP1 gene in human lung cancer cell linesand lung cancer tissuesrdquo Biochemical and Biophysical ResearchCommunications vol 373 no 1 pp 151ndash154 2008
[70] P Zhang A Singh S Yegnasubramanian et al ldquoLoss ofkelch-like ECH-associated protein 1 function in prostate cancercells causes chemoresistance and radioresistance and promotestumor growthrdquoMolecular Cancer Therapeutics vol 9 no 2 pp336ndash346 2010
[71] N Hanada T Takahata Q Zhou et al ldquoMethylation of theKEAP1 gene promoter region in human colorectal cancerrdquoBMCCancer vol 12 article 66 2012
[72] L A Muscarella R Barbano V DrsquoAngelo et al ldquoRegulationof KEAP1 expression by promoter methylation in malignantgliomas and association with patientrsquos outcomerdquo Epigeneticsvol 6 no 3 pp 317ndash325 2011
[73] S Yu T O Khor K L Cheung et al ldquoNRF2 expressionis regulated by epigenetic mechanisms in prostate cancer ofTRAMP micerdquo PLoS ONE vol 5 no 1 Article ID e8579 2010
[74] T O Khor Y Huang T Y Wu L Shu J Lee and A N KongldquoPharmacodynamics of curcumin as DNA hypomethylationagent in restoring the expression of NRF2 via promoter CpGsdemethylationrdquo Biochemical Pharmacology vol 82 no 9 pp1073ndash1078 2011
[75] I M Fingerman L McDaniel X Zhang et al ldquoNCBI epige-nomics a new public resource for exploring epigenomic datasetsrdquo Nucleic Acids Research vol 39 supplemen 1 pp D908ndashD912 2011
[76] I M Fingerman X Zhang W Ratzat N Husain R F Cohenand G D Schuler ldquoNCBI epigenomics whatrsquos new for 2013rdquoNucleic Acids Research vol 41 no 1 pp D221ndashD225 2013
[77] J H Lee T O Khor L Shu Z Y Su F Fuentes andA N Kong ldquoDietary phytochemicals and cancer preventionNRF2 signaling epigenetics and cell death mechanisms inblocking cancer initiation and progressionrdquo Pharmacology ampTherapeutics vol 137 no 2 pp 153ndash171 2012
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
2 Oxidative Medicine and Cellular Longevity
Carcinogenesis
Mammary carcinoma (+)Urinary bladder carcinogenesis (+)
Skin tumor (+)
Kidney and bladder
Diabetic nephropathy (+)As toxicity (+)
Brain and neuromuscular systemIschemic brain injury (+)
Cerebral ischemia and stroke (+)Hearing loss and ototoxicity (+)
AirwaysAcute lung injury (+)COPDemphysema (+)Allergy and asthma (+)Pulmonary fibrosis (+)
Steroid resistance (+)
Inflammationimmunity and developmentSepsis (+)Autoimmune encephalomyelitis (+)Rheumatoid arthritis and cartilage destruction (+)
Heart and circulation
Hemolytic anemia (+)
MetabolismLipid homeostasis (+)
Glucose homeostasis (+)
Skin and eyeSkin healing (+)
Uveitis (+)
GastrointestineAlcoholic liver injury (+)
Acetaminophen hepatotoxicity (+)Bile acid homeostasis (+)
Colitis (+)
Various xenobiotic toxicity (+)Cholestatic liver injury (+)
Steatohepatitis (+)Gastroparesis (+)Liver fibrosis (+)
Acute intestinal mucosal injury (+)
Traumatic brain injury (+)
Spinal cord injury complications (+)
Brain leukoencephalopathy with astrogliosis (+)Gliosis and dopaminergic nigrostriatal degeneration (+)Intracerebral hemorrhage-induced brain injury (+)
Cardiac hypertrophy (+)
Ischemia-induced neovascularization (+)Atherosclerosis (minus)
Lupus-like autoimmune nephritis (+)Spermatogenesis (+)
Gastric neoplasia (+)Colorectal cancer (+)
Lung adenomas (minus)
Pseudomonas aeruginosa infection (+)
Bronchopulmonary displasia (+)Respiratory syncytial virus disease (+)
High-fat-diet induced obesity (+)
UV-induced skin aging (+)
Retinal ischemia-reperfusion (+)Retinopathy of prematurity (+)
Ischemic nephrotoxicity (+)Cisplatin nephrotoxicity (+)
Pollutant (diesel exhaust O3) toxicity (+)
Parkinsonrsquos disease (+)
Nrf 2
Cardiomyocyte damage (+)
Figure 1 Role of Nrf2 in human pathogenesis learned from model studies using mice genetically deficient in Nrf2 (+) indicates protectiveor beneficial effects and (minus) indicates aberrant roles
NRF2 [11]) However modifications of KEAP1 (eg cysteineresidues) and NRF2 (eg serine residues) under stressedconditions activate NRF2 by liberating it from a ldquohinge andlatchrdquo NRF2-KEAP1 affinity binding allowing its nucleartranslocation [12]
In the current review I address genetic aspects of humanNRF2 and its homologs in other vertebrate species Sequencevariations in humanNRF2 andmurineNrf2 including single-nucleotide polymorphisms (SNPs)were collected frompublicdatabases and compiled Mutations that have been associatedwith disease risks are defined Nongenetic variations includ-ing somatic mutations and epigenetic modifications are alsodescribed Although the current review does not deal withmutations in other species recent characterization of nrf2mutant zebrafishwhichwere hypersensitive to environmentaltoxicants [13] also provides a useful investigational tool
2 Sequence of NF-E2-Related Factor 2and Cross-Species Homology
Homology scores of gene (coding DNA sequences cds) andprotein across 10 species were compared with human NRF2The highest sequence homology (98-99) was with chim-panzee and rhesus monkey while the lowest similarity wasfound with zebrafish (Table 1) While there is approximately
about 83 homology in cds and protein sequences of humansand rodents 51015840-untranslated regions (51015840-UTR UTR-5) ofthese strains extend differentially (114 bp in human 233 bp inmouse and 82 bp in rat) and the human 51015840-UTR does notshare significant sequence homology with either rat ormouse(the rat is 94 homologous with the 31015840 portion of mouse 51015840-UTR)
Human NRF2 is located in the cytogenetic band 2q312of chromosome 2 spanning 178095031ndash178129859 bp as acomplementary sequence (gene ID 4780 Table 1) MurineNrf2 maps as a complementary sequence to chromosome2 C3 (4475 centimorgan) and spans 75675519ndash75704641(gene ID 18024 Table 1) The complete cds of NRF2 is2859 bp and there are 14 transcript variants reported(httpuseastensemblorgHomo sapiens) Mouse Nrf2mRNA spans 2469 bp and another variant has been re-ported (httpuseastensemblorgMus musculus) The hu-man NRF2 protein (ID NP 006155) contains 605 amino acid(aa) residues with molecular weight of 677 kDa (isoform1) and total 12 isoforms are published (the National Centerfor Biotechnology Information NCBI httpwwwncbinlmnihgovrefseqrsg eEnsemble httpuseastensemblorgindexhtml UniProt Consortium httpwwwuniprotorguniprotQ16236 httpwwwuniprotorguniprotQ60795)Mouse Nrf2 protein (ID NP 035032) comprises 597 aa at668 kDa (Table 1) Structurally there are 6 NRF2-ECH
Oxidative Medicine and Cellular Longevity 3
Table1Geneo
rtho
logy
ofNF-E2
-related
factor
2acrossthes
pecies
Species
Hum
anHom
osapiens
Cat
Felis
catus
Chim
panzee
Pantro
glodytes
Dog
Canislupus
familiaris
Cattle
Bostaurus
Officialfullname
nucle
arfactor
(erythroid-derived
2)-like
2Antho
rnam
enu
clear
factor
erythroid2-related
factor
2NF-E2
-related
factor
2NFE
2-related
factor
2Antho
rnam
eHEB
P1mdash
mdashmdash
mdashGenes
ynon
yms
NFE
2L2NRF
2NFE
2L2NRF
2NFE
2L2NRF
2NFE
2L2NRF
2NFE
2L2NRF
2Ch
romosom
emap
2q312
C12B
362
Genom
esequence
(NCB
IBuild)
NC
0000
0211
(GRC
h37p
10)
NC
1087301
(Feliscatus-62)
NC
0064703
(Pan
troglod
ytes-214)
NC
0066183
(CanFam31)
AC00
01591
(Bos
taurus
UMD
31)
Prim
arysource
HGNC
7782
mdashmdash
mdashBO
S1716
GeneID
mapping
(orie
ntationlowast
)
4780
178095031minus178129859(minus)
1010
9881
2165356974minus
165392728(minus)
7426
22181721941minus181756837(minus)
4788
1320989206minus21021893
(minus)
4970
2419659540minus19692048
Ensembleg
eneID
(mapping
)EN
SG00
00011604
4(178092323minus178257425)
mdashEN
SPTR
G00
000012677
(181721949minus181756285)
ENSC
AFG
0000
0013506
(20987569minus
21022230)
ENSB
TAG00
000019255
(19659540minus19692048)
RefSeq
IDm
RNA
size
NM
006164
4(G
I372620346)2859b
p
XM0039908931
(GI410968909)
predicted2394
bp
XM00114
58762
(GI332814815)predicted
2850
bp
XM5359753
(GI345797173)
predicted2570
bp
NM
0010116
782
(GI147904941)240
9bp
ProteinID
(aaMW
sect )NP00
61552
(60567696)
XP0039909421
(60667797)
XP00114
58762
(60567686)
XP5359751
(60167087)
NP0010116
782
(60767813)
UniProt
IDQ16236
mdashA2T
6Y9
mdashQ5N
UA62
Varia
nts
14transcrip
ts12
isoform
s3transcrip
ts3iso
form
s5transcrip
ts5iso
form
s2transcrip
ts2iso
form
smdash
Hom
olog
ygeneprotein
100100
893914
999999
893888
905891
4 Oxidative Medicine and Cellular Longevity
Table1Con
tinued
Species
Rhesus
mon
key
Macacamulatta
Chicken
Gallusgallus
Zebrafish
Daniorerio
Mou
seMus
musculus
Rat
Rattu
snorvegicus
Officialfullname
nucle
arfactor
(erythroid-derived
2)-like
2nu
clear
factorerythroid-derived
2lik
e2Antho
rnam
enu
clear
factor
erythroid2-related
factor
2NF-E2
-related
factor
2NFE
2-related
factor
2Antho
rnam
eHEB
P1mdash
mdashmdash
mdash
Genes
ynon
yms
NFE
2L2NRF
2Mmu966
NFE
2L2NRF
2nfe2l2N
rf2N
rf2a
wufc15g09wufj6
7e03
Nfe2
l2N
rf2A
I194320
Nfe2
l2N
rf2
Chromosom
emap
127
92C
3(4475
cM)
3q23
Genom
esequence
(NCB
IBuild)
NC
0078691
NC
0060
943
NC
0071205
(Zv9)
NC
0000
687
(GRC
m38p1)
NC
0051023
(Rno
r50)
Prim
arySource
mdashCG
NC
4960
4ZF
INZ
DB-GEN
E-030723-2
MGI108420
RGD620360
GeneIDmapping
(orie
ntationlowast
)70
7606
40848940minus40
8846
00(minus)
3960
1415304546minus15320356
3601
491643746minus
1670288
1802
475675519minus757046
41(minus)
8361
969041647minus69069070
(minus)
Ensembleg
eneID
(mapping
)EN
SMMUG00
0000
01861
(40848948minus
40884174)
ENSG
ALG
0000
0009240
(16897956minus
16922637)
ENSD
ARG
0000
0042824
(1643631minus1672715)
ENSM
USG
0000
0015839
(75675513minus
757046
41)
ENSR
NOG00
0000
01548
(58366
693minus
58394118)
RefSeq
IDm
RNA
size
NM
0012576071
(GI383872375)2335b
pNM
2051171
(GI45384113)2555
bpNM
1828891
(GI33504557)2149
bpNM
0109023
(GI76573877)2469
bpNM
0317892
(GI402692377)2305b
pProteinID
(aaMW
sect )NP001244
5361
(606
67703)
NP9904
481
(58265160)
NP8783091
(58665757)
NP0350321
(59766770)
NP1139771
(59766825)
UniProt
IDF7GPD
8Q90834
Q8JIM
1Q60795
O54968
Varia
nts
2transcrip
ts2iso
form
s1transcript1isoform
1transcript1isoform
1transcript
1transcript1isoform
Hom
olog
ygeneprotein
988990
726674
546491
834825
838832
Referto
httpwwwncbinlm
nihgovhom
ologene2412
and
reference
[14]forcross-speciesho
molog
yDetails
ofho
molog
yscores
and
sequ
encescan
bealso
obtained
byblastagainsthu
man
sequ
ence
(http
blastn
cbin
lmnihgov)
Furtherinform
ation
ontranscrip
tvaria
ntsand
protein
isoform
sareview
able
bygene
IDat
NCB
I(http
wwwncbinlm
nihgovrefseqrsg)
and
eEn
semble
(http
useastensembleo
rgin
dexhtml)or
byproteinID
atUniProt(http
wwwun
iproto
rg)
sect MWpredicted
molecularweightfrom
NCB
I(MW
inUniProtvarie
sslightly)Re
fSeqreference
sequ
enceaaam
ino
acidsandlowastgene
incomplem
ento
rientation
Oxidative Medicine and Cellular Longevity 5
Table 2 Protein domains of NF-E2-related factor 2
Domains Amino acid positionslowast Predicted functionsHuman (605 aa) Mouse (597 aa)
Neh216ndash89
DLG motif 17ndash32ETGF motif 77ndash82
16ndash89 KEAP1 repression through DLGETGF motif-DC motif binding fastredox-sensitive proteasomal degradation
Neh4 111ndash134 111ndash134 Translocation and transactivation Phosphorylation or CBP bindingNeh5 182ndash209 172ndash201
Neh6 337ndash394(or 338ndash388) 328ndash385 Degron motif-associated constitutive turnover slow redox-insensitive
(Keap1-independent)
Neh1435ndash568
basic motif 503ndash518leucine zipper 525ndash539
427ndash560basic motif 494ndash509leucine zipper 517ndash531
Dimerization for nuclear translocation DNA binding through basicmotif-leucine zipper
Neh3 569ndash605 561ndash597 CHD6 binding stability or transactivationlowastVaries slightly among publications
homology (Neh) domains configuring the protein sequenceof either species (Table 2) and the potential functions of eachregion particularly the highly conserved KEAP1-bindingNeh2 and DNA- (ARE-) binding Neh1 domains have beenintensively investigated [12 14 15]
3 Genetic Variation of NF-E2-Related Factor 2in Human and Mouse
31 Evolution Genome Sequence and Polymorphism Dis-covery in Human and Mouse While rare and monogenicMendelian diseases are inheritable mutations in a single gene[16] many common diseases are complex traits and thedisease phenotypes are affected by variants inmultiple geneticloci Recent advancements in high-throughput technologyhave enabled sequencing of entire mammalian genomes[17ndash19] and information on DNA sequence and variationhas facilitated the study of complex traits of human dis-orders Genome-wide association studies (GWAS) examinewhether SNPs are associated with important disease traitsand ascertains ldquoat-riskrdquo genotypes that are significantly moreprevalent in the affected group than in the nonaffected groupThe HapMap Project (httphapmapncbinlmnihgov) hasmapped combinations of alleles at specific loci (haplotypes)that is common patterns of sequence variation in severalhuman populations It has supported efficient mapping ofmultiple loci for complex traits in GWAS Candidate geneapproaches based on findings from GWAS of similar dis-orders have also been useful for determining the potentialgenetic mechanisms of diseases
The evolutionary divergence of human and mouse lin-eages occurred for roughly 75million years and their genomesequences have been altered by nearly one substitution (ordeletion or addition) for every twonucleotides [20]Howeverthis slow evolution process resulted in a high degree ofconservation across the two species which allows alignmentof orthologous sequences gt90 of the human and mousegenomes is partitioned into corresponding regions of con-served synteny and at the nucleotide level approximately 40of the human genome is aligned to themouse [20]Due to this
fact biomedical studies of human genes are complemented byexperimental manipulation of corresponding mouse genesand they have aided functional understanding of genesin human health Following the 2003 completion of theHuman Genome Project of approximately 31 giga base pairs(Gbp) the Mouse Genome Project assembled the completegenome sequence of one strain (C57BL6J 2716965481 bp)in 2011 Using this reference strain whole genome sequenc-ing data across 16 additional inbred strains were done(httpwwwsangeracukresourcesmousegenomes [21])Discovery of high-density SNPs in the mouse genome sup-ports evolutionary history of the strain and provides a toolto investigate models of human disease processes that cannotoften be practically achieved through direct human studies
32 GeneticMutations in HumanNRF2 HumanNRF2 codesthree major isoforms of protein (Figure 2) Transcript variant2 (NM 0011454122 2746 bp) has an alternate promoter 51015840-UTR and a downstream start codon compared to variant 1(NM 0061644 2859 bp) It encodes an isoform 2 missing N-terminal 16 aa (NP 001138884 or Q16236-2 589 aa) relativeto isoform 1 (NP 0061552 or Q16236 605 aa) Isoform 3(NP 0011388851 or Q16236-3 582 aa) is encoded by tran-script variant 3 (NM 0011454132 2725 bp) and lacks aninternal segment relative to isoform 2 due to an alternate in-frame splice site in the 31015840 coding region In public databasesmore than 583 sequence mutations are reported in NRF2(34827 bp) and 7000 bp upstream (Table S1 data acquired asof December 2012) (See Table S1 in Supplementary Materialavailable on line at httpdxdoiorg1011552013286524)NRF2 locates on 178130354-178129304 bp of GRCh37p10PrimaryAssembly and Figure 2 shows sequences of proximalpromoter (minus1 to minus500) partial mRNA variant 1 including 51015840-UTR (exon 1 up to TSS) and protein isoform 1 (NP 006155encoded by variant 1 NM 0061644 556ndash2373 bp) Basedon the current assembly and sequence update previouspromoter positions minus686 [22]minus653 [23] are identified asminus214 minus684 [22]minus651 [23] as minus212 and minus650 [22]minus617 [23]asminus178Overall frequency ofNRF2 SNPs and othermutationsis about 1 per 72 bp The genetic mutations include 37 in
6 Oxidative Medicine and Cellular Longevity
Translation start site for variants 2 and 3
1 missed in isoforms 2 and 3pA72T pS99P pQ121H missed in isoform 3
pP406S pE423D pK428R pR437W pR449C
minus400
minus500
minus50minus100
minus200
minus300
minus450
minus350
minus250
minus150
g minus
g
pE152K pV155I pL177V
pV268M
pR517K pH453L
pS365I pS387N pK389R pL309F pI317M pD341V
pK219R pP233S pK237 = fs
51101151201251301351401451501551601
Protein isoform 1 (NP 006155)
g minus66minus65insC
Termination codon (c +2371 +2373)
Translation start site (c +1)
151
101151201251301351401451501551601
2351
frame stop codonc minus156 154 in-old + 1 [23]
old + 1 [22]
g minus1 (178130354)
c minus96delC
c minus438 minus437delTT
minus89 minus87delGCC
transcription start site)+1 (c minus555
p R43Q p R43W
Genomic DNA (reversed contig GRCh37 p10 primary assemblyNM 0061644)minus477
minus214 g minus212gminus188
c 9minus95
g minus178
c minus304
c 8minus13
c 2minus16
TgtA
CgtT
GgtA
GgtA
GgtA
GgtT
GgtCAgtG
AgtCAgtG
Figure 2 DNA (variant 1 partial promoter and exons 1 and 5) and protein (isoform 1) sequence of Human NRF2 SNPs and amino acidresidues for nonsynonymous SNPs are marked
the 51015840 flanking promoter and 59 in exons (Tables 3 and S1)Among exon SNPs 26 are nonsynonymous (Cns) mutationsA triplet repeat variation (rs143406266 GCC
4versus GCC
5
previously published as minus20minus6) in the 51015840-UTR was uniquelyidentified in Asian populations [22 24]
33 SNPs Haplotypes and Association with Disease RiskThe use of gene knockout mice in model systems hasprovided potential insights into the role of NRF2 in thepathogenesis of various human disorders (see Figure 1)Recent epidemiological and association studies have revealedsignificant associations of NRF2 sequence variations withdisease risks which further supportsNRF2 as a susceptibilitygene Most of the phenotype-associated variants are in thepromoter region and presumed to be involved in NRF2 generegulation Table 4summarizes NRF2 SNP andor haplotypealleles that have been associated with oxidant-related diseaserisks Interestingly there is no evidence for exon SNPs as at-risk alleles For convenience and consistency intronic and31015840 distal SNP alleles are presented as chromosome contig(HGVS) alleles while promoter and exon SNPs are presentedas reversed contig alleles throughout the text
Pulmonary Diseases NRF2 SNPs in the promoter and intron1 sequences have been investigated for their potential asso-ciations with risk of pulmonary critical disorders includingacute lung injury (ALI) cigarette smoke-induced chronicobstructive pulmonary disease (COPD) and asthma Aheterozygous CA SNP at minus178 position (rs6721961TgtCor TgtG previously minus617 or minus650) significantly increasedthe risk for developing ALI following major trauma inEuropean and African-American populations (odds ratioOR 644 95 confidence interval CI 134ndash308 allelicfrequency = 119 at 21180) [23] Promoter activity ofthe A allele (AC or AA) determined in vivo and invitro was significantly lower than CC allele at that locus(minus178 in an ARE-like motif) indicating that it is a func-tional SNP for autoregulation [23] The minus178GG werealso nominally associated with ALI-related 28-day mor-tality following systemic inflammatory response syndrome[37] In a Japanese cohort SNP haplotype (rs2001350Trs6726395Ars1962142Ars2364722Ars6721961T) co-tainingthe minus178AA homozygote was associated with an annualdecline of rapid forced expiratory volume in one second(FEV1) in relation to cigarette-smoking status [34] In addi-
tion a promoter and 51015840-UTR SNP haplotype consistingof minus214G allele (52 rs35652124 previously minus686minus653)
Oxidative Medicine and Cellular Longevity 7Ta
ble3Geneticmutations
inprom
oter
andexon
sofh
uman
NRF
2
IDMap
onchromosom
e2lowast
(HGVS
name)
Positionin
Nfe2
l2
(HGVS
name)
Region
s(position
from
mRN
A)
Varia
tioncla
ssand
consequences
(HGVS
name)dagger
Minor
allelefrequ
ency
(MAF)
MAcoun
tsDagger(coh
ort
size)
MAFsources
rs16865105
g178136629AgtC
cminus555minus
6770Tgt
G51015840Flanking
(minus6770)
SNP
C=01928421
1000
Genom
esrs7557529
g178135097CgtT
cminus555minus
5238GgtA
51015840Flanking
(minus5238)
SNP
nars6750320
g178131796CgtT
cminus555minus
1937GgtA
51015840Flanking
(minus1937)
SNP
T=0030266
1000
Genom
esrs18116
2518
g178131774TgtC
cminus555minus
1915AgtG
51015840Flanking
(minus1915)
SNP
C=000
051
1000
Genom
esrs1900
44775
g178131746CgtT
cminus555minus
1887GgtA
51015840Flanking
(minus1887)
SNP
T=000
051
1000
Genom
esrs185117338
g178131704AgtG
cminus555minus
1845Tgt
C51015840Flanking
(minus1845)
SNP
G=000
051
1000
Genom
esrs149947189
g178131697CgtT
cminus555minus
1838GgtA
51015840Flanking
(minus1838)
SNP
T=000
092
1000
Genom
esrs139771244
g178131625AgtG
cminus555minus
1766Tgt
C51015840Flanking
(minus1766)
SNP
G=000
092
1000
Genom
esrs6747203
g17813160
4CgtG
cminus555minus
1745GgtC
51015840Flanking
(minus1745)
SNP
G=000
613
1000
Genom
esrs193101749
g178131504TgtC
cminus555minus
1645AgtG
51015840Flanking
(minus1645)
SNP
C=000
4610
1000
Genom
esrs190630762
g178131495GgtC
cminus555minus
1636Cgt
G51015840Flanking
(minus1636)
SNP
C=000
092
1000
Genom
esrs183764
402
g178131366CgtA
cminus555minus
1507GgtT
51015840Flanking
(minus1507)
SNP
A=000
051
1000
Genom
esrs19122296
4g178131211GgtA
cminus555minus
1352Cgt
T51015840Flanking
(minus1352)
SNP
A=000
092
1000
Genom
esrs187137522
g178131165TgtG
cminus555minus
1306AgtC
51015840Flanking
(minus1306)
SNP
G=000
092
1000
Genom
esrs182620359
g178131158AgtG
cminus555minus
1299Tgt
C51015840Flanking
(minus1299)
SNP
G=000
051
1000
Genom
esrs4893819
(rs61433302)
g178131134CgtT
cminus555minus
1275GgtA
51015840Flanking
(minus1275)
SNP
C=04263931
1000
Genom
es
rs191547130
g178131017CgtT
cminus555minus
1158GgtA
51015840Flanking
(minus1158)
SNP
T=000
051
1000
Genom
esrs188422217
g178131003AgtG
cminus555minus
1144TgtC
51015840Flanking
(minus114
4)SN
PG=000143
1000
Genom
esrs143047764
g178130865AgtG
cminus555minus
1006Tgt
C51015840Flanking
(minus1006)
SNP
G=000
6915
1000
Genom
esrs74432849
g178130766CgtA
cminus555minus
907GgtT
51015840Flanking
(minus907)
SNP
nars116
79252
g178130691CgtG
cminus555minus
832GgtC
51015840Flanking
(minus832)
SNP
nars12993217
g178130516AgtG
cminus555minus
657TgtC
51015840Flanking
(minus657)
SNP
nars115644
826
g17813044
2TgtA
cminus555minus
583AgtT
51015840Flanking
(minus583)
SNP
A=0015133
1000
Genom
esrs140803524
g178130431GgtA
cminus555minus
572CgtT
51015840Flanking
(minus572)
SNP
A=000
4610
1000
Genom
es
rs776844
20g178130427TgtC
cminus555minus
568AgtG
51015840Flanking
(minus568)
SNP
C=0033974
C=0190(84)
1000
Genom
es[24]
rs183651094
g178130336AgtT
cminus555minus
477TgtA
51015840Flanking
(minus477)
SNP
T=000184
1000
Genom
es
rs35652124
sect
(rs57695243)
g178130073TgtC
cminus555minus
214AgtG
51015840Flanking
(minus214)
SNP
C=03512767
T=0429(84)
T=0413181Dagger
C=033827Dagger
C=03517
69
1000
Genom
es[24]
[22]
[23]
[25]
rs6706649
sectg178130071CgtT
cminus555minus
212GgtA
51015840Flanking
(minus212)
SNP
T=0078170
T=004
8(84)
T=004
821Dagger
T=00753Dagger
1000
Genom
es[24]
[22]
[23]
rs1506
48896
g178130047CgtG
cminus555minus
188GgtC
51015840Flanking
(minus188)
SNP
G=000235
G=000
6(84)
1000
Genom
es[24]
rs6721961
sect
(rs117801448)
g178130037TgtC
TgtG
cminus555minus
178AgtC
AgtG
51015840Flanking
(minus178)
SNP
T=0150328
T=0283124Dagger
T=031325Dagger
T=0321(84)
1000
Genom
es[22]
[23]
[24]
8 Oxidative Medicine and Cellular Longevity
Table3Con
tinued
IDMap
onchromosom
e2lowast
(HGVS
name)
Positionin
Nfe2
l2
(HGVS
name)
Region
s(position
from
mRN
A)
Varia
tioncla
ssand
consequences
(HGVS
name)dagger
Minor
allelefrequ
ency
(MAF)
MAcoun
tsDagger(coh
ort
size)
MAFsources
rs20134560
4g178129924178129925insG
cminus555-66minus555-65insC
51015840Flanking
(minus66minus65)
Insertion
G=0017939
1000
Genom
es
rs200432479
g178129741178
129742delAA
cminus438minus437delT
Texon
1UTR
-51015840(118minus119
)Dele
tion
-=000378
-=000
6(84)
1000
Genom
es[24]
rs75485459
g178129608CgtA
cminus304GgtT
Exon
1UTR
-51015840(252)
SNP
nars192086766
g17812946
6CgtT
cminus162GgtA
Exon
1UTR
-51015840(394)
SNP
T=002248
1000
Genom
esmdash
g17812944
2GgtA
cminus138CgtT
Exon
1UTR
-51015840(418)
SNP
A=0012(84)
[24]
rs7166
8246
g17812940
0delG
cminus96delC
Exon
1UTR
-51015840(460)
Dele
tion
nars187291840
g178129399CgtT
cminus95GgtA
Exon
1UTR
-51015840(461)
SNP
T=0054912
01000
Genom
es
rs143406266
g178129391178129393delGGC
cminus89minus87delGCC
Exon
1UTR
-51015840(467minus469)
Dele
tion
-=064
4282Dagger
-=0589(84)
[22]
[24]
rs182428269
g178098918GgtA
c127Cgt
TEx
on2(682)
Cns(pArg43Trp)
A=000
051
1000
Genom
esrs35248500
g178098917CgtT
c128GgtA
Exon
2(683)
Cns(pArg43Gln)
T=000
613
1000
Genom
esrs1135118
g178098831CgtT
c214GgtA
Exon
2(769)
Cns(pAla72Th
r)TDagger
[23]
rs19969166
0g178098829AgtT
c216Tgt
AEx
on2(771)
Cs(pAla72=)
namdash
g178098769GgtA
c276Cgt
TEx
on2(831)
Cs(pIle92=)
TDagger[23]
rs5031039
g178098750AgtG
c295Tgt
CEx
on2(850)
Cns(pSer99P
ro)
G=0Dagger
[23]
rs200239262
g178098017CgtG
c363GgtC
Exon
3(918)
Cns(pGln121H
is)na
rs183034165
g178098008TgtC
c372GgtA
Exon
3(927)
Cs(pAla124=
)T=000
092
T=000
6(84)
1000
Genom
es[24]
rs199970826
g178097996CgtT
c384GgtA
Exon
3(939)
Cs(pPro128=
)T=000
051
1000
Genom
esrs201992337
g178097260CgtT
c454GgtA
Exon
4(100
9)Cn
s(pGlu152Lys)
nars201589693
g178097251CgtT
c463GgtA
Exon
4(1018)
Cns(pV
al155Ile)
T=000
051
1000
Genom
es
rs35577826
g178097185AgtC
c529Tgt
GEx
on4(1084)
Cns(pLeu177Va
l)C=000143
Alt0005Dagger
1000
Genom
es[23]
rs181513314
g178096710CgtT
c621GgtA
Exon
5(1176)
Cn(pLeu207=
)T=000
051
1000
Genom
esrs60132461
g178096675TgtC
c656AgtG
Exon
5(1211)
Cns(pLys219Arg)
C=000184
1000
Genom
es
rs139187151
g178096634GgtA
c697Cgt
TEx
on5(1252)
Cns(pPro233Ser)
A=000
051
A=0012Dagger
(84)
1000
Genom
es[24]
rs35557421
g178096620d
elT
c711delA
Exon
5(1266)
Fram
eshiftdeletio
n(pLys237=fs)
na
rs34154613
g178096529CgtT
c802GgtA
Exon
5(1357)
Cns(pV
al268M
et)
T=000184
1000
Genom
esrs141363120
g178096
406GgtA
c925Cgt
TEx
on5(14
80)
Cns(pLeu309Ph
e)A=000378
1000
Genom
esrs201661476
g178096380AgtC
c951Tgt
GEx
on5(1506)
Cns(pIle
317M
et)
na
Oxidative Medicine and Cellular Longevity 9
Table3Con
tinued
IDMap
onchromosom
e2lowast
(HGVS
name)
Positionin
Nfe2
l2
(HGVS
name)
Region
s(position
from
mRN
A)
Varia
tioncla
ssand
consequences
(HGVS
name)dagger
Minor
allelefrequ
ency
(MAF)
MAcoun
tsDagger(coh
ort
size)
MAFsources
rs199673454
g178096309TgtA
c1022AgtT
Exon
5(1577)
Cns(pAs
p341Va
l)A=000
051
1000
Genom
esrs35007548
g178096299GgtA
c1032CgtT
Exon
5(1587)
Cs(pSer344=
)A=000
092
1000
Genom
esrs200209692
g178096287TgtC
c104
4AgtG
Exon
5(1599)
Cs(pLeu348=
)C=000
051
1000
Genom
es
mdashg178096237CgtA
c1094GgtT
Exon
5(1649)
Cns(pSer365Ile
)A=0125273
A=000
6Dagger(84)
PubM
ed[24]
rs201214197
g178096171CgtT
c1160GgtA
Exon
5(1715)
Cns(pSer387Asn)
T=000
051
1000
Genom
esrs200494292
g178096165TgtC
c1166AgtG
Exon
5(1721)
Cns(pLys389Arg)
nars186171287
g178096115GgtA
c1216CgtT
Exon
5(1771)
Cns(pPro4
06Ser)
A=000
051
1000
Genom
esrs182276775
g178096062CgtA
c1269GgtT
Exon
5(1824)
Cns(pGlu423A
sp)
A=000
051
1000
Genom
esrs201560221
g17809604
8TgtC
c1283AgtG
Exon
5(1838)
Cns(pLys428Arg)
nars189238236
g178096043AgtG
c1288TgtC
Exon
5(1843)
Cs(pLeu430=
)G=000
051
1000
Genom
esrs184287392
g178096022GgtA
c1309CgtT
Exon
5(1864)
Cns(pArg437Trp)
A=000
051
1000
Genom
esrs201871588
g178095986GgtA
c1345CgtT
Exon
5(19
00)
Cns(pAg449Cy
s)na
rs181294188
g178095985TgtC
c1346GgtA
Exon
5(19
01)
Cns(pArg44
9His)
T=000
092
1000
Genom
esrs20169046
6g178095973TgtA
c1358AgtT
Exon
5(19
13)
Cns(pHis4
53Leu)
A=000
051
1000
Genom
esrs105704
4(rs52789869)
g178095781CgtT
c1550GgtA
Exon
5(2105)
Cns(pArg517Lys)
TDagger[23]
rs200750800
g178095603AgtG
c1728TgtC
Exon
5(2283)
Cs(pTyr576=
)na
rs200175942
g178095567AgtG
c1764TgtC
Exon
5(2319)
Cs(pAsp588=
)G=000
051
1000
Genom
esrs77547666
g178095495GgtC
clowast18Cgt
GEx
on5UTR
-31015840(2391)
SNP
C=000
6915
1000
Genom
esrs73031353
g178095425TgtC
clowast88AgtG
Exon
5UTR
-31015840(2461)
SNP
C=000184
1000
Genom
esrs675944
3g178095345TgtC
clowast168AgtG
Exon
5UTR
-31015840(2541)
SNP
C=000
419
1000
Genom
esrs188674558
g178095279CgtA
clowast234GgtT
Exon
5UTR
-31015840(2607)
SNP
A=000
051
1000
Genom
esrs77685897
g178095247AgtG
clowast266TgtC
Exon
5UTR
-31015840(2639)
SNP
G=000
092
1000
Genom
esrs1057092
g178095162TgtG
clowast351AgtC
Exon
5UTR
-31015840(2724)
SNP
nars3197704
g178095162TgtG
clowast351AgtC
Exon
5UTR
-31015840(2724)
SNP
nars184701151
g178095159TgtC
clowast354AgtG
Exon
5UTR
-31015840(2727)
SNP
C=000276
1000
Genom
esrs11543307
g178095153AgtG
clowast360TgtC
Exon
5UTR
-31015840(2733)
SNP
nars111874043
g178095146AgtG
clowast367TgtC
Exon
5UTR
-31015840(2740)
SNP
nars34012004
g178095102AgtC
clowast411TgtG
Exon
5UTR
-31015840(2784)
SNP
C=0071
[24]
10 Oxidative Medicine and Cellular Longevity
Table3Con
tinued
IDMap
onchromosom
e2lowast
(HGVS
name)
Positionin
Nfe2
l2
(HGVS
name)
Region
s(position
from
mRN
A)
Varia
tioncla
ssand
consequences
(HGVS
name)dagger
Minor
allelefrequ
ency
(MAF)
MAcoun
tsDagger(coh
ort
size)
MAFsources
rs201481890
g178095090178095091in
sTclowast
422lowast423insA
Exon
5UTR
-31015840
(2795minus
2796)
Insertion
na
rs3082500
g178095089178095090d
elTT
delTTinsT
clowast423lowast424d
elAAinsA
Exon
5UTR
-31015840
(2796minus
2797)
Dele
tion
insertion
na
rs71792546
(rs71796710)
g178095079d
elT
clowast425delA
Exon
5UTR
-31015840(2798)
Dele
tion
na
rs1057106
g178095078AgtC
AgtT
clowast435TgtA
TgtG
Exon
5UTR
-31015840(2808)
SNP
na
rs34176791
g178095076AgtC
clowast437TgtG
Exon
5UTR
-31015840(2810)
SNP
C=000235
1000
Genom
esrs35911553
g178095045CgtT
clowast46
8GgtA
Exon
5UTR
-31015840(2841)
SNP
T=000
6915
1000
Genom
esSequ
ence
varia
tions
inup
stream
andexon
sofh
uman
NRF
2fro
m655varia
tions
availablein
publicdatabase
asof
Decem
ber2012
(583
activ
esomeSN
Psmergedge14
SNPs
citedin
PubM
ed)lowastNCB
Ireference
sequ
ence
NC
0000
0211
(Hom
osapiens
chromosom
e2G
RCh37p
10prim
arya
ssem
bly)spanning
178095033ndash1781298
59bp
(com
plem
ent34827
bpforexons
andintro
ns)51015840-Flank
ingregions
startat17812
9860
bp(minus1)in
transcrip
tvariant
1HGVS
Hum
anGenom
eVa
riatio
nSo
ciety
Position
sinvaria
nt1(NM
006164
4)2859
bpsectSN
Pscitedin
PubM
edE
xon11781298
59ndash17812
9260
(600
bpT
TS=1781293
04)exon
2178098999ndash
178098733(267
bp)exon
3178098067ndash1780979
78(90b
p)exon41780973
11ndash178097120
(192b
p)and
exon
5178096736ndash
178095033(1704b
p)daggerProteinam
inoacid
(aa)
resid
uesiniso
form
1(N
P00
61552605
aa)Cn
scoding
-non
syno
nymou
sCs
cod
ing-syno
nymou
sDaggerheterozygositydetectednano
tavailable
Oxidative Medicine and Cellular Longevity 11
Table4Hum
anNRF
2SN
Psassociated
with
diseaser
isk
IDMap
onchromosom
e2 (H
GVS
name)
Region
class
Dise
asea
ssociatio
nandreferences
Risk
allelesa
ndstatistics
Ethn
icgrou
p(num
bero
fcase)
rs7557529
g178135097CgtT
minus5238
Ggt
AParkinsonrsquos
disease(2010
[26])
G=0718252Dagger
inhaplotype
OR=090
or040
CI=
060ndash140or
03ndash06
Swedish
Polish
Caucasian
(357)
rs2886162
g178133165AgtG
minus3306
TgtC
Breastcancer
survival(2012[27])
TT=0324
OR=16
87C
I=1105ndash275
FinlandKB
CP(452)
Chronicg
astritisgastric
ulcer(2007
[28])
GDagger
GDaggerin
haplotypefor
ulcer(OR=
252C
I=119ndash
545)
Japanese
(159)
P14methylationin
gastric
cancer
Hpyloriinfectio
n(2008[29])
Gin
haplotype
OR=290C
I=114ndash
736
Japanese
(209)
Gastriccancer
inHpylori-n
egative
cases(2008
[30])
Ain
haplotype
119875=0022
Japanese
(209)
rs35652124
(rs57695243)
g178130073TgtC
minus214Agt
G(previou
slyminus653[23]
orminus686[22])
Ulcerativec
olitis(2008
[31])
AG
(OR=045C
I=022ndash0
93)
G(O
R=257C
I=10
1ndash660)
Japanese
(89)
Lupu
swith
neph
ritisin
female(2010
[25])
GA
OR=18
1CI
=10
4ndash312
Mexican
mestizo(362)
Parkinsonrsquos
disease(2010
[26])
A=0884312Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
COPD
(2010[32])
G=052Dagger
hazard
ratio
=095C
I=091ndash0
99
(Haplotype)
German
(69)
Gastriculcer(2007
[28])
GDaggerin
haplotype
OR=252C
I=119ndash
545
Japanese
(159)
P14methylationin
gastric
cancer
H
pyloriinfection(2008[29])
Gin
haplotype
OR=290C
I=114ndash
736
Japanese
(209)
Gastriccancer
inHpylori-negative
cases(2008
[30])
Gin
haplotype119875=0022
Japanese
(209)
rs6706
649
g178130071CgtT
minus212Ggt
A(previou
slyminus651[23]
orminus684[22])
Ulcerativec
olitis(2008
[31])
AG
(OR=045C
I=022ndash0
93)
G(O
R=257C
I=10
1ndash660)
Japanese
(89)
Maternalacetaminop
henandasthma
(2010[33])
A=02321137Dagger
OR=17
3CI
=12
2245
UKALSPA
C(gt40
00mothersgt
5000
child
ren)
12 Oxidative Medicine and Cellular Longevity
Table4Con
tinued
IDMap
onchromosom
e2 (H
GVS
name)
Region
class
Dise
asea
ssociatio
nandreferences
Risk
allelesa
ndstatistics
Ethn
icgrou
p(num
bero
fcase)
COPD
(2010[32])
G=098Daggerin
haplotype
hazard
ratio
=095C
I=091ndash0
99
German
(69)
Parkinsonrsquos
disease(2010
[26])
G=0972343Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
03ndash06
Swedish
Polish
Caucasian
(357)
Acutelun
ginjury
follo
wingtrauma
(2007[23])
CA=0119Dagger
OR=644
CI=
134ndash
3080
Caucasia
nAfrican-
American
(164
)
Ann
ualF
EV1decline
(2011[34])
A=0082in
haplotype
Japanese
(915)
Vitiligo(2008[35])
CAA
AOR=17
24C
I=13
5ndash221
Chinese(300)
COPD
(2010[32])
G=073Daggerin
haplotype
hazard
ratio
=095C
I=091ndash0
99
German
(69)
rs6721961
(rs117801448)
g178130037TgtC
TgtG
minus178Agt
GAgt
C(previou
slyminus617[23]
orminus650[22])
Parkinsonrsquos
disease(2010
[26])
C=0980346Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
Postm
enop
ausalvenou
sthrombo
embo
lism
(2011[36])
A=033311
OR=25CI
=370ndash8570
Caucasia
n(161)
Breastcancer
survivalandNRF
2proteinexpressio
n(2012[27])
AA
OR=4656CI
=13
5ndash1606
FinlandKB
CP(452)
Acutelun
ginjury-related
mortality
followingsyste
micinflammatory
respon
sesynd
rome(2012
[37])
GG
OR=97
3CI
=12
7ndash7480
Caucasia
n(750)
Infection-indu
cedasthma(
2012
[38])
AC
OR=0437CI
=028ndash0
80
Hun
garia
nCaucasia
nGypsy
(307)
rs143406266
g178129391178
129393delGGC
Exon
1(467ndash4
69G
CC)
COPD
(2010[32])
GCC
4=053Dagger
hazard
ratio
=095C
I=091ndash0
99
Taiwanese(69)
rs2886161
g178127839Tgt
ClowastIntro
n1
Parkinsonrsquos
disease(2010
[26])
T=0878309Dagger
inhaplotype
OR=09or
04CI
=06ndash
14or
03ndash06
Swedish
Polish
Caucasian
(357)
rs2364723
g178126546Ggt
CIntro
n1
Basaland
smoker
FEV
1(200
9[39])
CI=minus6360simminus1780C
=0525Dagger
Also
ashaplotype
Netherla
nd(2542)
rs2364722
g178124787Agt
GIntro
n1
Ann
ualF
EV1decline
(2011[34])
A=0082in
haplotype
Japanese
(915)
rs13001694
g178118990Agt
GIntro
n1
Basaland
smoker
FEV
1(200
9[39])
G=040
11578Dagger
inhaplotype
Netherla
nd(2542)
Oxidative Medicine and Cellular Longevity 13
Table4Con
tinued
IDMap
onchromosom
e2 (H
GVS
name)
Region
class
Dise
asea
ssociatio
nandreferences
Risk
allelesa
ndstatistics
Ethn
icgrou
p(num
bero
fcase)
Breastcancer
[40]
Twith
NQO1N
OS3H
O1risk
allelesOR=15
6CI
=097ndash251
Caucasia
nandothers(505)
rs1806
649
(rs58745895)
g178118152Cgt
TIntro
n1
Basaland
smoker
FEV
1(200
9[39])
T=02631119Daggerin
haplotype
CI=minus8730ndash
(minus17
0)Netherla
nd(2542)
Parkinsonrsquos
disease(2010
[26])
T=0422148Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
Particulatem
attera
ndasthmaCO
PDadmiss
ion(2012[41])
Cwith
lowvitamin
Clevel(OR=
31CI
=15
0ndash630)
UK(209)
rs4243387
(rs6003846
4)g178117765Cgt
TIntro
n1
Basaland
smoker
FEV
1(200
9[39])
T=00914
25Daggerin
haplotype
Netherla
nd(2542)
rs1962142
(rs58448508)
g178113484Agt
GIntro
n1
Ann
ualF
EV1decline
(2011[34])
A=0082in
haplotype
Japanese
(915)
Breastcancer
NRF
2andARE
expressio
n(2012[27])
A(119875=0036)
FinlandKB
CP(452)
rs6726395
(rs57309289)
g178103229Agt
GIntro
n1
Smok
ing-relatedFE
V1decline
and
annu
alFE
V1decline
(2011[34])
G=0884Dagger
A=0082in
haplotype
Japanese
(915)
Basaland
smoker
FEV
1(200
9[39])
G=046
41764Daggerin
haplotype
Netherla
nd(2542)
rs2001350
(rs17515179
rs60883775)
g178100
425Cgt
TIntro
n1
Ann
ualF
EV1decline
(2011[34])
T=0082in
haplotype
Japanese
(915)
Parkinsonrsquos
disease(2010
[26])
T=0986350Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
rs10183914
(rs58731187
rs61374844
)g17809766
6Cgt
TIntro
n3
Parkinsonrsquos
disease(2010
[26])
T=0536188Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
rs2706110
g178092162Tgt
C31015840Flanking
Breastcancer
(2012[27])
TT
OR=2079CI
=118ndash368
FinlandKB
CP(452)
rs2588882
g178087165Ggt
T31015840Flanking
Infection-indu
cedasthma(
2012
[38])
TG
OR=0290CI
=013ndash0
62
Hun
garia
nCaucasia
nGypsy
(307)
minus686in
reference[22]
=minus653in
reference[23]
=currentlyminus214minus684in
reference[22]
=minus651inreference[23]
=currentlyminus212minus651inreference[22]
=minus617in
reference[23]
=currentlyminus178Ch
romosom
econtig(in
tron
31015840flank
ing)
orreversed
(51015840flank
ingprom
oter)a
llelesinbo
ldhave
been
used
inthetextand
TableOR
odds
ratio
CI95confi
denceinterval
14 Oxidative Medicine and Cellular Longevity
minus212GG (98 rs67006649 previously minus684minus651) minus178Callele (73) and GCC
4(53) was predicted to increase
respiratory failure development (hazard ratio = 095 CI091ndash099) in German COPD patients [32] Significant inter-action was also identified between an intronic SNP Gallele (rs6726395 g178103229AgtG 884 frequency) andsmoking status on FEV
1decline relative to the reference
AA allele in the above Japanese cohort [34] Siedlinskiet al [39] reported that the CC genotype of anotherintronic SNP (rs2364723 g178126546GgtC) was associatedwith a lower FEV
1level compared to the wild-type geno-
type (GG) in two Netherland cohorts (CI minus636minus178frequency = 0525 and pooled cohort size = 2542) ThisSNP alone or as a haplotype with 4 more intronic SNPs(rs13001694Grs1806649Trs4243387Trs6726395G)was alsoassociated with high FEV
1levels in individuals that ever
smoked [39] In a Hungarian population of childhoodasthma SNPs at minus178 (CA) and 31015840 flanking (rs2588882TG)loci were inversely associated with infection-induced asthma(OR 0437 CI 028ndash080 OR 0290 CI 013ndash062 resp) andthese SNPs significantly influenced an asthma-environmentalpollution interaction [38]The intronic SNP rs1806649 (CgtT)was associated but not significantly with an increased risk ofhospitalization during high-level particulate matter (PM
10)
periods in asthma or COPD patients (119899 = 209) of the UnitedKingdom (UK) [41] Asthma andCOPDadmission rateswererelated to the increase in environmental PM
10concentration
Importantly effects of interaction between prenatal stressand NRF2 SNPs on descendant pulmonary health wereinvestigated by the Avalon Longitudinal Study in the UKmaternal smoking during pregnancy was not associated withlung function change determined by maximum mild expi-ratory flow (FEF
25ndash75) or with asthma incidence in school-aged children and this relation was not modified by NRF2SNP genotypes [42]However early gestation acetaminophenexposure significantly influenced the risk of asthma andwheezing at the age of 7 years in gt4000 mothers and gt5000children [33] When maternal copies of the minus212A allele werepresent association with asthma (11374891 OR 173 CI122ndash245) and wheezing (11494949 OR 153 95 CI 106ndash220) was significantly increased [33]
Gastrointestinal Disorders While there was no evidence inlung cancer cases studies in Japanese populations suggesteda potential association of NRF2 variations with gastro-intestinal tumorigenesisHelicobacter pylori (H pylori) causesgastritis which can lead to gastric atrophy and cancer Ingastric epithelium from the Japanese cancer cohorts (39gastric cancers 46 controls) H pylori infection was posi-tively correlated with aberrant CpG island methylation oftumor suppressor genes (eg p14) and minus214Gminus212G orminus214Aminus212G NRF2 haplotype was significantly associatedwith increased (OR 290 95 CI 114ndash736) or decreased(OR 033 95 CI 013ndash088) risk of the CpG methyla-tion respectively in the H pylori-infected patients [29]Further study from the same investigators determined thatminus214Aminus212G allele carriers had significantly (119875 = 0022)reduced risk of gastric cancer inH pylori-negative cases [30]The minus214AGminus212AG genotypes were negatively associated
(OR 045 CI 022ndash093) and the minus214Gminus212G genotypeswere positively associated (chronic continuous phenotypeOR 257 CI 101ndash660) with ulcerative colitis (89 patients 141controls) in a Japanese population [31]
Autoimmune Disorders Systemic lupus erythematosus (SLE)is a long-term autoimmune disease more frequently found infemales than in males It affects organs including skin jointskidneys and brain and nephritis is an aggressive character-istic in some patients Genome-wide association studies inhumans identified a suggestive quantitative trait locus nearNRF2 [43] A study of a Mexican Mestizo population (362patients with childhood-onset SLE 379 controls and 212nephritis diagnosed) determined that lupus with nephritiswas significantly (OR 181 CI 104ndash312) associated with theminus214GA SNP in females [25] The same SNPs were notclosely associated with SLE risk in a Japanese cohort [22]Vitiligo is a skin condition in which there is a loss of browncolor (pigment) from areas of skin resulting in irregularwhite patches It is thought to be an autoimmune diseasecaused by loss of cells (melanocytes) that produce brownpigment A study indicated that theminus178A allele increased therisk of vitiligo dose-dependently (OR 1724 95 CI 135ndash221for CA OR 2902 CI 162ndash519 for AA) [35]
Female Disorders It is well known that estrogen metabolites(eg catechols) cause ROS formation suggesting correla-tion of NRF2 and downstream effectors in postmenopausalmammary cancer In a study of a Finish population (KuopioBreast Cancer Project 119899 = 452 patients 370 controls)the minus178AA homozygous genotype (OR 4656 CI = 135ndash1606) and 31015840 flanking rs2706110 (TT OR 2079 CI 118ndash368) genotype were associated with increased risk of breastcancer while the 51015840 flanking minus3306TT homozygous allelewas significantly associated with lower survival (frequency =71219 OR 1687 CI 1105ndash275) [27] suggesting that NRF2genetic polymorphisms affect susceptibility and outcome ofthe patients The minus178A allele carriers together with intronicrs1962142A allele carriers were associated with lowered tissuelevels of NRF2 proteins [27] In postmenopausal women theminus178A allele (OR 179 95CI 370ndash8570) appeared tomodifythe risk of venous thromboembolism caused by oral estrogentherapy (AA or AC frequency = 333) as demonstratedby the French ESTHER study (161 cases 474 controls) [36]An intronic rs1806649CgtT SNP did not associate with breastcancer risk in postmenopausal women [40] However whenthis SNP and other at-risk alleles of ARE-responsive genes(NQO1 HO-1 NOS3) were combined there was a significantgene-dose effect on the breast cancer risk [40] Althoughcoding region SNPs in NRF2 and KEAP1 were identifiedin the Japanese endometrial adenocarcinoma patients noassociation of NRF2 SNPs with the disease was found [44]
Neurodegenerative Diseases Oxidative stress is known to beinvolved in Parkinsonrsquos disease (PD) presumably due toproduction of ROS fromhigh-dopaminemetabolism and lowlevels of antioxidants in the substantia nigra of the brainInvestigators found a protective NRF2 haplotype consistingof four 51015840 flanking SNPs (minus5238Gminus214Aminus212Gminus178C)
Oxidative Medicine and Cellular Longevity 15
and 4 intronic SNPs (rs2886161Ars1806649Ars2001350Ars10183914A) from Swedish (OR 09 CI 060ndash140) and Polish(OR 04 CI 030ndash060) populations (total 165+192 PD cases190+192 controls) [26]The investigators also suggested thatNRF2 haplotype alleles were associated with 2 years earlierage of Alzheimerrsquos disease (AD) onset 4 years earlier age ofposterior subcapsular cataract surgery and 4 years later ageof cortical cataract surgery while they were not significantlyrelated to AD or age-related cataract risk [45]
34 Genetic Mutations in Mouse Nrf2 Tsang et al [46]compiled 673 SNPs in 55 mouse strains and constructedtheir phylogenetic tree to correlate and clarify the originsof strains based on the assembled mouse genome sequenceand SNP data [20 47 48] Recently using the completegenome sequence of C57BL6J (B6) mouse as a referencehigh-density SNP screening in other laboratory strains orin panels of strains has been published (see [17]) Althoughmillions of mouse SNPs (gt10089892 as of December 2012)and haplotype mappings from more than 120 strains havebeen published as valuable references for dissecting thegenetic basis of complex traits [49ndash51] little attention hasbeen paid to polymorphisms of Nrf2 and their correlationwith disease phenotypes
Figure 3 demonstrates the proximal promoter region(minus1 to minus950)51015840-UTR (exon 1 up to TSS) and proteinsequence of mouse Nrf2 based on GRCm38p1 PrimaryAssembly (75704641ndash75675513 bp) mRNA variant1 and protein (NP 035032 encoded by NM 010902234ndash2027 bp) sequences Genetic variations in the Nrf2genome of inbred strains collected from public databasesare listed in Tables S2 and 5 (See Supplementary TableS2) Overall 968 genetic mutations are compiled forNrf2 gene and 5 kb upstream2 kb downstream regions785 SNPs between B6 and another 16 strains wereacquired from the Mouse Phenome Database (MPDhttpphenomejaxorg dbqrtn=snpret1) and additionalSNPs and other mutations were acquired from NCBI dbSNP(httpwwwncbinlmnihgovsnpterm=mus+musculus20nfe2l2) In total 132 mutations are in the promoter (37in proximal 1 kb) 49 in exons (38 in coding region 19 Cns)727 in introns and 60 in the 31015840 flanking region Excludingmutations in the 51015840 and 31015840 flanking sequences murine Nrf2sequences appear to be more highly variable (1 variationper 375 bp) than much of the mouse genome which has anapproximate frequency of one SNP per every 245 bp (httpwwwinformaticsjaxorgmgihomehomepagesstatsallstatsshtmlallstats snp)
Nrf2 was found to be a susceptibility gene from genome-wide linkage analysis in a murine model of hyperoxia-induced ALI [52] A promoter SNP minus103TgtC (previouslypublished as minus336TgtC) in Nrf2 was found and predicted toadd an additional Sp1 binding site in hyperoxia-susceptibleB6 mice but not in resistant C3HHeJ mice [52] Genotypesfrom the SNP and from simple-sequence length polymor-phism markers of the Nrf2 locus (D2Mit248 and D2Mit94)cosegregated in the B6C3F
2mouse cohort [52] and Nrf2
deficient mice were significantly more susceptible to ALI
sub-phenotypes caused by hyperoxia than similarly exposedwild-type mice supporting Nrf2 as a contributor to thephenotypic traits [53] Although no other functional analyseson Nrf2 SNPs or haplotype association studies have beenconducted in inbred mice strains bearing haplotypes suchas multiple Cns in functional domains (eg F71L L451VH543Q and L575M) may be useful to elucidate the role ofNrf2 in differential susceptibility to oxidative diseases
4 Oncogenic Somatic Mutations in HumanNF-E2-Related Factor 2
Somatic mutation is a change in the DNA of somatic cellsthat affects derived cells but is not inherited by offspringEfforts to discover somatic mutations have provided insightinto mutagenesis and cancer development Lung cancerparticularly non-small cell lung cancer (NSCLC) is theleading cause of cancer death worldwide Somatic mutationsof NRF2 and KEAP1 discovered in lung cancer patients havedetermined the oncogenic potential ofNRF2 [54 55] KEAP1somatic mutations were associated with its reduced proteinlevels in lung cancer tissues and cells [56 57] Investigationsof NSCLC in various ethnic populations as well as cancersin gastrointestine breast and prostate have coordinatelydemonstrated that multiple Cns somatic mutations in KEAP1cause dysfunction of the translated protein and in turnconstitutive activation of NRF2 increasing risk of neoplasiaand chemoresistance [12 55 58 59] Somatic mutations ofNRF2 have been detected in various cancer tissues (largelysquamous cell carcinomas) in Asian populations (Table 6)NRF2 mutations were significantly associated with NSCLCcases (squamous cell lung carcinoma adenocarcinoma) ofthe Japanese (107 [54]) the Chinese (23 [60]) andthe Koreans (8 [61]) as well as with lung cancer celllines Smoking history was also correlated with mutationoccurrence in all of the studies [54 60 61] In addition tolung cancers laryngeal squamous carcinoma (13 in [61])esophageal squamous cancer (ESC 22 in [60] 114 in[61]) head and neck cancers (25 in [54]) skin (117 casein [61]) and oral cancer cell lines had somatic changesin NRF2 In contrast to wide-spread KEAP1 mutationsmutations in NRF2 were clustered in DLGETGE motifs ofthe Neh2 domain which are critical in the ldquohinge and latchrdquomodel of KEAP1 binding [12] Similar to KEAP1 somaticmutations it has been postulated that NRF2 mutations incancer cells lead to NRF2 accumulation by suppressing itsubiquitination or KEAP1 binding which eventually confersmalignant potential and resistance to chemotherapy
Most variable sites in NRF2 included aa residues 29 (AspD) 31 (Gly G) 77 (Asp D) and 79 (Glu E) (Table 6)Residue 33 (Ser S) in the Neh2 domain is mutated by eithergenetic or somatic processes (Figure 4) Cns in the EDGFmotif of NRF2 was experimentally determined to impairrecognition of KEAP1 [54] NRF2 mutations were signif-icantly correlated with increased (25-fold) copy number(31 of mutants versus 3 wild types) in Japanese NSCLCcases [63] Aberrant mutation of NRF2 also led to increasedexpression of downstream effectors includingRagD known to
16 Oxidative Medicine and Cellular Longevity
pL575M
pF71L
pH543QpL451V
pA151P pA151V pN159D pQ198delpV105D
pA204T pS224I225S226del pT253S pS278ApD305G
pM409V pR410HpP388L pA389G pT395I
Genomic DNA (reversed contig GRCm38 p1 C57BL6JNM 0109023)
Protein (NP 035032)1
51101151201251301351401451501551
minus400
minus500
minus50
minus100
minus200
minus300
minus450
minus350
minus250
minus150
minus950
minus900
minus850
minus800
minus750
minus700
151
101151201
2001
g minus470gg minusg minus
c minus41c minus
c
c
c c
gg minus 6C
g minus125insTTC g minus118insC
g minus281delC
g minus63delCg minus1 (75704642 c minus234)
Translation start site (c +1)Termination codon (c +2025 +2027)
c minus136 minus134in-frame stop codonc minus8585
g minus41
246
440
minus381
minus11
minus91 minus90
minus332
minus153
minus98
c minus197c minus209 minus202
gminus942 minus899 (cminus1175 minus1132) CpG islands [71]
g 460CgtT
g minus103CCgtT [51]
CgtTCgtG
g minus320CgtT
CgtT
CgtT
CgtTg minus426GgtA
GgtACgtA
CgtA
GgtA
g minus238GgtTTgtC
TgtC
TgtC
TgtCTgtG
g minus229AgtG
CgtG
CgtG
GgtC
gminus826 minus654 (cminus1059 ) CpG islands [71]minus887
Figure 3 DNA (partial promoter and exons 1 and 5) and protein sequence ofmouseNrf2 SNPs and amino acid residues for non-synonymousSNPs are marked Promoter regions bearing 5CpG islands are underlined
151
201251301351401451501551601
Genetic mutation (Table 3 and Figure 2)Somatic mutation (Table 6)
Neh2
Neh3
Neh4Neh5
Neh6
Neh1
Protein isoform 1 (NP 006155)
101151
Figure 4 Genetic and somatic mutation loci in human NRF2 protein
Oxidative Medicine and Cellular Longevity 17
Table5Mou
seup
stream
andexon
varia
tions
ofNfe2
l2locusin17
inbred
strainsR
eference
sequ
ence
isC5
7BL6J
(B6strain
1)geno
me(GI14933824975547698ndash75513576)SNPalleleand
geno
typesareshow
nas
chromosom
econ
tigsequ
enceStrains
2129S1S
vlmJ3AJ
4AKR
J5BA
LBcJ6C3
HH
eJ7C
57BL
6NJ8CA
STEiJ9CB
AJ
10D
BA2J11FVBNJ12LPJ
13N
ODShiLtJ14N
ZOH
ILtJ
15P
WK
PhJ16SPR
ETEiJ
17W
SBEiJ
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs2566
08517
75705565
GA
(CT)
51015840Flankingminus924
NC
GG
GG
GG
GG
GG
GG
GG
AA
Grs47274959
75705562
AT
(TA
)51015840Flankingminus921
NC
AT
TA
TT
AA
TT
AT
AT
TT
Ars216940398
75705554ndash75705555
Tadd(A
add)
51015840Flankingminus913minus914
NC
CGrs239114134
75705540
CT(G
A)
51015840Flankingminus899
NC
CT
TC
TT
CC
TT
CT
CT
CT
Crs46
461765
75705528
TG(A
C)oA
C
51015840Flankingminus887
NC
TG
GT
GG
TT
GG
TG
TG
GG
Trs5144
9853
75705525
CA(G
T)
51015840Flankingminus884
NC
CA
AC
AA
CC
AA
CA
CA
CC
Crs249093111
75705512
CT(G
A)
51015840Flankingminus871
NC
CT
TC
TT
CC
TT
CT
CT
TT
Crs263745200
75705510
CG(G
C)
51015840Flankingminus869
NC
GG
GG
GG
GG
GG
GG
GG
CG
Grs45651867
75705498
TG(A
C)
51015840Flankingminus857
NC
TG
GT
GG
TT
GG
TG
TG
GG
Trs219997531
75705495
GA
(CT)
51015840Flankingminus854
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs251918379
75705451
TA(A
T)
51015840Flankingminus810
NC
TT
TT
TT
TT
TT
TT
TT
TA
Trs27978306
75705449
TA(A
T)
51015840Flankingminus808
NC
TA
AT
AA
TT
AA
TA
TA
TT
Trs216087412
75705429
TG(A
C)lowast
51015840Flankingminus788
NC
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
GTlowast
Tlowast
rs261229914
75705423ndash75705424
AGdel(CT
del)
51015840Flankingminus783minus784
NC
AGrs27978307
75705400
CT(G
A)
51015840Flankingminus759
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs243167395
75705359
GA
(CT)
51015840Flankingminus718
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs27978308
75705307
GA
(CT)
51015840Flankingminus66
6NC
GG
GG
GG
GG
GG
GG
GG
AG
Grs254744
09875705294
AT
(TA
)51015840Flankingminus653
NC
AA
AA
AA
AA
AA
AA
AA
AT
Ars228133419
75705179
GT
(CA
)51015840Flankingminus538
NC
GG
GG
GG
GG
GG
GG
GG
GT
Grs214719520
75705111
GA
(CT)
51015840Flankingminus470
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs2440
87730
75705101
GA
(CT)
51015840Flankingminus46
0NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs227781699
75705081
CG(G
C)
51015840Flankingminus44
0NC
CC
CC
CC
CC
CC
CC
CC
GC
Crs257870353
75705067
CT(G
A)
51015840Flankingminus426
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs24487744
075705022
AG
(TC)
51015840Flankingminus381
NC
AA
AA
AA
AA
AA
AA
AA
AG
Ars234628138
75704973
AC
(TG
)51015840Flankingminus332
NC
AA
AA
AA
AA
AA
AA
AA
AC
A
18 Oxidative Medicine and Cellular Longevity
Table5Con
tinued
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs247275247
75704961
GA
(CT)
51015840Flankingminus320
NC
GG
GG
GG
GG
GG
GG
AG
GG
Grs247519047
75704922
Gdel(Cdel)
51015840Flankingminus281
NC
Grs27978309
(rs51915758)
75704887
GA
C(C
TG
)51015840Flankingminus246
NC
GA
AG
AA
GG
AA
GA
GA
CC
G
rs218139102
75704879
CA(G
T)
51015840Flankingminus238
NC
CC
CC
CC
CC
CC
CC
CC
AC
Crs251990355
75704870
TC(A
G)
51015840Flankingminus229
NC
TT
TT
TT
TT
TT
TT
TT
TC
Trs238746955
75704794
AG
(TC)
51015840Flankingminus153
NC
AA
AA
AA
AA
AA
AA
AA
GA
Ars221664
405
75704786
AG
(TC)
51015840Flankingminus145
NC
AA
AA
AA
AA
AA
AA
AA
AG
Ars217054035
75704766
ndash75704767
GAAadd(TTC
add)
51015840Flankingminus125minus126
NC
GA
rs248182931
75704759ndash75704760
Gadd(C
add)
51015840Flankingminus118
minus119
NC
AGrs217197904
75704744
GA
(CT)
51015840Flankingminus103
(+)S
p1[23]
GA
AG
AA
GG
AA
GA
AA
GG
Ars2644
9364975704704
Gdel(Cdel)
51015840Flankingminus63
NC
Grs2404
81112
757046
82CT(G
A)
51015840Flankingminus41
NC
CC
CC
CC
CC
CC
CC
CC
CT
Crs27978310
75704617
AG
(TC)
UTR
-525
NC
AA
AA
AA
AA
AA
AA
AA
GG
Ars27978311
75704610
CG(G
C)
UTR
-532
NC
GG
GG
GG
GG
GG
GG
GG
GG
Crs27978312
757046
05GA
(CT)
UTR
-537
NC
GA
AG
AA
GG
AA
GA
GA
GG
Grs214784220
757044
99AG
(TC)
UTR
-5143
NC
AA
AA
AA
AA
AA
AA
AA
GG
Ars244146318
757044
98GT
(CA
)UTR
-5144
NC
GG
GG
GG
GG
GG
GG
GG
GT
Grs27978313
757044
97GC
(CG
)UTR
-5145
NC
GC
CG
CC
GC
CC
GC
CC
CC
Crs215431944
757044
93CT(G
A)
UTR
-5149
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs257015697
757044
49GA
(CT)
UTR
-5193
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs252234782
757044
19GT
(CA
)UTR
-5223
NC
Grs1346
0861
75679262
AG
(TC)
Exon
244
6Cn
sF71L
AG
GA
GG
AG
GG
AG
AG
GG
Grs1346
0859
75679202
GA
(CT)lowast
Exon
2506
CsH91=
GA
AG
AA
GG
AA
GA
GA
GG
Grs27978436
75679187
GA
(CT)lowast
Exon
2521
CsT9
6=G
GG
GG
GG
AG
GG
GG
GA
GG
rs226131070
75679178
GA
(CT)
Exon
2530
CsS99=
GG
GG
GG
GG
GG
GG
GG
GA
Grs227683834
75678576
AT
(TA
)Ex
on3
547
CnsV
105D
AA
AA
AA
AA
AA
AA
AA
AT
Ars257321187
75678506
TC(A
G)
Exon
3617
CsP128=
TT
TT
TT
TC
TT
TT
TT
TT
Trs27978444
75678500
TA(A
T)
Exon
3623
CsV130=
TT
TT
TT
TA
TT
TT
TT
AT
Trs227743136
75677686
GA
(CT)
Exon
4662
CsH143=
GG
GG
GG
GG
GG
GG
GG
GA
Grs215327202
75677664
CG(G
C)
Exon
4684
CnsA
151P
CC
CC
CC
CG
CC
CC
CC
CC
Crs247539755
75677663
GA
(CT)
Exon
4685
CnsA
151V
GG
GG
GG
GA
GG
GG
GG
GG
Grs233164
668
75677640
TC(A
G)
Exon
4708
CnsN
159D
TT
TT
TT
TT
TT
TT
TT
TC
T
rs2340
95816
75677162ndash75677160
GCT
del
(AGCdel)
Exon
5826minus
828
Cds-in
delQ
198
GCT
Oxidative Medicine and Cellular Longevity 19
Table5Con
tinued
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs27978452
75677145
CT(G
A)
Exon
5843
CnsA
204T
CC
CC
CC
CC
CC
CC
CC
CC
T
rs225047937
75677086ndash75677078
GAG
ATCG
ATdel
(ATG
CATC
TCdel)
Exon
5902minus
910
Cds-in
del
(S224I225S226)
GAG
ATC
GAT
rs241469868
75676998
TA(G
T)
Exon
5990
CnT2
53S
TT
TT
TT
TT
TT
TT
TT
TA
Trs221602571
75676923
AC
(TG
)Ex
on5
1065
CnsS
278A
AA
AA
AA
AA
AA
AA
AA
AC
Ars252576472
75676876
GA
(CT)
Exon
51112
CsS293=
GG
GG
GG
GG
GG
GG
GG
GA
Grs250802933
75676841
TC(A
G)
Exon
5114
7Cn
sD305G
TT
TT
TT
TT
TT
TT
TT
TC
Trs225425698
75676792
CT(G
A)
Exon
5119
6Cs
P321=
CC
CC
CC
CC
CC
CC
CC
CT
Crs13459064
75676732
CT(G
A)
Exon
51256
CsT3
41=
CT
TC
TT
CT
TT
CT
CT
TC
Crs27978453
75676717
CT(G
A)
Exon
51271
CsA346=
CC
CC
CC
CC
CC
CC
CC
TC
Crs214335034
75676636
AG
(TC)
Exon
51352
CsD373=
AA
AA
AA
AA
AA
AA
AA
AG
Ars24958364
4756766
03AC
(TG
)Ex
on5
1385
CsP3
84=
AA
AA
AA
AA
AA
AA
AA
AC
Ars27978454
75676592
GA
(CT)
Exon
51396
CnsP
388L
GG
GG
GG
GG
GG
GG
GG
AG
Grs215384328
75676589
GC
(CG
)Ex
on5
1399
CnsA
389G
GG
GG
GG
GG
GG
GG
GG
GC
Grs251728286
75676571
GA
(CT)
Exon
51417
CnsT
395I
Grs247602334
75676567
TC(A
G)
Exon
51421
CsV396=
TT
TT
TT
TT
TT
TT
TT
TC
Trs234216231
75676530
TC(A
G)
Exon
51458
CnsM
409V
TT
TT
TT
TT
TT
TT
TT
TC
Trs212904337
75676526
CT(G
A)
Exon
51462
CnsR
410H
CC
CC
CC
CC
CC
CC
CC
CT
Crs252650779
75676522
TC(A
G)
Exon
51466
CsE4
11=
TT
TT
TT
TT
TT
TT
TT
TC
Trs231273560
75676516
TC(A
G)
Exon
51472
CsQ413=
TT
TT
TT
TT
TT
TT
TT
TC
Trs257886949
756764
13GT
(CA
)Ex
on5
1575
CsR4
48=
GG
GG
GG
GG
GG
GG
GG
GT
Grs241537608
756764
04GC
(CG
)Ex
on5
1584
CnsL
451V
GG
GG
GG
GG
GG
GG
GG
GC
Grs227619071
75676312
TC(A
G)
Exon
51676
CsQ481=
TT
TT
TT
TT
TT
TT
TT
TC
Trs258913831
75676290
AG
(TC)
Exon
51698
CsL4
89=
AA
AA
AA
AA
AA
AA
AA
AG
Ars225593319
75676126
AT
(TA
)Ex
on5
1862
CnsH
543Q
Ars4223233
75676105
GA
(CT)
Exon
51883
CsS550=
GG
GG
GG
GA
GG
GG
GG
GG
Grs4223232
75676032
GT
(CA
)Ex
on5
1956
CnsL
575M
Grs4223231
75675816
CG
UTR
-32172
NC
GG
GG
GG
GG
GG
GG
GG
GC
Grs1346
860
75675682
AT
UTR
-32306
NC
ASequ
ence
varia
tions
inmou
seNr
f2wereob
tained
from
Mou
sePh
enom
eDatabase(http
ph
enom
ejaxorgSNP)
andNCB
ISNPdatabase
(http
wwwncbinlm
nihgovSNP)N
CBIreference
sequ
ence
isMus
Musculusstra
inC5
7BL6J
chromosom
e2GRC
m38p1(NC
0000
687
GI37209910875704641ndash756755192
9123
bp)To
tal968
genetic
mutations
arerepo
rted
inNr
f2gene
and5k
bup
stream
(ge75704
642minus
1)and2k
bdo
wnstre
am(le75675512)sequences
asof
Janu
ary2003A
llSN
Psandallelesa
representedas
geno
miccontig
(reversedsequ
ence
indicated)E
xon175704
641ndash75704364(278
bpT
TS=75704
408)
exon
275679431ndash756791
65(267
bp)exon
375678579ndash75678490(90b
p)exon475677713ndash756775
46(168
bp)andexon
57567718
4ndash75678849
(1666
bp)Proteinam
inoacid
(aa)
resid
uesinNP00
61552
(597
aa)Cs
cod
ing-syno
nymou
sCn
scoding
-non
syno
nymou
sAminoacid
A-alanineD-aspartic
acidE
-glutamicacidF-phenylalanineG
-glycineH
-histidineI-iso
leucineL-leu
cineand
M-m
ethion
ineN-
asparagineP-prolin
eQ-glutamineR-arginineS-serineT-threon
ineandV-valin
elowastErrorsin
databasesfi
xedNC
notcon
firmed
20 Oxidative Medicine and Cellular Longevity
Table 6 NRF2 somatic mutations revealed in various human cancers
Domain Amino acid residues DNA mutation Cancer types (cases) ReferencesLocus Wild type Mutant
DLG motif
24 W (Trp) C (Cys) c72GgtCGgtT NSCLC neck ESC [54 62]K (Lys) c72TgtC ESC [62]
26 Q (Gln) E (Glu) c76CgtG NSCLC ESC [54 62]
27 D (Asp) G (Gly)lowast c80GgtA NSCLC [60]Y (Tyr) c79GgtT ESC [61]
28 I (Ile) T (Thr) c83CgtT NSCLC [54]
29 D (Asp) G (Gly) c86AgtG Head and neck ESC [54 62]H (His) c85GgtC NSCLC laynx [60 61]
30 L (Leu) F (Phe) c88CgtT NSCLC ESC [54 62]
31 G (Gly) A (Ala) c92GgtC NSCLC ESC skin [54 60ndash62]
32 V (Val) T (Thr) c95TgtG NSCLC [54]del c93 95delAGT ESC [61]
33ndash36 S-R-E-V S-R-E-V-S-R-E-Vlowast c97 108dupAGTCGAGCCGTA ESC [61]
34 R (Arg) Q (Gln) c101GgtA NCSCL [54 60 61]P (Pro) c101GgtC NSCLC [63]
ETGF motif
75 Q (Gln) H (His) c225AgtC Head and neck ESC [54 62]
77 D (Asp)
V (Val) c230AgtT NSCLC ESC [54 60 62]G (Gly) c230AgtG ESC [62]A (Ala) c230AgtC NSCLC [61]N (Asn) c229GgtA Larynx [61]
78 E (Glu) K (Lys) c232GgtA NSCLC ESC [54 62]
79 E (Glu)
K (Lys) c235GgtA NSCLC ESC [54 61 62]Q (Gln) c235GgtC NSCLC ESC [54 60ndash62]G (Gly) c236AgtG Larynx [61]E-E c234 236dupAGA ESC [61]
80 T (Thr) K (Lys) c239CgtACgtG NSCLC ESC [54 61 62]
80 T (Thr)P (Phe) c238AgtC ESC [62]I (Ile) c239CgtT Head and neck [54]A (Ala) c238AgtG NSCLC [63]
81 G (Gly) V (Val) c242GgtT ESC [61]D (Asp) c242GgtA NSCLC ESC [60ndash62]
82 E (Glu)
D (Asp) c246AgtT ESC oral cancer cell line [54 62]G (Gly) c245AgtG NSCLC [54]Q (Gln)lowast c244GgtC NSCLC ESC [60 61]V (Val)lowast c245AgtT ESC [61]
83 F (Phe) L (Leu)lowast c247TgtC NSCLC [60]NSCLC non-small cell lung cancer ESC esophageal squamous cancer and lowasterrors in reference fixed Number of cases 82 NSCLC and 10 ESC in [62] 125NSCLC 70 ESC 23 larynx and 17 skin in [61] 103 NSCLC and 12 head and neck in [54] 90 NSCLC in [63] 103 NSCLC in [60]
be involved in squamous lung cancer cell proliferation [64]suggesting that the mutation is functional and overcomesKEAP1 inhibition Singh et al [65] determined in vitrothat RNAi-mediated depletion of NRF2 in lung cancer cellsenhanced ROS production and susceptibility to cell deathby ionizing radiation These studies support the concept thatelevated NRF2 and ARE responsiveness provides cancer cellswith proliferative advantage for malignant transformation
and undue protection from anti-cancer therapy Onco-genic epidermal growth factor receptor (EGFR) signaling isrecently found to be critical in NRF2-mediated proliferationof NSCLC cells [66]
Collectively ldquogain of functionrdquo mutations in NRF2 thatreduce KEAP1 recognition are suggested to be predictivemarkers for poor responsiveness to chemotherapy and radi-ation therapy Although NRF2-mediated cellular defense
Oxidative Medicine and Cellular Longevity 21
processes are essential in the initiation stage enhancedNRF2-ARE activity in advanced stages of cancer developmentmay create a favorable intracellular environment for tumorcell growth and survival [67 68] In this context NRF2may be a potential molecular target for the treatment ofradio-resistance cancers especially those that have ldquoloss offunctionrdquo mutations in EGFR KRAS or KEAP1 as well asldquogain of functionrdquo mutations in NRF2
5 Epigenetic Alterations of NF-E2-RelatedFactor 2
Epigenetic modifications are alterations of molecules inter-acting with genes without changes to the primary DNAsequence They include post-translational modification ofhistones DNA methylation events chromatin conforma-tional changes and alterations to noncoding regulatoryRNAs Epigenetic alterations are stable and often inheritablebut are reversible and may affect expression of the geneDysregulation or defects in epigenetic processes particularlyhypermethylation of tumor suppressor gene promoters (egCpG islands) or histone modifications are thought to beassociated with carcinogenesis Investigators have reportedhypermethylation in CpG islands of KEAP1 which wereassociated with reduced KEAP1 expression in human cancersfrom lung prostate colon and so forth [69ndash72] Similar tosomatic mutations epigenetic changes on KEAP1 impairedthe function of its encoded protein leading to constitutiveNRF2 activation
Supporting the role of ldquopathogenic mutationsrdquo in NRF2expression of Nrf2 and downstream Nqo1 was suppressedin prostate tumors of mice (transgenic adenocarcinomaof mouse prostate TRAMP) Among 15 promoter CpGislands located between minus942 and minus654 (cminus1175 cminus1132and cminus1059 cminus887 gap in cminus1131 cminus1060 see Figure 3)hypermethylation of the first 5 CpG islands (minus942 minus899 andcminus1175 cminus1132) was significantly associated with tumorige-nesis [73] Moreover treatment with inhibitors for DNAmethyltransferase and histone deacetylase restored Nrf2expression in these tumor cells [73] A dietary phytochemicalcurcumin known as a DNA hypomethylation agent restoredepigenetically silent Nrf2 expression through CpG demethy-lation in carcinogen-induced mouse tumor cells [74]
The whole genome epigenetic datasets for 5 species arepublicly accessible at NCBI Epigenomics [75 76]The humanNRF2 epigenome of primary cells (breast penis) and H1stem cell line as well as mouse Nrf2 CpG island methylationdata for sperm blood and cerebellum are currently avail-able (httpwwwncbinlmnihgovepigenomics) Althoughno direct evidence of disease-associated epigenetic modu-lation has been identified in human NRF2 various phyto-chemical NRF2 agonists such as sulforaphane and curcuminhave shown their roles in DNA methylation and histonemodification (see reviews by Lee and colleagues eg [77])Taken together epigenetic modifications of theNRF2KEAP1axle are predicted to cause dysregulation of ARE-mediatedcellular defense leading to deleterious health effects and
phytochemical antioxidants as epigenetic modulators forNRF2 are suggested to be useful in cancer prevention
6 Conclusions
NRF2 is evolutionally conserved with high-sequence homol-ogy in many species However it is a highly mutable geneand numerous genetic variants have been discovered inhuman ethnic groups Importantly certain SNPs or haplo-types have been identified in various diseases as ldquoat-riskrdquoalleles and are related to functional alterations In additionto genetic variations multiple somatic mutations identifiedin the KEAP1 recognition domain of NRF2 in cancer cellshave been found to be oncogenic due to dysregulation ofNRF2 homeostasis by its excess ldquogain of functionrdquo Epigeneticalteration of theNRF2 is under investigation and is predictedto have pathogenic influences as learned from mouse andphytochemical agonist studies Continuous updates of Nrf2allelic variants in inbred mouse strains will provide a usefultool for effective experimental designs formodels of oxidativedisorders to provide insight into the disease mechanisms andintervention strategies
Abbreviations
AD Alzheimerrsquos diseaseALI acute lung injuryARE antioxidant response elementbZIP basic leucine zippercds coding DNA sequencesCI confidence intervalCOPD chronic obstructive pulmonary diseaseESC esophageal squamous cancerFEV1 forced expiratory volume in one second
Gbp giga base pairsGST glutathione S-transferaseGWAS genome-wide association studyHGVS Human Genome Variation SocietyHO-1 heme oxygenase-1119867 119901119910119897119900119903119894 119867119890119897119894119888119900119887119886119888119905119890119903 119901119910119897119900119903119894KEAP1 Kelch-like ECH activating protein 1MPD Mouse Phenome DatabaseMRP multidrug resistance proteinNCBI National Center for Biotechnology InformationNeh NRF2-ECH homologyNfe2l2 nuclear factor (erythroid-derived 2)-like 2Nrf2 NF-E2-related factor 2NSCLC nonsmall cell lung cancerNQO1 NAD(P)Hquinone oxidoreductase 1OR odds ratioPD Parkinsonrsquos diseasePM particulate matterROS reactive oxygen speciesSLE systemic lupus erythematosusSNP single nucleotide polymorphismSOD superoxide dismutaseURT untranslated region
22 Oxidative Medicine and Cellular Longevity
Disclosure
Authorrsquos contribution to the Work was done as part of theAuthorrsquos official duties as a NIH employee and is a Work ofthe United States Government Therefore copyright may notbe established in the United States
Conflict of Interests
The author declares that there is no conflict of interests
Acknowledgments
The research related to this paper was supported by theIntramural Research Program of the National Institutes ofHealth of the National Institute of Environmental HealthSciences (NIEHS) Drs Steven Kleeberger and StephanieLondon at the NIEHS provided excellent critical review ofthis paper The author thanks Mrs Jacqui Marzec for herhelpful comments and English editing
References
[1] J Y Chan M C Cheung P Moi K Chan and Y W KanldquoChromosomal localization of the humanNF-E2 family of bZIPtranscription factors by fluorescence in situ hybridizationrdquoHuman Genetics vol 95 no 3 pp 265ndash269 1995
[2] K Itoh K Igarashi N Hayashi M Nishizawa and MYamamoto ldquoCloning and characterization of a novel erythroidcell-derived CNC family transcription factor heterodimerizingwith the small Maf family proteinsrdquo Molecular and CellularBiology vol 15 no 8 pp 4184ndash4193 1995
[3] W O Osburn and T W Kensler ldquoNRF2 signaling an adaptiveresponse pathway for protection against environmental toxicinsultsrdquoMutation Research vol 659 no 1-2 pp 31ndash39 2008
[4] D Papp K Lenti D Modos D Fazekas Z Dul D Tureiet al ldquoThe NRF2-related interactome and regulome containmultifunctional proteins and fine-tuned autoregulatory loopsrdquoFEBS Letters vol 586 no 13 pp 1795ndash1802 2012
[5] X Wang D J Tomso B N Chorley et al ldquoIdentificationof polymorphic antioxidant response elements in the humangenomerdquo Human Molecular Genetics vol 16 no 10 pp 1188ndash1200 2007
[6] D Malhotra E Portales-Casamar A Singh et al ldquoGlobalmapping of binding sites for NRF2 identifies novel targets incell survival response through ChIP-Seq profiling and networkanalysisrdquo Nucleic Acids Research vol 38 no 17 pp 5718ndash57342010
[7] K Chan R Lu J C Chang and Y W Kan ldquoNRF2 a memberof the NFE2 family of transcription factors is not essential formurine erythropoiesis growth and developmentrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 93 no 24 pp 13943ndash13948 1996
[8] K Itoh T Chiba S Takahashi et al ldquoAn NRF2small Mafheterodimer mediates the induction of phase II detoxifyingenzyme genes through antioxidant response elementsrdquo Bio-chemical and Biophysical Research Communications vol 236no 2 pp 313ndash322 1997
[9] FMartin J M vanDeursen R A Shivdasani CW Jackson AG Troutman and P A Ney ldquoErythroid maturation and globin
gene expression inmicewith combined deficiency ofNF-E2 andNrf-2rdquo Blood vol 91 no 9 pp 3459ndash3466 1998
[10] K Itoh N Wakabayashi Y Katoh et al ldquoKEAP1 repressesnuclear activation of antioxidant responsive elements by NRF2through binding to the amino-terminal Neh2 domainrdquo Genesand Development vol 13 no 1 pp 76ndash86 1999
[11] A Kobayashi M I Kang H Okawa et al ldquoOxidative stresssensor KEAP1 functions as an adaptor for Cul3-based E3 ligaseto regulate proteasomal degradation of NRF2rdquo Molecular andCellular Biology vol 24 no 16 pp 7130ndash7139 2004
[12] K Taguchi H Motohashi and M Yamamoto ldquoMolecularmechanisms of the KEAP1-NRF2 pathway in stress responseand cancer evolutionrdquo Genes to Cells vol 16 no 2 pp 123ndash1402011
[13] K Mukaigasa L T Nguyen L Li H Nakajima M Yamamotoand M Kobayashi ldquoGenetic evidence of an evolutionarilyconserved role for NRF2 in the protection against oxidativestressrdquoMolecular and Cellular Biology vol 32 no 21 pp 4455ndash4461 2012
[14] JMaher andMYamamoto ldquoThe rise of antioxidant signalingmdashthe evolution and hormetic actions of NRF2rdquo Toxicology andApplied Pharmacology vol 244 no 1 pp 4ndash15 2010
[15] S C Lo X Li M T Henzl L J Beamer and M HanninkldquoStructure of the KEAP1NRF2 interface provides mechanisticinsight intoNRF2 signalingrdquoThe EMBO Journal vol 25 no 15pp 3605ndash3617 2006
[16] V A McKusick ldquoMendelian inheritance in man and its onlineversion OMIMrdquo American Journal of Human Genetics vol 80no 4 pp 588ndash604 2007
[17] B Yalcin D J Adams J Flint and T M Keane ldquoNext-generation sequencing of experimental mouse strainsrdquo Mam-malian Genome vol 23 no 9-10 pp 490ndash498 2012
[18] D R Bentley S Balasubramanian H P Swerdlow et alldquoAccurate whole human genome sequencing using reversibleterminator chemistryrdquo Nature vol 456 no 7218 pp 53ndash592008
[19] P S G Chain D V Grafham R S Fulton et al ldquoGenomeproject standards in a new era of sequencingrdquo Science vol 326no 5950 pp 236ndash237 2009
[20] RHWaterstonK Lindblad-Toh E Birney J Rogers J F AbrilP Agarwal et al ldquoInitial sequencing and comparative analysis ofthe mouse genomerdquo Nature vol 420 pp 520ndash562 2002
[21] T M Keane L Goodstadt P Danecek M A White K WongB Yalcin et al ldquoMouse genomic variation and its effect onphenotypes and gene regulationrdquoNature vol 477 pp 289ndash2942011
[22] T Yamamoto K Yoh A Kobayashi et al ldquoIdentification ofpolymorphisms in the promoter region of the human NRF2generdquo Biochemical and Biophysical Research Communicationsvol 321 no 1 pp 72ndash79 2004
[23] J M Marzec J D Christie S P Reddy et al ldquoFunctionalpolymorphisms in the transcription factor NRF2 in humansincrease the risk of acute lung injuryrdquo The FASEB Journal vol21 no 9 pp 2237ndash2246 2007
[24] H Fukushima-Uesaka Y Saito K Maekawa et al ldquoGeneticvariations and haplotype structures of transcriptional factorNRF2 and its cytosolic reservoir protein KEAP1 in JapaneserdquoDrug Metabolism and Pharmacokinetics vol 22 no 3 pp 212ndash219 2007
[25] E J Cordova R Velazquez-Cruz F Centeno V Baca and LOrozco ldquoThe NRF2 gene variant -653GA is associated with
Oxidative Medicine and Cellular Longevity 23
nephritis in childhood-onset systemic lupus erythematosusrdquoLupus vol 19 no 10 pp 1237ndash1242 2010
[26] M von Otter S Landgren S Nilsson et al ldquoAssociation ofNRF2-encoding NFE2L2 haplotypes with Parkinsonrsquos diseaserdquoBMCMedical Genetics vol 11 no 1 article 36 2010
[27] JMHartikainenM TengstromVMKosmaV L Kinnula AMannermaa andY Soini ldquoGenetic polymorphisms and proteinexpression of NRF2 and sulfiredoxin predict survival outcomesin breast cancerrdquo Cancer Research vol 72 pp 5537ndash5546 2012
[28] T Arisawa T Tahara T Shibata et al ldquoThe relationshipbetween Helicobacter pylori infection and promoter polymor-phism of the NRF2 gene in chronic gastritisrdquo InternationalJournal of Molecular Medicine vol 19 no 1 pp 143ndash148 2007
[29] T Arisawa T Tahara T Shibata et al ldquoThe influence ofpromoter polymorphism of nuclear factor-erythroid 2-relatedfactor 2 gene on the aberrant DNA methylation in gastricepitheliumrdquo Oncology Reports vol 19 no 1 pp 211ndash216 2008
[30] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene pro-moter polymorphism and gastric carcinogenesisrdquo Hepato-Gastroenterology vol 55 no 82-83 pp 750ndash754 2008
[31] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene promoterpolymorphism is associated with ulcerative colitis in a Japanesepopulationrdquo Hepato-Gastroenterology vol 55 no 82-83 pp394ndash397 2008
[32] C C Hua L C Chang J C Tseng C M Chu Y C Liu andW B Shieh ldquoFunctional haplotypes in the promoter region oftranscription factor NRF2 in chronic obstructive pulmonarydiseaserdquo Disease Markers vol 28 no 3 pp 185ndash193 2010
[33] S O Shaheen R B Newson S M Ring M J Rose-ZerilliJ W Holloway and A J Henderson ldquoPrenatal and infantacetaminophen exposure antioxidant gene polymorphismsand childhood asthmardquo The Journal of Allergy and ClinicalImmunology vol 126 no 6 pp 1141e7ndash1148e7 2010
[34] H Masuko T Sakamoto Y Kaneko et al ldquoLower FEV1 innon-COPD nonasthmatic subjects association with smokingannual decline in FEV1 total IgE levels and TSLP genotypesrdquoInternational Journal of Chronic Obstructive Pulmonary Diseasevol 6 pp 181ndash189 2011
[35] C P Guan M N Zhou A E Xu et al ldquoThe susceptibilityto vitiligo is associated with NF-E2-related factor 2 (NRF2)gene polymorphisms a study on Chinese Han populationrdquoExperimental Dermatology vol 17 no 12 pp 1059ndash1062 2008
[36] J Bouligand O Cabaret M Canonico et al ldquoEffect of NFE2L2genetic polymorphism on the association between oral estrogentherapy and the risk of venous thromboembolism in post-menopausal womenrdquo Clinical Pharmacology amp Therapeuticsvol 89 no 1 pp 60ndash64 2011
[37] D S OrsquoMahony B J Glavan T D Holden C Fong R A BlackG Rona et al ldquoInflammation and immune-related candidategene associations with acute lung injury susceptibility andseverity a validation studyrdquo PLoS ONE vol 7 no 12 Article IDe51104 2012
[38] I Ungvari E Hadadi V Virag et al ldquoRelationship between airpollutionNFE2L2 gene polymorphisms and childhood asthmain aHungarian populationrdquo Journal of CommunityGenetics vol3 no 1 pp 25ndash33 2012
[39] M SiedlinskiD S Postma JMA Boer et al ldquoLevel and courseof FEV1 in relation to polymorphisms inNFE2L2 andKEAP1 inthe general populationrdquo Respiratory Research vol 10 article 732009
[40] C C Hong C B Ambrosone J Ahn et al ldquoGenetic variabilityin iron-related oxidative stress pathways (NRF2 NQ01 NOS3
and HO-1) iron intake and risk of postmenopausal breastcancerrdquo Cancer Epidemiology Biomarkers and Prevention vol16 no 9 pp 1784ndash1794 2007
[41] C Canova C Dunster F J Kelly C Minelli P L Shah CCaneja et al ldquoPM10-induced hospital admissions for asthmaand chronic obstructive pulmonary disease the modifyingeffect of individual characteristicsrdquo Epidemiology vol 23 no 4pp 607ndash615 2012
[42] A J Henderson R B Newson M Rose-Zerilli S M RingJ W Holloway and S O Shaheen ldquoMaternal NRF2 andgluthathione-S-transferase polymorphisms donotmodify asso-ciations of prenatal tobacco smoke exposure with asthma andlung function in school-aged childrenrdquo Thorax vol 65 no 10pp 897ndash902 2010
[43] C Xing A L Sestak J A Kelly et al ldquoLocalization andreplication of the systemic lupus erythematosus linkage signalat 4p16 interaction with 2p11 12q24 and 19q13 in EuropeanAmericansrdquo Human Genetics vol 120 no 5 pp 623ndash631 2007
[44] T F Wong K Yoshinaga Y Monma K Ito H NiikuraS Nagase et al ldquoAssociation of KEAP1 and NRF2 geneticmutations and polymorphisms with endometrioid endometrialadenocarcinoma survivalrdquo International Journal of Gynecologi-cal Cancer vol 21 no 8 pp 1428ndash1435 2011
[45] M von Otter S Landgren S Nilsson et al ldquoNRF2-encodingNFE2L2 haplotypes influence disease progression but not riskin Alzheimerrsquos disease and age-related cataractrdquoMechanisms ofAgeing and Development vol 131 no 2 pp 105ndash110 2010
[46] S Tsang Z Sun B Luke et al ldquoA comprehensive SNP-basedgenetic analysis of inbred mouse strainsrdquoMammalian Genomevol 16 no 7 pp 476ndash480 2005
[47] C M Wade E J Kulbokas III A W Kirby et al ldquoThe mosaicstructure of variation in the laboratory mouse genomerdquoNaturevol 420 no 6915 pp 574ndash578 2002
[48] T Wiltshire M T Pletcher S Batalov et al ldquoGenome-widesingle-nucleotide polymorphism analysis defines haplotypepatterns in mouserdquo Proceedings of the National Academy ofSciences of the United States of America vol 100 no 6 pp 3380ndash3385 2003
[49] K A Frazer E Eskin H M Kang et al ldquoA sequence-basedvariation map of 827 million SNPs in inbred mouse strainsrdquoNature vol 448 no 7157 pp 1050ndash1053 2007
[50] T P Maddatu S C Grubb C J Bult andM A Bogue ldquoMousephenome database (MPD)rdquo Nucleic Acids Research vol 40 ppD887ndashD894 2007
[51] A Kirby H M Kang C M Wade et al ldquoFine mapping in 94inbred mouse strains using a high-density haplotype resourcerdquoGenetics vol 185 no 3 pp 1081ndash1095 2010
[52] H Y Cho A E Jedlicka S P M Reddy L Y Zhang T WKensler and S R Kleeberger ldquoLinkage analysis of susceptibilityto hyperoxia NRF2 is a candidate generdquo American Journal ofRespiratory Cell and Molecular Biology vol 26 no 1 pp 42ndash512002
[53] H Y Cho A E Jedlicka S P M Reddy et al ldquoRole of NRF2in protection against hyperoxic lung injury in micerdquo AmericanJournal of Respiratory Cell and Molecular Biology vol 26 no 2pp 175ndash182 2002
[54] T Shibata T Ohta K I Tong et al ldquoCancer related mutationsin NRF2 impair its recognition by KEAP1-Cul3 E3 ligase andpromote malignancyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 36 pp13568ndash13573 2008
24 Oxidative Medicine and Cellular Longevity
[55] A Singh V Misra R K Thimmulappa et al ldquoDysfunctionalKEAP1-NRF2 interaction in non-small-cell lung cancerrdquo PLoSMedicine vol 3 no 10 article e420 2006
[56] T Ohta K Iijima M Miyamoto et al ldquoLoss of KEAP1 functionactivates NRF2 and provides advantages for lung cancer cellgrowthrdquo Cancer Research vol 68 no 5 pp 1303ndash1309 2008
[57] B Padmanabhan K I Tong T Ohta et al ldquoStructural basisfor defects of KEAP1 activity provoked by its point mutationsin lung cancerrdquoMolecular Cell vol 21 no 5 pp 689ndash700 2006
[58] T Shibata A Kokubu M Gotoh et al ldquoGenetic alteration ofKEAP1 confers constitutive NRF2 activation and resistance tochemotherapy in gallbladder cancerrdquoGastroenterology vol 135no 4 pp 1358e4ndash1368e4 2008
[59] N J Yoo H R Kim Y R Kim C H An and S H LeeldquoSomatic mutations of the KEAP1 gene in common solidcancersrdquo Histopathology vol 60 no 6 pp 943ndash952 2012
[60] Y Hu Y Ju D Lin Z Wang Y Huang S Zhang et alldquoMutation of the NRF2 gene in non-small cell lung cancerrdquoMolecular Biology Reports vol 39 no 4 pp 4743ndash4747 2012
[61] Y R Kim J EOhM S Kim et al ldquoOncogenicNRF2mutationsin squamous cell carcinomas of oesophagus and skinrdquo TheJournal of Pathology vol 220 no 4 pp 446ndash451 2010
[62] T Shibata A Kokubu S Saito et al ldquoNRF2 mutation confersmalignant potential and resistance to chemoradiation therapyin advanced esophageal squamous cancerrdquo Neoplasia vol 13pp 864ndash873 2011
[63] H Sasaki M Shitara K Yokota Y Hikosaka S MoriyamaM Yano et al ldquoIncreased NRF2 gene (NFE2L2) copy numbercorrelates with mutations in lung squamous cell carcinomasrdquoMolecular Medicine Reports vol 6 no 2 pp 391ndash394 2012
[64] H Sasaki M Shitara K Yokota Y Hikosaka S Moriyama MYano et al ldquoRagD gene expression andNRF2mutations in lungsquamous cell carcinomasrdquo Oncology Letters vol 4 pp 1167ndash1170 2012
[65] A Singh M Bodas N Wakabayashi F Bunz and S BiswalldquoGain of NRF2 function in non-small-cell lung cancer cellsconfers radioresistancerdquo Antioxidants and Redox Signaling vol13 no 11 pp 1627ndash1637 2010
[66] T Yamadori Y Ishii SHommaYMorishimaKKurishimaKItoh et al ldquoMolecular mechanisms for the regulation of NRF2-mediated cell proliferation in non-small-cell lung cancersrdquoOncogene vol 31 pp 4768ndash4777 2012
[67] A K Bauer H Y Cho L Miller-Degraff C Walker K HelmsJ Fostel et al ldquoTargeted deletion of NRF2 reduces urethane-induced lung tumor development in micerdquo PLoS ONE vol 6no 10 Article ID e26590 2011
[68] H Y Cho and S R Kleeberger ldquoNRF2 protects against airwaydisordersrdquo Toxicology and Applied Pharmacology vol 244 no1 pp 43ndash56 2010
[69] R Wang J An F Ji H Jiao H Sun and D Zhou ldquoHyperme-thylation of the KEAP1 gene in human lung cancer cell linesand lung cancer tissuesrdquo Biochemical and Biophysical ResearchCommunications vol 373 no 1 pp 151ndash154 2008
[70] P Zhang A Singh S Yegnasubramanian et al ldquoLoss ofkelch-like ECH-associated protein 1 function in prostate cancercells causes chemoresistance and radioresistance and promotestumor growthrdquoMolecular Cancer Therapeutics vol 9 no 2 pp336ndash346 2010
[71] N Hanada T Takahata Q Zhou et al ldquoMethylation of theKEAP1 gene promoter region in human colorectal cancerrdquoBMCCancer vol 12 article 66 2012
[72] L A Muscarella R Barbano V DrsquoAngelo et al ldquoRegulationof KEAP1 expression by promoter methylation in malignantgliomas and association with patientrsquos outcomerdquo Epigeneticsvol 6 no 3 pp 317ndash325 2011
[73] S Yu T O Khor K L Cheung et al ldquoNRF2 expressionis regulated by epigenetic mechanisms in prostate cancer ofTRAMP micerdquo PLoS ONE vol 5 no 1 Article ID e8579 2010
[74] T O Khor Y Huang T Y Wu L Shu J Lee and A N KongldquoPharmacodynamics of curcumin as DNA hypomethylationagent in restoring the expression of NRF2 via promoter CpGsdemethylationrdquo Biochemical Pharmacology vol 82 no 9 pp1073ndash1078 2011
[75] I M Fingerman L McDaniel X Zhang et al ldquoNCBI epige-nomics a new public resource for exploring epigenomic datasetsrdquo Nucleic Acids Research vol 39 supplemen 1 pp D908ndashD912 2011
[76] I M Fingerman X Zhang W Ratzat N Husain R F Cohenand G D Schuler ldquoNCBI epigenomics whatrsquos new for 2013rdquoNucleic Acids Research vol 41 no 1 pp D221ndashD225 2013
[77] J H Lee T O Khor L Shu Z Y Su F Fuentes andA N Kong ldquoDietary phytochemicals and cancer preventionNRF2 signaling epigenetics and cell death mechanisms inblocking cancer initiation and progressionrdquo Pharmacology ampTherapeutics vol 137 no 2 pp 153ndash171 2012
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
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Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Oxidative Medicine and Cellular Longevity 3
Table1Geneo
rtho
logy
ofNF-E2
-related
factor
2acrossthes
pecies
Species
Hum
anHom
osapiens
Cat
Felis
catus
Chim
panzee
Pantro
glodytes
Dog
Canislupus
familiaris
Cattle
Bostaurus
Officialfullname
nucle
arfactor
(erythroid-derived
2)-like
2Antho
rnam
enu
clear
factor
erythroid2-related
factor
2NF-E2
-related
factor
2NFE
2-related
factor
2Antho
rnam
eHEB
P1mdash
mdashmdash
mdashGenes
ynon
yms
NFE
2L2NRF
2NFE
2L2NRF
2NFE
2L2NRF
2NFE
2L2NRF
2NFE
2L2NRF
2Ch
romosom
emap
2q312
C12B
362
Genom
esequence
(NCB
IBuild)
NC
0000
0211
(GRC
h37p
10)
NC
1087301
(Feliscatus-62)
NC
0064703
(Pan
troglod
ytes-214)
NC
0066183
(CanFam31)
AC00
01591
(Bos
taurus
UMD
31)
Prim
arysource
HGNC
7782
mdashmdash
mdashBO
S1716
GeneID
mapping
(orie
ntationlowast
)
4780
178095031minus178129859(minus)
1010
9881
2165356974minus
165392728(minus)
7426
22181721941minus181756837(minus)
4788
1320989206minus21021893
(minus)
4970
2419659540minus19692048
Ensembleg
eneID
(mapping
)EN
SG00
00011604
4(178092323minus178257425)
mdashEN
SPTR
G00
000012677
(181721949minus181756285)
ENSC
AFG
0000
0013506
(20987569minus
21022230)
ENSB
TAG00
000019255
(19659540minus19692048)
RefSeq
IDm
RNA
size
NM
006164
4(G
I372620346)2859b
p
XM0039908931
(GI410968909)
predicted2394
bp
XM00114
58762
(GI332814815)predicted
2850
bp
XM5359753
(GI345797173)
predicted2570
bp
NM
0010116
782
(GI147904941)240
9bp
ProteinID
(aaMW
sect )NP00
61552
(60567696)
XP0039909421
(60667797)
XP00114
58762
(60567686)
XP5359751
(60167087)
NP0010116
782
(60767813)
UniProt
IDQ16236
mdashA2T
6Y9
mdashQ5N
UA62
Varia
nts
14transcrip
ts12
isoform
s3transcrip
ts3iso
form
s5transcrip
ts5iso
form
s2transcrip
ts2iso
form
smdash
Hom
olog
ygeneprotein
100100
893914
999999
893888
905891
4 Oxidative Medicine and Cellular Longevity
Table1Con
tinued
Species
Rhesus
mon
key
Macacamulatta
Chicken
Gallusgallus
Zebrafish
Daniorerio
Mou
seMus
musculus
Rat
Rattu
snorvegicus
Officialfullname
nucle
arfactor
(erythroid-derived
2)-like
2nu
clear
factorerythroid-derived
2lik
e2Antho
rnam
enu
clear
factor
erythroid2-related
factor
2NF-E2
-related
factor
2NFE
2-related
factor
2Antho
rnam
eHEB
P1mdash
mdashmdash
mdash
Genes
ynon
yms
NFE
2L2NRF
2Mmu966
NFE
2L2NRF
2nfe2l2N
rf2N
rf2a
wufc15g09wufj6
7e03
Nfe2
l2N
rf2A
I194320
Nfe2
l2N
rf2
Chromosom
emap
127
92C
3(4475
cM)
3q23
Genom
esequence
(NCB
IBuild)
NC
0078691
NC
0060
943
NC
0071205
(Zv9)
NC
0000
687
(GRC
m38p1)
NC
0051023
(Rno
r50)
Prim
arySource
mdashCG
NC
4960
4ZF
INZ
DB-GEN
E-030723-2
MGI108420
RGD620360
GeneIDmapping
(orie
ntationlowast
)70
7606
40848940minus40
8846
00(minus)
3960
1415304546minus15320356
3601
491643746minus
1670288
1802
475675519minus757046
41(minus)
8361
969041647minus69069070
(minus)
Ensembleg
eneID
(mapping
)EN
SMMUG00
0000
01861
(40848948minus
40884174)
ENSG
ALG
0000
0009240
(16897956minus
16922637)
ENSD
ARG
0000
0042824
(1643631minus1672715)
ENSM
USG
0000
0015839
(75675513minus
757046
41)
ENSR
NOG00
0000
01548
(58366
693minus
58394118)
RefSeq
IDm
RNA
size
NM
0012576071
(GI383872375)2335b
pNM
2051171
(GI45384113)2555
bpNM
1828891
(GI33504557)2149
bpNM
0109023
(GI76573877)2469
bpNM
0317892
(GI402692377)2305b
pProteinID
(aaMW
sect )NP001244
5361
(606
67703)
NP9904
481
(58265160)
NP8783091
(58665757)
NP0350321
(59766770)
NP1139771
(59766825)
UniProt
IDF7GPD
8Q90834
Q8JIM
1Q60795
O54968
Varia
nts
2transcrip
ts2iso
form
s1transcript1isoform
1transcript1isoform
1transcript
1transcript1isoform
Hom
olog
ygeneprotein
988990
726674
546491
834825
838832
Referto
httpwwwncbinlm
nihgovhom
ologene2412
and
reference
[14]forcross-speciesho
molog
yDetails
ofho
molog
yscores
and
sequ
encescan
bealso
obtained
byblastagainsthu
man
sequ
ence
(http
blastn
cbin
lmnihgov)
Furtherinform
ation
ontranscrip
tvaria
ntsand
protein
isoform
sareview
able
bygene
IDat
NCB
I(http
wwwncbinlm
nihgovrefseqrsg)
and
eEn
semble
(http
useastensembleo
rgin
dexhtml)or
byproteinID
atUniProt(http
wwwun
iproto
rg)
sect MWpredicted
molecularweightfrom
NCB
I(MW
inUniProtvarie
sslightly)Re
fSeqreference
sequ
enceaaam
ino
acidsandlowastgene
incomplem
ento
rientation
Oxidative Medicine and Cellular Longevity 5
Table 2 Protein domains of NF-E2-related factor 2
Domains Amino acid positionslowast Predicted functionsHuman (605 aa) Mouse (597 aa)
Neh216ndash89
DLG motif 17ndash32ETGF motif 77ndash82
16ndash89 KEAP1 repression through DLGETGF motif-DC motif binding fastredox-sensitive proteasomal degradation
Neh4 111ndash134 111ndash134 Translocation and transactivation Phosphorylation or CBP bindingNeh5 182ndash209 172ndash201
Neh6 337ndash394(or 338ndash388) 328ndash385 Degron motif-associated constitutive turnover slow redox-insensitive
(Keap1-independent)
Neh1435ndash568
basic motif 503ndash518leucine zipper 525ndash539
427ndash560basic motif 494ndash509leucine zipper 517ndash531
Dimerization for nuclear translocation DNA binding through basicmotif-leucine zipper
Neh3 569ndash605 561ndash597 CHD6 binding stability or transactivationlowastVaries slightly among publications
homology (Neh) domains configuring the protein sequenceof either species (Table 2) and the potential functions of eachregion particularly the highly conserved KEAP1-bindingNeh2 and DNA- (ARE-) binding Neh1 domains have beenintensively investigated [12 14 15]
3 Genetic Variation of NF-E2-Related Factor 2in Human and Mouse
31 Evolution Genome Sequence and Polymorphism Dis-covery in Human and Mouse While rare and monogenicMendelian diseases are inheritable mutations in a single gene[16] many common diseases are complex traits and thedisease phenotypes are affected by variants inmultiple geneticloci Recent advancements in high-throughput technologyhave enabled sequencing of entire mammalian genomes[17ndash19] and information on DNA sequence and variationhas facilitated the study of complex traits of human dis-orders Genome-wide association studies (GWAS) examinewhether SNPs are associated with important disease traitsand ascertains ldquoat-riskrdquo genotypes that are significantly moreprevalent in the affected group than in the nonaffected groupThe HapMap Project (httphapmapncbinlmnihgov) hasmapped combinations of alleles at specific loci (haplotypes)that is common patterns of sequence variation in severalhuman populations It has supported efficient mapping ofmultiple loci for complex traits in GWAS Candidate geneapproaches based on findings from GWAS of similar dis-orders have also been useful for determining the potentialgenetic mechanisms of diseases
The evolutionary divergence of human and mouse lin-eages occurred for roughly 75million years and their genomesequences have been altered by nearly one substitution (ordeletion or addition) for every twonucleotides [20]Howeverthis slow evolution process resulted in a high degree ofconservation across the two species which allows alignmentof orthologous sequences gt90 of the human and mousegenomes is partitioned into corresponding regions of con-served synteny and at the nucleotide level approximately 40of the human genome is aligned to themouse [20]Due to this
fact biomedical studies of human genes are complemented byexperimental manipulation of corresponding mouse genesand they have aided functional understanding of genesin human health Following the 2003 completion of theHuman Genome Project of approximately 31 giga base pairs(Gbp) the Mouse Genome Project assembled the completegenome sequence of one strain (C57BL6J 2716965481 bp)in 2011 Using this reference strain whole genome sequenc-ing data across 16 additional inbred strains were done(httpwwwsangeracukresourcesmousegenomes [21])Discovery of high-density SNPs in the mouse genome sup-ports evolutionary history of the strain and provides a toolto investigate models of human disease processes that cannotoften be practically achieved through direct human studies
32 GeneticMutations in HumanNRF2 HumanNRF2 codesthree major isoforms of protein (Figure 2) Transcript variant2 (NM 0011454122 2746 bp) has an alternate promoter 51015840-UTR and a downstream start codon compared to variant 1(NM 0061644 2859 bp) It encodes an isoform 2 missing N-terminal 16 aa (NP 001138884 or Q16236-2 589 aa) relativeto isoform 1 (NP 0061552 or Q16236 605 aa) Isoform 3(NP 0011388851 or Q16236-3 582 aa) is encoded by tran-script variant 3 (NM 0011454132 2725 bp) and lacks aninternal segment relative to isoform 2 due to an alternate in-frame splice site in the 31015840 coding region In public databasesmore than 583 sequence mutations are reported in NRF2(34827 bp) and 7000 bp upstream (Table S1 data acquired asof December 2012) (See Table S1 in Supplementary Materialavailable on line at httpdxdoiorg1011552013286524)NRF2 locates on 178130354-178129304 bp of GRCh37p10PrimaryAssembly and Figure 2 shows sequences of proximalpromoter (minus1 to minus500) partial mRNA variant 1 including 51015840-UTR (exon 1 up to TSS) and protein isoform 1 (NP 006155encoded by variant 1 NM 0061644 556ndash2373 bp) Basedon the current assembly and sequence update previouspromoter positions minus686 [22]minus653 [23] are identified asminus214 minus684 [22]minus651 [23] as minus212 and minus650 [22]minus617 [23]asminus178Overall frequency ofNRF2 SNPs and othermutationsis about 1 per 72 bp The genetic mutations include 37 in
6 Oxidative Medicine and Cellular Longevity
Translation start site for variants 2 and 3
1 missed in isoforms 2 and 3pA72T pS99P pQ121H missed in isoform 3
pP406S pE423D pK428R pR437W pR449C
minus400
minus500
minus50minus100
minus200
minus300
minus450
minus350
minus250
minus150
g minus
g
pE152K pV155I pL177V
pV268M
pR517K pH453L
pS365I pS387N pK389R pL309F pI317M pD341V
pK219R pP233S pK237 = fs
51101151201251301351401451501551601
Protein isoform 1 (NP 006155)
g minus66minus65insC
Termination codon (c +2371 +2373)
Translation start site (c +1)
151
101151201251301351401451501551601
2351
frame stop codonc minus156 154 in-old + 1 [23]
old + 1 [22]
g minus1 (178130354)
c minus96delC
c minus438 minus437delTT
minus89 minus87delGCC
transcription start site)+1 (c minus555
p R43Q p R43W
Genomic DNA (reversed contig GRCh37 p10 primary assemblyNM 0061644)minus477
minus214 g minus212gminus188
c 9minus95
g minus178
c minus304
c 8minus13
c 2minus16
TgtA
CgtT
GgtA
GgtA
GgtA
GgtT
GgtCAgtG
AgtCAgtG
Figure 2 DNA (variant 1 partial promoter and exons 1 and 5) and protein (isoform 1) sequence of Human NRF2 SNPs and amino acidresidues for nonsynonymous SNPs are marked
the 51015840 flanking promoter and 59 in exons (Tables 3 and S1)Among exon SNPs 26 are nonsynonymous (Cns) mutationsA triplet repeat variation (rs143406266 GCC
4versus GCC
5
previously published as minus20minus6) in the 51015840-UTR was uniquelyidentified in Asian populations [22 24]
33 SNPs Haplotypes and Association with Disease RiskThe use of gene knockout mice in model systems hasprovided potential insights into the role of NRF2 in thepathogenesis of various human disorders (see Figure 1)Recent epidemiological and association studies have revealedsignificant associations of NRF2 sequence variations withdisease risks which further supportsNRF2 as a susceptibilitygene Most of the phenotype-associated variants are in thepromoter region and presumed to be involved in NRF2 generegulation Table 4summarizes NRF2 SNP andor haplotypealleles that have been associated with oxidant-related diseaserisks Interestingly there is no evidence for exon SNPs as at-risk alleles For convenience and consistency intronic and31015840 distal SNP alleles are presented as chromosome contig(HGVS) alleles while promoter and exon SNPs are presentedas reversed contig alleles throughout the text
Pulmonary Diseases NRF2 SNPs in the promoter and intron1 sequences have been investigated for their potential asso-ciations with risk of pulmonary critical disorders includingacute lung injury (ALI) cigarette smoke-induced chronicobstructive pulmonary disease (COPD) and asthma Aheterozygous CA SNP at minus178 position (rs6721961TgtCor TgtG previously minus617 or minus650) significantly increasedthe risk for developing ALI following major trauma inEuropean and African-American populations (odds ratioOR 644 95 confidence interval CI 134ndash308 allelicfrequency = 119 at 21180) [23] Promoter activity ofthe A allele (AC or AA) determined in vivo and invitro was significantly lower than CC allele at that locus(minus178 in an ARE-like motif) indicating that it is a func-tional SNP for autoregulation [23] The minus178GG werealso nominally associated with ALI-related 28-day mor-tality following systemic inflammatory response syndrome[37] In a Japanese cohort SNP haplotype (rs2001350Trs6726395Ars1962142Ars2364722Ars6721961T) co-tainingthe minus178AA homozygote was associated with an annualdecline of rapid forced expiratory volume in one second(FEV1) in relation to cigarette-smoking status [34] In addi-
tion a promoter and 51015840-UTR SNP haplotype consistingof minus214G allele (52 rs35652124 previously minus686minus653)
Oxidative Medicine and Cellular Longevity 7Ta
ble3Geneticmutations
inprom
oter
andexon
sofh
uman
NRF
2
IDMap
onchromosom
e2lowast
(HGVS
name)
Positionin
Nfe2
l2
(HGVS
name)
Region
s(position
from
mRN
A)
Varia
tioncla
ssand
consequences
(HGVS
name)dagger
Minor
allelefrequ
ency
(MAF)
MAcoun
tsDagger(coh
ort
size)
MAFsources
rs16865105
g178136629AgtC
cminus555minus
6770Tgt
G51015840Flanking
(minus6770)
SNP
C=01928421
1000
Genom
esrs7557529
g178135097CgtT
cminus555minus
5238GgtA
51015840Flanking
(minus5238)
SNP
nars6750320
g178131796CgtT
cminus555minus
1937GgtA
51015840Flanking
(minus1937)
SNP
T=0030266
1000
Genom
esrs18116
2518
g178131774TgtC
cminus555minus
1915AgtG
51015840Flanking
(minus1915)
SNP
C=000
051
1000
Genom
esrs1900
44775
g178131746CgtT
cminus555minus
1887GgtA
51015840Flanking
(minus1887)
SNP
T=000
051
1000
Genom
esrs185117338
g178131704AgtG
cminus555minus
1845Tgt
C51015840Flanking
(minus1845)
SNP
G=000
051
1000
Genom
esrs149947189
g178131697CgtT
cminus555minus
1838GgtA
51015840Flanking
(minus1838)
SNP
T=000
092
1000
Genom
esrs139771244
g178131625AgtG
cminus555minus
1766Tgt
C51015840Flanking
(minus1766)
SNP
G=000
092
1000
Genom
esrs6747203
g17813160
4CgtG
cminus555minus
1745GgtC
51015840Flanking
(minus1745)
SNP
G=000
613
1000
Genom
esrs193101749
g178131504TgtC
cminus555minus
1645AgtG
51015840Flanking
(minus1645)
SNP
C=000
4610
1000
Genom
esrs190630762
g178131495GgtC
cminus555minus
1636Cgt
G51015840Flanking
(minus1636)
SNP
C=000
092
1000
Genom
esrs183764
402
g178131366CgtA
cminus555minus
1507GgtT
51015840Flanking
(minus1507)
SNP
A=000
051
1000
Genom
esrs19122296
4g178131211GgtA
cminus555minus
1352Cgt
T51015840Flanking
(minus1352)
SNP
A=000
092
1000
Genom
esrs187137522
g178131165TgtG
cminus555minus
1306AgtC
51015840Flanking
(minus1306)
SNP
G=000
092
1000
Genom
esrs182620359
g178131158AgtG
cminus555minus
1299Tgt
C51015840Flanking
(minus1299)
SNP
G=000
051
1000
Genom
esrs4893819
(rs61433302)
g178131134CgtT
cminus555minus
1275GgtA
51015840Flanking
(minus1275)
SNP
C=04263931
1000
Genom
es
rs191547130
g178131017CgtT
cminus555minus
1158GgtA
51015840Flanking
(minus1158)
SNP
T=000
051
1000
Genom
esrs188422217
g178131003AgtG
cminus555minus
1144TgtC
51015840Flanking
(minus114
4)SN
PG=000143
1000
Genom
esrs143047764
g178130865AgtG
cminus555minus
1006Tgt
C51015840Flanking
(minus1006)
SNP
G=000
6915
1000
Genom
esrs74432849
g178130766CgtA
cminus555minus
907GgtT
51015840Flanking
(minus907)
SNP
nars116
79252
g178130691CgtG
cminus555minus
832GgtC
51015840Flanking
(minus832)
SNP
nars12993217
g178130516AgtG
cminus555minus
657TgtC
51015840Flanking
(minus657)
SNP
nars115644
826
g17813044
2TgtA
cminus555minus
583AgtT
51015840Flanking
(minus583)
SNP
A=0015133
1000
Genom
esrs140803524
g178130431GgtA
cminus555minus
572CgtT
51015840Flanking
(minus572)
SNP
A=000
4610
1000
Genom
es
rs776844
20g178130427TgtC
cminus555minus
568AgtG
51015840Flanking
(minus568)
SNP
C=0033974
C=0190(84)
1000
Genom
es[24]
rs183651094
g178130336AgtT
cminus555minus
477TgtA
51015840Flanking
(minus477)
SNP
T=000184
1000
Genom
es
rs35652124
sect
(rs57695243)
g178130073TgtC
cminus555minus
214AgtG
51015840Flanking
(minus214)
SNP
C=03512767
T=0429(84)
T=0413181Dagger
C=033827Dagger
C=03517
69
1000
Genom
es[24]
[22]
[23]
[25]
rs6706649
sectg178130071CgtT
cminus555minus
212GgtA
51015840Flanking
(minus212)
SNP
T=0078170
T=004
8(84)
T=004
821Dagger
T=00753Dagger
1000
Genom
es[24]
[22]
[23]
rs1506
48896
g178130047CgtG
cminus555minus
188GgtC
51015840Flanking
(minus188)
SNP
G=000235
G=000
6(84)
1000
Genom
es[24]
rs6721961
sect
(rs117801448)
g178130037TgtC
TgtG
cminus555minus
178AgtC
AgtG
51015840Flanking
(minus178)
SNP
T=0150328
T=0283124Dagger
T=031325Dagger
T=0321(84)
1000
Genom
es[22]
[23]
[24]
8 Oxidative Medicine and Cellular Longevity
Table3Con
tinued
IDMap
onchromosom
e2lowast
(HGVS
name)
Positionin
Nfe2
l2
(HGVS
name)
Region
s(position
from
mRN
A)
Varia
tioncla
ssand
consequences
(HGVS
name)dagger
Minor
allelefrequ
ency
(MAF)
MAcoun
tsDagger(coh
ort
size)
MAFsources
rs20134560
4g178129924178129925insG
cminus555-66minus555-65insC
51015840Flanking
(minus66minus65)
Insertion
G=0017939
1000
Genom
es
rs200432479
g178129741178
129742delAA
cminus438minus437delT
Texon
1UTR
-51015840(118minus119
)Dele
tion
-=000378
-=000
6(84)
1000
Genom
es[24]
rs75485459
g178129608CgtA
cminus304GgtT
Exon
1UTR
-51015840(252)
SNP
nars192086766
g17812946
6CgtT
cminus162GgtA
Exon
1UTR
-51015840(394)
SNP
T=002248
1000
Genom
esmdash
g17812944
2GgtA
cminus138CgtT
Exon
1UTR
-51015840(418)
SNP
A=0012(84)
[24]
rs7166
8246
g17812940
0delG
cminus96delC
Exon
1UTR
-51015840(460)
Dele
tion
nars187291840
g178129399CgtT
cminus95GgtA
Exon
1UTR
-51015840(461)
SNP
T=0054912
01000
Genom
es
rs143406266
g178129391178129393delGGC
cminus89minus87delGCC
Exon
1UTR
-51015840(467minus469)
Dele
tion
-=064
4282Dagger
-=0589(84)
[22]
[24]
rs182428269
g178098918GgtA
c127Cgt
TEx
on2(682)
Cns(pArg43Trp)
A=000
051
1000
Genom
esrs35248500
g178098917CgtT
c128GgtA
Exon
2(683)
Cns(pArg43Gln)
T=000
613
1000
Genom
esrs1135118
g178098831CgtT
c214GgtA
Exon
2(769)
Cns(pAla72Th
r)TDagger
[23]
rs19969166
0g178098829AgtT
c216Tgt
AEx
on2(771)
Cs(pAla72=)
namdash
g178098769GgtA
c276Cgt
TEx
on2(831)
Cs(pIle92=)
TDagger[23]
rs5031039
g178098750AgtG
c295Tgt
CEx
on2(850)
Cns(pSer99P
ro)
G=0Dagger
[23]
rs200239262
g178098017CgtG
c363GgtC
Exon
3(918)
Cns(pGln121H
is)na
rs183034165
g178098008TgtC
c372GgtA
Exon
3(927)
Cs(pAla124=
)T=000
092
T=000
6(84)
1000
Genom
es[24]
rs199970826
g178097996CgtT
c384GgtA
Exon
3(939)
Cs(pPro128=
)T=000
051
1000
Genom
esrs201992337
g178097260CgtT
c454GgtA
Exon
4(100
9)Cn
s(pGlu152Lys)
nars201589693
g178097251CgtT
c463GgtA
Exon
4(1018)
Cns(pV
al155Ile)
T=000
051
1000
Genom
es
rs35577826
g178097185AgtC
c529Tgt
GEx
on4(1084)
Cns(pLeu177Va
l)C=000143
Alt0005Dagger
1000
Genom
es[23]
rs181513314
g178096710CgtT
c621GgtA
Exon
5(1176)
Cn(pLeu207=
)T=000
051
1000
Genom
esrs60132461
g178096675TgtC
c656AgtG
Exon
5(1211)
Cns(pLys219Arg)
C=000184
1000
Genom
es
rs139187151
g178096634GgtA
c697Cgt
TEx
on5(1252)
Cns(pPro233Ser)
A=000
051
A=0012Dagger
(84)
1000
Genom
es[24]
rs35557421
g178096620d
elT
c711delA
Exon
5(1266)
Fram
eshiftdeletio
n(pLys237=fs)
na
rs34154613
g178096529CgtT
c802GgtA
Exon
5(1357)
Cns(pV
al268M
et)
T=000184
1000
Genom
esrs141363120
g178096
406GgtA
c925Cgt
TEx
on5(14
80)
Cns(pLeu309Ph
e)A=000378
1000
Genom
esrs201661476
g178096380AgtC
c951Tgt
GEx
on5(1506)
Cns(pIle
317M
et)
na
Oxidative Medicine and Cellular Longevity 9
Table3Con
tinued
IDMap
onchromosom
e2lowast
(HGVS
name)
Positionin
Nfe2
l2
(HGVS
name)
Region
s(position
from
mRN
A)
Varia
tioncla
ssand
consequences
(HGVS
name)dagger
Minor
allelefrequ
ency
(MAF)
MAcoun
tsDagger(coh
ort
size)
MAFsources
rs199673454
g178096309TgtA
c1022AgtT
Exon
5(1577)
Cns(pAs
p341Va
l)A=000
051
1000
Genom
esrs35007548
g178096299GgtA
c1032CgtT
Exon
5(1587)
Cs(pSer344=
)A=000
092
1000
Genom
esrs200209692
g178096287TgtC
c104
4AgtG
Exon
5(1599)
Cs(pLeu348=
)C=000
051
1000
Genom
es
mdashg178096237CgtA
c1094GgtT
Exon
5(1649)
Cns(pSer365Ile
)A=0125273
A=000
6Dagger(84)
PubM
ed[24]
rs201214197
g178096171CgtT
c1160GgtA
Exon
5(1715)
Cns(pSer387Asn)
T=000
051
1000
Genom
esrs200494292
g178096165TgtC
c1166AgtG
Exon
5(1721)
Cns(pLys389Arg)
nars186171287
g178096115GgtA
c1216CgtT
Exon
5(1771)
Cns(pPro4
06Ser)
A=000
051
1000
Genom
esrs182276775
g178096062CgtA
c1269GgtT
Exon
5(1824)
Cns(pGlu423A
sp)
A=000
051
1000
Genom
esrs201560221
g17809604
8TgtC
c1283AgtG
Exon
5(1838)
Cns(pLys428Arg)
nars189238236
g178096043AgtG
c1288TgtC
Exon
5(1843)
Cs(pLeu430=
)G=000
051
1000
Genom
esrs184287392
g178096022GgtA
c1309CgtT
Exon
5(1864)
Cns(pArg437Trp)
A=000
051
1000
Genom
esrs201871588
g178095986GgtA
c1345CgtT
Exon
5(19
00)
Cns(pAg449Cy
s)na
rs181294188
g178095985TgtC
c1346GgtA
Exon
5(19
01)
Cns(pArg44
9His)
T=000
092
1000
Genom
esrs20169046
6g178095973TgtA
c1358AgtT
Exon
5(19
13)
Cns(pHis4
53Leu)
A=000
051
1000
Genom
esrs105704
4(rs52789869)
g178095781CgtT
c1550GgtA
Exon
5(2105)
Cns(pArg517Lys)
TDagger[23]
rs200750800
g178095603AgtG
c1728TgtC
Exon
5(2283)
Cs(pTyr576=
)na
rs200175942
g178095567AgtG
c1764TgtC
Exon
5(2319)
Cs(pAsp588=
)G=000
051
1000
Genom
esrs77547666
g178095495GgtC
clowast18Cgt
GEx
on5UTR
-31015840(2391)
SNP
C=000
6915
1000
Genom
esrs73031353
g178095425TgtC
clowast88AgtG
Exon
5UTR
-31015840(2461)
SNP
C=000184
1000
Genom
esrs675944
3g178095345TgtC
clowast168AgtG
Exon
5UTR
-31015840(2541)
SNP
C=000
419
1000
Genom
esrs188674558
g178095279CgtA
clowast234GgtT
Exon
5UTR
-31015840(2607)
SNP
A=000
051
1000
Genom
esrs77685897
g178095247AgtG
clowast266TgtC
Exon
5UTR
-31015840(2639)
SNP
G=000
092
1000
Genom
esrs1057092
g178095162TgtG
clowast351AgtC
Exon
5UTR
-31015840(2724)
SNP
nars3197704
g178095162TgtG
clowast351AgtC
Exon
5UTR
-31015840(2724)
SNP
nars184701151
g178095159TgtC
clowast354AgtG
Exon
5UTR
-31015840(2727)
SNP
C=000276
1000
Genom
esrs11543307
g178095153AgtG
clowast360TgtC
Exon
5UTR
-31015840(2733)
SNP
nars111874043
g178095146AgtG
clowast367TgtC
Exon
5UTR
-31015840(2740)
SNP
nars34012004
g178095102AgtC
clowast411TgtG
Exon
5UTR
-31015840(2784)
SNP
C=0071
[24]
10 Oxidative Medicine and Cellular Longevity
Table3Con
tinued
IDMap
onchromosom
e2lowast
(HGVS
name)
Positionin
Nfe2
l2
(HGVS
name)
Region
s(position
from
mRN
A)
Varia
tioncla
ssand
consequences
(HGVS
name)dagger
Minor
allelefrequ
ency
(MAF)
MAcoun
tsDagger(coh
ort
size)
MAFsources
rs201481890
g178095090178095091in
sTclowast
422lowast423insA
Exon
5UTR
-31015840
(2795minus
2796)
Insertion
na
rs3082500
g178095089178095090d
elTT
delTTinsT
clowast423lowast424d
elAAinsA
Exon
5UTR
-31015840
(2796minus
2797)
Dele
tion
insertion
na
rs71792546
(rs71796710)
g178095079d
elT
clowast425delA
Exon
5UTR
-31015840(2798)
Dele
tion
na
rs1057106
g178095078AgtC
AgtT
clowast435TgtA
TgtG
Exon
5UTR
-31015840(2808)
SNP
na
rs34176791
g178095076AgtC
clowast437TgtG
Exon
5UTR
-31015840(2810)
SNP
C=000235
1000
Genom
esrs35911553
g178095045CgtT
clowast46
8GgtA
Exon
5UTR
-31015840(2841)
SNP
T=000
6915
1000
Genom
esSequ
ence
varia
tions
inup
stream
andexon
sofh
uman
NRF
2fro
m655varia
tions
availablein
publicdatabase
asof
Decem
ber2012
(583
activ
esomeSN
Psmergedge14
SNPs
citedin
PubM
ed)lowastNCB
Ireference
sequ
ence
NC
0000
0211
(Hom
osapiens
chromosom
e2G
RCh37p
10prim
arya
ssem
bly)spanning
178095033ndash1781298
59bp
(com
plem
ent34827
bpforexons
andintro
ns)51015840-Flank
ingregions
startat17812
9860
bp(minus1)in
transcrip
tvariant
1HGVS
Hum
anGenom
eVa
riatio
nSo
ciety
Position
sinvaria
nt1(NM
006164
4)2859
bpsectSN
Pscitedin
PubM
edE
xon11781298
59ndash17812
9260
(600
bpT
TS=1781293
04)exon
2178098999ndash
178098733(267
bp)exon
3178098067ndash1780979
78(90b
p)exon41780973
11ndash178097120
(192b
p)and
exon
5178096736ndash
178095033(1704b
p)daggerProteinam
inoacid
(aa)
resid
uesiniso
form
1(N
P00
61552605
aa)Cn
scoding
-non
syno
nymou
sCs
cod
ing-syno
nymou
sDaggerheterozygositydetectednano
tavailable
Oxidative Medicine and Cellular Longevity 11
Table4Hum
anNRF
2SN
Psassociated
with
diseaser
isk
IDMap
onchromosom
e2 (H
GVS
name)
Region
class
Dise
asea
ssociatio
nandreferences
Risk
allelesa
ndstatistics
Ethn
icgrou
p(num
bero
fcase)
rs7557529
g178135097CgtT
minus5238
Ggt
AParkinsonrsquos
disease(2010
[26])
G=0718252Dagger
inhaplotype
OR=090
or040
CI=
060ndash140or
03ndash06
Swedish
Polish
Caucasian
(357)
rs2886162
g178133165AgtG
minus3306
TgtC
Breastcancer
survival(2012[27])
TT=0324
OR=16
87C
I=1105ndash275
FinlandKB
CP(452)
Chronicg
astritisgastric
ulcer(2007
[28])
GDagger
GDaggerin
haplotypefor
ulcer(OR=
252C
I=119ndash
545)
Japanese
(159)
P14methylationin
gastric
cancer
Hpyloriinfectio
n(2008[29])
Gin
haplotype
OR=290C
I=114ndash
736
Japanese
(209)
Gastriccancer
inHpylori-n
egative
cases(2008
[30])
Ain
haplotype
119875=0022
Japanese
(209)
rs35652124
(rs57695243)
g178130073TgtC
minus214Agt
G(previou
slyminus653[23]
orminus686[22])
Ulcerativec
olitis(2008
[31])
AG
(OR=045C
I=022ndash0
93)
G(O
R=257C
I=10
1ndash660)
Japanese
(89)
Lupu
swith
neph
ritisin
female(2010
[25])
GA
OR=18
1CI
=10
4ndash312
Mexican
mestizo(362)
Parkinsonrsquos
disease(2010
[26])
A=0884312Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
COPD
(2010[32])
G=052Dagger
hazard
ratio
=095C
I=091ndash0
99
(Haplotype)
German
(69)
Gastriculcer(2007
[28])
GDaggerin
haplotype
OR=252C
I=119ndash
545
Japanese
(159)
P14methylationin
gastric
cancer
H
pyloriinfection(2008[29])
Gin
haplotype
OR=290C
I=114ndash
736
Japanese
(209)
Gastriccancer
inHpylori-negative
cases(2008
[30])
Gin
haplotype119875=0022
Japanese
(209)
rs6706
649
g178130071CgtT
minus212Ggt
A(previou
slyminus651[23]
orminus684[22])
Ulcerativec
olitis(2008
[31])
AG
(OR=045C
I=022ndash0
93)
G(O
R=257C
I=10
1ndash660)
Japanese
(89)
Maternalacetaminop
henandasthma
(2010[33])
A=02321137Dagger
OR=17
3CI
=12
2245
UKALSPA
C(gt40
00mothersgt
5000
child
ren)
12 Oxidative Medicine and Cellular Longevity
Table4Con
tinued
IDMap
onchromosom
e2 (H
GVS
name)
Region
class
Dise
asea
ssociatio
nandreferences
Risk
allelesa
ndstatistics
Ethn
icgrou
p(num
bero
fcase)
COPD
(2010[32])
G=098Daggerin
haplotype
hazard
ratio
=095C
I=091ndash0
99
German
(69)
Parkinsonrsquos
disease(2010
[26])
G=0972343Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
03ndash06
Swedish
Polish
Caucasian
(357)
Acutelun
ginjury
follo
wingtrauma
(2007[23])
CA=0119Dagger
OR=644
CI=
134ndash
3080
Caucasia
nAfrican-
American
(164
)
Ann
ualF
EV1decline
(2011[34])
A=0082in
haplotype
Japanese
(915)
Vitiligo(2008[35])
CAA
AOR=17
24C
I=13
5ndash221
Chinese(300)
COPD
(2010[32])
G=073Daggerin
haplotype
hazard
ratio
=095C
I=091ndash0
99
German
(69)
rs6721961
(rs117801448)
g178130037TgtC
TgtG
minus178Agt
GAgt
C(previou
slyminus617[23]
orminus650[22])
Parkinsonrsquos
disease(2010
[26])
C=0980346Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
Postm
enop
ausalvenou
sthrombo
embo
lism
(2011[36])
A=033311
OR=25CI
=370ndash8570
Caucasia
n(161)
Breastcancer
survivalandNRF
2proteinexpressio
n(2012[27])
AA
OR=4656CI
=13
5ndash1606
FinlandKB
CP(452)
Acutelun
ginjury-related
mortality
followingsyste
micinflammatory
respon
sesynd
rome(2012
[37])
GG
OR=97
3CI
=12
7ndash7480
Caucasia
n(750)
Infection-indu
cedasthma(
2012
[38])
AC
OR=0437CI
=028ndash0
80
Hun
garia
nCaucasia
nGypsy
(307)
rs143406266
g178129391178
129393delGGC
Exon
1(467ndash4
69G
CC)
COPD
(2010[32])
GCC
4=053Dagger
hazard
ratio
=095C
I=091ndash0
99
Taiwanese(69)
rs2886161
g178127839Tgt
ClowastIntro
n1
Parkinsonrsquos
disease(2010
[26])
T=0878309Dagger
inhaplotype
OR=09or
04CI
=06ndash
14or
03ndash06
Swedish
Polish
Caucasian
(357)
rs2364723
g178126546Ggt
CIntro
n1
Basaland
smoker
FEV
1(200
9[39])
CI=minus6360simminus1780C
=0525Dagger
Also
ashaplotype
Netherla
nd(2542)
rs2364722
g178124787Agt
GIntro
n1
Ann
ualF
EV1decline
(2011[34])
A=0082in
haplotype
Japanese
(915)
rs13001694
g178118990Agt
GIntro
n1
Basaland
smoker
FEV
1(200
9[39])
G=040
11578Dagger
inhaplotype
Netherla
nd(2542)
Oxidative Medicine and Cellular Longevity 13
Table4Con
tinued
IDMap
onchromosom
e2 (H
GVS
name)
Region
class
Dise
asea
ssociatio
nandreferences
Risk
allelesa
ndstatistics
Ethn
icgrou
p(num
bero
fcase)
Breastcancer
[40]
Twith
NQO1N
OS3H
O1risk
allelesOR=15
6CI
=097ndash251
Caucasia
nandothers(505)
rs1806
649
(rs58745895)
g178118152Cgt
TIntro
n1
Basaland
smoker
FEV
1(200
9[39])
T=02631119Daggerin
haplotype
CI=minus8730ndash
(minus17
0)Netherla
nd(2542)
Parkinsonrsquos
disease(2010
[26])
T=0422148Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
Particulatem
attera
ndasthmaCO
PDadmiss
ion(2012[41])
Cwith
lowvitamin
Clevel(OR=
31CI
=15
0ndash630)
UK(209)
rs4243387
(rs6003846
4)g178117765Cgt
TIntro
n1
Basaland
smoker
FEV
1(200
9[39])
T=00914
25Daggerin
haplotype
Netherla
nd(2542)
rs1962142
(rs58448508)
g178113484Agt
GIntro
n1
Ann
ualF
EV1decline
(2011[34])
A=0082in
haplotype
Japanese
(915)
Breastcancer
NRF
2andARE
expressio
n(2012[27])
A(119875=0036)
FinlandKB
CP(452)
rs6726395
(rs57309289)
g178103229Agt
GIntro
n1
Smok
ing-relatedFE
V1decline
and
annu
alFE
V1decline
(2011[34])
G=0884Dagger
A=0082in
haplotype
Japanese
(915)
Basaland
smoker
FEV
1(200
9[39])
G=046
41764Daggerin
haplotype
Netherla
nd(2542)
rs2001350
(rs17515179
rs60883775)
g178100
425Cgt
TIntro
n1
Ann
ualF
EV1decline
(2011[34])
T=0082in
haplotype
Japanese
(915)
Parkinsonrsquos
disease(2010
[26])
T=0986350Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
rs10183914
(rs58731187
rs61374844
)g17809766
6Cgt
TIntro
n3
Parkinsonrsquos
disease(2010
[26])
T=0536188Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
rs2706110
g178092162Tgt
C31015840Flanking
Breastcancer
(2012[27])
TT
OR=2079CI
=118ndash368
FinlandKB
CP(452)
rs2588882
g178087165Ggt
T31015840Flanking
Infection-indu
cedasthma(
2012
[38])
TG
OR=0290CI
=013ndash0
62
Hun
garia
nCaucasia
nGypsy
(307)
minus686in
reference[22]
=minus653in
reference[23]
=currentlyminus214minus684in
reference[22]
=minus651inreference[23]
=currentlyminus212minus651inreference[22]
=minus617in
reference[23]
=currentlyminus178Ch
romosom
econtig(in
tron
31015840flank
ing)
orreversed
(51015840flank
ingprom
oter)a
llelesinbo
ldhave
been
used
inthetextand
TableOR
odds
ratio
CI95confi
denceinterval
14 Oxidative Medicine and Cellular Longevity
minus212GG (98 rs67006649 previously minus684minus651) minus178Callele (73) and GCC
4(53) was predicted to increase
respiratory failure development (hazard ratio = 095 CI091ndash099) in German COPD patients [32] Significant inter-action was also identified between an intronic SNP Gallele (rs6726395 g178103229AgtG 884 frequency) andsmoking status on FEV
1decline relative to the reference
AA allele in the above Japanese cohort [34] Siedlinskiet al [39] reported that the CC genotype of anotherintronic SNP (rs2364723 g178126546GgtC) was associatedwith a lower FEV
1level compared to the wild-type geno-
type (GG) in two Netherland cohorts (CI minus636minus178frequency = 0525 and pooled cohort size = 2542) ThisSNP alone or as a haplotype with 4 more intronic SNPs(rs13001694Grs1806649Trs4243387Trs6726395G)was alsoassociated with high FEV
1levels in individuals that ever
smoked [39] In a Hungarian population of childhoodasthma SNPs at minus178 (CA) and 31015840 flanking (rs2588882TG)loci were inversely associated with infection-induced asthma(OR 0437 CI 028ndash080 OR 0290 CI 013ndash062 resp) andthese SNPs significantly influenced an asthma-environmentalpollution interaction [38]The intronic SNP rs1806649 (CgtT)was associated but not significantly with an increased risk ofhospitalization during high-level particulate matter (PM
10)
periods in asthma or COPD patients (119899 = 209) of the UnitedKingdom (UK) [41] Asthma andCOPDadmission rateswererelated to the increase in environmental PM
10concentration
Importantly effects of interaction between prenatal stressand NRF2 SNPs on descendant pulmonary health wereinvestigated by the Avalon Longitudinal Study in the UKmaternal smoking during pregnancy was not associated withlung function change determined by maximum mild expi-ratory flow (FEF
25ndash75) or with asthma incidence in school-aged children and this relation was not modified by NRF2SNP genotypes [42]However early gestation acetaminophenexposure significantly influenced the risk of asthma andwheezing at the age of 7 years in gt4000 mothers and gt5000children [33] When maternal copies of the minus212A allele werepresent association with asthma (11374891 OR 173 CI122ndash245) and wheezing (11494949 OR 153 95 CI 106ndash220) was significantly increased [33]
Gastrointestinal Disorders While there was no evidence inlung cancer cases studies in Japanese populations suggesteda potential association of NRF2 variations with gastro-intestinal tumorigenesisHelicobacter pylori (H pylori) causesgastritis which can lead to gastric atrophy and cancer Ingastric epithelium from the Japanese cancer cohorts (39gastric cancers 46 controls) H pylori infection was posi-tively correlated with aberrant CpG island methylation oftumor suppressor genes (eg p14) and minus214Gminus212G orminus214Aminus212G NRF2 haplotype was significantly associatedwith increased (OR 290 95 CI 114ndash736) or decreased(OR 033 95 CI 013ndash088) risk of the CpG methyla-tion respectively in the H pylori-infected patients [29]Further study from the same investigators determined thatminus214Aminus212G allele carriers had significantly (119875 = 0022)reduced risk of gastric cancer inH pylori-negative cases [30]The minus214AGminus212AG genotypes were negatively associated
(OR 045 CI 022ndash093) and the minus214Gminus212G genotypeswere positively associated (chronic continuous phenotypeOR 257 CI 101ndash660) with ulcerative colitis (89 patients 141controls) in a Japanese population [31]
Autoimmune Disorders Systemic lupus erythematosus (SLE)is a long-term autoimmune disease more frequently found infemales than in males It affects organs including skin jointskidneys and brain and nephritis is an aggressive character-istic in some patients Genome-wide association studies inhumans identified a suggestive quantitative trait locus nearNRF2 [43] A study of a Mexican Mestizo population (362patients with childhood-onset SLE 379 controls and 212nephritis diagnosed) determined that lupus with nephritiswas significantly (OR 181 CI 104ndash312) associated with theminus214GA SNP in females [25] The same SNPs were notclosely associated with SLE risk in a Japanese cohort [22]Vitiligo is a skin condition in which there is a loss of browncolor (pigment) from areas of skin resulting in irregularwhite patches It is thought to be an autoimmune diseasecaused by loss of cells (melanocytes) that produce brownpigment A study indicated that theminus178A allele increased therisk of vitiligo dose-dependently (OR 1724 95 CI 135ndash221for CA OR 2902 CI 162ndash519 for AA) [35]
Female Disorders It is well known that estrogen metabolites(eg catechols) cause ROS formation suggesting correla-tion of NRF2 and downstream effectors in postmenopausalmammary cancer In a study of a Finish population (KuopioBreast Cancer Project 119899 = 452 patients 370 controls)the minus178AA homozygous genotype (OR 4656 CI = 135ndash1606) and 31015840 flanking rs2706110 (TT OR 2079 CI 118ndash368) genotype were associated with increased risk of breastcancer while the 51015840 flanking minus3306TT homozygous allelewas significantly associated with lower survival (frequency =71219 OR 1687 CI 1105ndash275) [27] suggesting that NRF2genetic polymorphisms affect susceptibility and outcome ofthe patients The minus178A allele carriers together with intronicrs1962142A allele carriers were associated with lowered tissuelevels of NRF2 proteins [27] In postmenopausal women theminus178A allele (OR 179 95CI 370ndash8570) appeared tomodifythe risk of venous thromboembolism caused by oral estrogentherapy (AA or AC frequency = 333) as demonstratedby the French ESTHER study (161 cases 474 controls) [36]An intronic rs1806649CgtT SNP did not associate with breastcancer risk in postmenopausal women [40] However whenthis SNP and other at-risk alleles of ARE-responsive genes(NQO1 HO-1 NOS3) were combined there was a significantgene-dose effect on the breast cancer risk [40] Althoughcoding region SNPs in NRF2 and KEAP1 were identifiedin the Japanese endometrial adenocarcinoma patients noassociation of NRF2 SNPs with the disease was found [44]
Neurodegenerative Diseases Oxidative stress is known to beinvolved in Parkinsonrsquos disease (PD) presumably due toproduction of ROS fromhigh-dopaminemetabolism and lowlevels of antioxidants in the substantia nigra of the brainInvestigators found a protective NRF2 haplotype consistingof four 51015840 flanking SNPs (minus5238Gminus214Aminus212Gminus178C)
Oxidative Medicine and Cellular Longevity 15
and 4 intronic SNPs (rs2886161Ars1806649Ars2001350Ars10183914A) from Swedish (OR 09 CI 060ndash140) and Polish(OR 04 CI 030ndash060) populations (total 165+192 PD cases190+192 controls) [26]The investigators also suggested thatNRF2 haplotype alleles were associated with 2 years earlierage of Alzheimerrsquos disease (AD) onset 4 years earlier age ofposterior subcapsular cataract surgery and 4 years later ageof cortical cataract surgery while they were not significantlyrelated to AD or age-related cataract risk [45]
34 Genetic Mutations in Mouse Nrf2 Tsang et al [46]compiled 673 SNPs in 55 mouse strains and constructedtheir phylogenetic tree to correlate and clarify the originsof strains based on the assembled mouse genome sequenceand SNP data [20 47 48] Recently using the completegenome sequence of C57BL6J (B6) mouse as a referencehigh-density SNP screening in other laboratory strains orin panels of strains has been published (see [17]) Althoughmillions of mouse SNPs (gt10089892 as of December 2012)and haplotype mappings from more than 120 strains havebeen published as valuable references for dissecting thegenetic basis of complex traits [49ndash51] little attention hasbeen paid to polymorphisms of Nrf2 and their correlationwith disease phenotypes
Figure 3 demonstrates the proximal promoter region(minus1 to minus950)51015840-UTR (exon 1 up to TSS) and proteinsequence of mouse Nrf2 based on GRCm38p1 PrimaryAssembly (75704641ndash75675513 bp) mRNA variant1 and protein (NP 035032 encoded by NM 010902234ndash2027 bp) sequences Genetic variations in the Nrf2genome of inbred strains collected from public databasesare listed in Tables S2 and 5 (See Supplementary TableS2) Overall 968 genetic mutations are compiled forNrf2 gene and 5 kb upstream2 kb downstream regions785 SNPs between B6 and another 16 strains wereacquired from the Mouse Phenome Database (MPDhttpphenomejaxorg dbqrtn=snpret1) and additionalSNPs and other mutations were acquired from NCBI dbSNP(httpwwwncbinlmnihgovsnpterm=mus+musculus20nfe2l2) In total 132 mutations are in the promoter (37in proximal 1 kb) 49 in exons (38 in coding region 19 Cns)727 in introns and 60 in the 31015840 flanking region Excludingmutations in the 51015840 and 31015840 flanking sequences murine Nrf2sequences appear to be more highly variable (1 variationper 375 bp) than much of the mouse genome which has anapproximate frequency of one SNP per every 245 bp (httpwwwinformaticsjaxorgmgihomehomepagesstatsallstatsshtmlallstats snp)
Nrf2 was found to be a susceptibility gene from genome-wide linkage analysis in a murine model of hyperoxia-induced ALI [52] A promoter SNP minus103TgtC (previouslypublished as minus336TgtC) in Nrf2 was found and predicted toadd an additional Sp1 binding site in hyperoxia-susceptibleB6 mice but not in resistant C3HHeJ mice [52] Genotypesfrom the SNP and from simple-sequence length polymor-phism markers of the Nrf2 locus (D2Mit248 and D2Mit94)cosegregated in the B6C3F
2mouse cohort [52] and Nrf2
deficient mice were significantly more susceptible to ALI
sub-phenotypes caused by hyperoxia than similarly exposedwild-type mice supporting Nrf2 as a contributor to thephenotypic traits [53] Although no other functional analyseson Nrf2 SNPs or haplotype association studies have beenconducted in inbred mice strains bearing haplotypes suchas multiple Cns in functional domains (eg F71L L451VH543Q and L575M) may be useful to elucidate the role ofNrf2 in differential susceptibility to oxidative diseases
4 Oncogenic Somatic Mutations in HumanNF-E2-Related Factor 2
Somatic mutation is a change in the DNA of somatic cellsthat affects derived cells but is not inherited by offspringEfforts to discover somatic mutations have provided insightinto mutagenesis and cancer development Lung cancerparticularly non-small cell lung cancer (NSCLC) is theleading cause of cancer death worldwide Somatic mutationsof NRF2 and KEAP1 discovered in lung cancer patients havedetermined the oncogenic potential ofNRF2 [54 55] KEAP1somatic mutations were associated with its reduced proteinlevels in lung cancer tissues and cells [56 57] Investigationsof NSCLC in various ethnic populations as well as cancersin gastrointestine breast and prostate have coordinatelydemonstrated that multiple Cns somatic mutations in KEAP1cause dysfunction of the translated protein and in turnconstitutive activation of NRF2 increasing risk of neoplasiaand chemoresistance [12 55 58 59] Somatic mutations ofNRF2 have been detected in various cancer tissues (largelysquamous cell carcinomas) in Asian populations (Table 6)NRF2 mutations were significantly associated with NSCLCcases (squamous cell lung carcinoma adenocarcinoma) ofthe Japanese (107 [54]) the Chinese (23 [60]) andthe Koreans (8 [61]) as well as with lung cancer celllines Smoking history was also correlated with mutationoccurrence in all of the studies [54 60 61] In addition tolung cancers laryngeal squamous carcinoma (13 in [61])esophageal squamous cancer (ESC 22 in [60] 114 in[61]) head and neck cancers (25 in [54]) skin (117 casein [61]) and oral cancer cell lines had somatic changesin NRF2 In contrast to wide-spread KEAP1 mutationsmutations in NRF2 were clustered in DLGETGE motifs ofthe Neh2 domain which are critical in the ldquohinge and latchrdquomodel of KEAP1 binding [12] Similar to KEAP1 somaticmutations it has been postulated that NRF2 mutations incancer cells lead to NRF2 accumulation by suppressing itsubiquitination or KEAP1 binding which eventually confersmalignant potential and resistance to chemotherapy
Most variable sites in NRF2 included aa residues 29 (AspD) 31 (Gly G) 77 (Asp D) and 79 (Glu E) (Table 6)Residue 33 (Ser S) in the Neh2 domain is mutated by eithergenetic or somatic processes (Figure 4) Cns in the EDGFmotif of NRF2 was experimentally determined to impairrecognition of KEAP1 [54] NRF2 mutations were signif-icantly correlated with increased (25-fold) copy number(31 of mutants versus 3 wild types) in Japanese NSCLCcases [63] Aberrant mutation of NRF2 also led to increasedexpression of downstream effectors includingRagD known to
16 Oxidative Medicine and Cellular Longevity
pL575M
pF71L
pH543QpL451V
pA151P pA151V pN159D pQ198delpV105D
pA204T pS224I225S226del pT253S pS278ApD305G
pM409V pR410HpP388L pA389G pT395I
Genomic DNA (reversed contig GRCm38 p1 C57BL6JNM 0109023)
Protein (NP 035032)1
51101151201251301351401451501551
minus400
minus500
minus50
minus100
minus200
minus300
minus450
minus350
minus250
minus150
minus950
minus900
minus850
minus800
minus750
minus700
151
101151201
2001
g minus470gg minusg minus
c minus41c minus
c
c
c c
gg minus 6C
g minus125insTTC g minus118insC
g minus281delC
g minus63delCg minus1 (75704642 c minus234)
Translation start site (c +1)Termination codon (c +2025 +2027)
c minus136 minus134in-frame stop codonc minus8585
g minus41
246
440
minus381
minus11
minus91 minus90
minus332
minus153
minus98
c minus197c minus209 minus202
gminus942 minus899 (cminus1175 minus1132) CpG islands [71]
g 460CgtT
g minus103CCgtT [51]
CgtTCgtG
g minus320CgtT
CgtT
CgtT
CgtTg minus426GgtA
GgtACgtA
CgtA
GgtA
g minus238GgtTTgtC
TgtC
TgtC
TgtCTgtG
g minus229AgtG
CgtG
CgtG
GgtC
gminus826 minus654 (cminus1059 ) CpG islands [71]minus887
Figure 3 DNA (partial promoter and exons 1 and 5) and protein sequence ofmouseNrf2 SNPs and amino acid residues for non-synonymousSNPs are marked Promoter regions bearing 5CpG islands are underlined
151
201251301351401451501551601
Genetic mutation (Table 3 and Figure 2)Somatic mutation (Table 6)
Neh2
Neh3
Neh4Neh5
Neh6
Neh1
Protein isoform 1 (NP 006155)
101151
Figure 4 Genetic and somatic mutation loci in human NRF2 protein
Oxidative Medicine and Cellular Longevity 17
Table5Mou
seup
stream
andexon
varia
tions
ofNfe2
l2locusin17
inbred
strainsR
eference
sequ
ence
isC5
7BL6J
(B6strain
1)geno
me(GI14933824975547698ndash75513576)SNPalleleand
geno
typesareshow
nas
chromosom
econ
tigsequ
enceStrains
2129S1S
vlmJ3AJ
4AKR
J5BA
LBcJ6C3
HH
eJ7C
57BL
6NJ8CA
STEiJ9CB
AJ
10D
BA2J11FVBNJ12LPJ
13N
ODShiLtJ14N
ZOH
ILtJ
15P
WK
PhJ16SPR
ETEiJ
17W
SBEiJ
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs2566
08517
75705565
GA
(CT)
51015840Flankingminus924
NC
GG
GG
GG
GG
GG
GG
GG
AA
Grs47274959
75705562
AT
(TA
)51015840Flankingminus921
NC
AT
TA
TT
AA
TT
AT
AT
TT
Ars216940398
75705554ndash75705555
Tadd(A
add)
51015840Flankingminus913minus914
NC
CGrs239114134
75705540
CT(G
A)
51015840Flankingminus899
NC
CT
TC
TT
CC
TT
CT
CT
CT
Crs46
461765
75705528
TG(A
C)oA
C
51015840Flankingminus887
NC
TG
GT
GG
TT
GG
TG
TG
GG
Trs5144
9853
75705525
CA(G
T)
51015840Flankingminus884
NC
CA
AC
AA
CC
AA
CA
CA
CC
Crs249093111
75705512
CT(G
A)
51015840Flankingminus871
NC
CT
TC
TT
CC
TT
CT
CT
TT
Crs263745200
75705510
CG(G
C)
51015840Flankingminus869
NC
GG
GG
GG
GG
GG
GG
GG
CG
Grs45651867
75705498
TG(A
C)
51015840Flankingminus857
NC
TG
GT
GG
TT
GG
TG
TG
GG
Trs219997531
75705495
GA
(CT)
51015840Flankingminus854
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs251918379
75705451
TA(A
T)
51015840Flankingminus810
NC
TT
TT
TT
TT
TT
TT
TT
TA
Trs27978306
75705449
TA(A
T)
51015840Flankingminus808
NC
TA
AT
AA
TT
AA
TA
TA
TT
Trs216087412
75705429
TG(A
C)lowast
51015840Flankingminus788
NC
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
GTlowast
Tlowast
rs261229914
75705423ndash75705424
AGdel(CT
del)
51015840Flankingminus783minus784
NC
AGrs27978307
75705400
CT(G
A)
51015840Flankingminus759
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs243167395
75705359
GA
(CT)
51015840Flankingminus718
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs27978308
75705307
GA
(CT)
51015840Flankingminus66
6NC
GG
GG
GG
GG
GG
GG
GG
AG
Grs254744
09875705294
AT
(TA
)51015840Flankingminus653
NC
AA
AA
AA
AA
AA
AA
AA
AT
Ars228133419
75705179
GT
(CA
)51015840Flankingminus538
NC
GG
GG
GG
GG
GG
GG
GG
GT
Grs214719520
75705111
GA
(CT)
51015840Flankingminus470
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs2440
87730
75705101
GA
(CT)
51015840Flankingminus46
0NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs227781699
75705081
CG(G
C)
51015840Flankingminus44
0NC
CC
CC
CC
CC
CC
CC
CC
GC
Crs257870353
75705067
CT(G
A)
51015840Flankingminus426
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs24487744
075705022
AG
(TC)
51015840Flankingminus381
NC
AA
AA
AA
AA
AA
AA
AA
AG
Ars234628138
75704973
AC
(TG
)51015840Flankingminus332
NC
AA
AA
AA
AA
AA
AA
AA
AC
A
18 Oxidative Medicine and Cellular Longevity
Table5Con
tinued
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs247275247
75704961
GA
(CT)
51015840Flankingminus320
NC
GG
GG
GG
GG
GG
GG
AG
GG
Grs247519047
75704922
Gdel(Cdel)
51015840Flankingminus281
NC
Grs27978309
(rs51915758)
75704887
GA
C(C
TG
)51015840Flankingminus246
NC
GA
AG
AA
GG
AA
GA
GA
CC
G
rs218139102
75704879
CA(G
T)
51015840Flankingminus238
NC
CC
CC
CC
CC
CC
CC
CC
AC
Crs251990355
75704870
TC(A
G)
51015840Flankingminus229
NC
TT
TT
TT
TT
TT
TT
TT
TC
Trs238746955
75704794
AG
(TC)
51015840Flankingminus153
NC
AA
AA
AA
AA
AA
AA
AA
GA
Ars221664
405
75704786
AG
(TC)
51015840Flankingminus145
NC
AA
AA
AA
AA
AA
AA
AA
AG
Ars217054035
75704766
ndash75704767
GAAadd(TTC
add)
51015840Flankingminus125minus126
NC
GA
rs248182931
75704759ndash75704760
Gadd(C
add)
51015840Flankingminus118
minus119
NC
AGrs217197904
75704744
GA
(CT)
51015840Flankingminus103
(+)S
p1[23]
GA
AG
AA
GG
AA
GA
AA
GG
Ars2644
9364975704704
Gdel(Cdel)
51015840Flankingminus63
NC
Grs2404
81112
757046
82CT(G
A)
51015840Flankingminus41
NC
CC
CC
CC
CC
CC
CC
CC
CT
Crs27978310
75704617
AG
(TC)
UTR
-525
NC
AA
AA
AA
AA
AA
AA
AA
GG
Ars27978311
75704610
CG(G
C)
UTR
-532
NC
GG
GG
GG
GG
GG
GG
GG
GG
Crs27978312
757046
05GA
(CT)
UTR
-537
NC
GA
AG
AA
GG
AA
GA
GA
GG
Grs214784220
757044
99AG
(TC)
UTR
-5143
NC
AA
AA
AA
AA
AA
AA
AA
GG
Ars244146318
757044
98GT
(CA
)UTR
-5144
NC
GG
GG
GG
GG
GG
GG
GG
GT
Grs27978313
757044
97GC
(CG
)UTR
-5145
NC
GC
CG
CC
GC
CC
GC
CC
CC
Crs215431944
757044
93CT(G
A)
UTR
-5149
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs257015697
757044
49GA
(CT)
UTR
-5193
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs252234782
757044
19GT
(CA
)UTR
-5223
NC
Grs1346
0861
75679262
AG
(TC)
Exon
244
6Cn
sF71L
AG
GA
GG
AG
GG
AG
AG
GG
Grs1346
0859
75679202
GA
(CT)lowast
Exon
2506
CsH91=
GA
AG
AA
GG
AA
GA
GA
GG
Grs27978436
75679187
GA
(CT)lowast
Exon
2521
CsT9
6=G
GG
GG
GG
AG
GG
GG
GA
GG
rs226131070
75679178
GA
(CT)
Exon
2530
CsS99=
GG
GG
GG
GG
GG
GG
GG
GA
Grs227683834
75678576
AT
(TA
)Ex
on3
547
CnsV
105D
AA
AA
AA
AA
AA
AA
AA
AT
Ars257321187
75678506
TC(A
G)
Exon
3617
CsP128=
TT
TT
TT
TC
TT
TT
TT
TT
Trs27978444
75678500
TA(A
T)
Exon
3623
CsV130=
TT
TT
TT
TA
TT
TT
TT
AT
Trs227743136
75677686
GA
(CT)
Exon
4662
CsH143=
GG
GG
GG
GG
GG
GG
GG
GA
Grs215327202
75677664
CG(G
C)
Exon
4684
CnsA
151P
CC
CC
CC
CG
CC
CC
CC
CC
Crs247539755
75677663
GA
(CT)
Exon
4685
CnsA
151V
GG
GG
GG
GA
GG
GG
GG
GG
Grs233164
668
75677640
TC(A
G)
Exon
4708
CnsN
159D
TT
TT
TT
TT
TT
TT
TT
TC
T
rs2340
95816
75677162ndash75677160
GCT
del
(AGCdel)
Exon
5826minus
828
Cds-in
delQ
198
GCT
Oxidative Medicine and Cellular Longevity 19
Table5Con
tinued
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs27978452
75677145
CT(G
A)
Exon
5843
CnsA
204T
CC
CC
CC
CC
CC
CC
CC
CC
T
rs225047937
75677086ndash75677078
GAG
ATCG
ATdel
(ATG
CATC
TCdel)
Exon
5902minus
910
Cds-in
del
(S224I225S226)
GAG
ATC
GAT
rs241469868
75676998
TA(G
T)
Exon
5990
CnT2
53S
TT
TT
TT
TT
TT
TT
TT
TA
Trs221602571
75676923
AC
(TG
)Ex
on5
1065
CnsS
278A
AA
AA
AA
AA
AA
AA
AA
AC
Ars252576472
75676876
GA
(CT)
Exon
51112
CsS293=
GG
GG
GG
GG
GG
GG
GG
GA
Grs250802933
75676841
TC(A
G)
Exon
5114
7Cn
sD305G
TT
TT
TT
TT
TT
TT
TT
TC
Trs225425698
75676792
CT(G
A)
Exon
5119
6Cs
P321=
CC
CC
CC
CC
CC
CC
CC
CT
Crs13459064
75676732
CT(G
A)
Exon
51256
CsT3
41=
CT
TC
TT
CT
TT
CT
CT
TC
Crs27978453
75676717
CT(G
A)
Exon
51271
CsA346=
CC
CC
CC
CC
CC
CC
CC
TC
Crs214335034
75676636
AG
(TC)
Exon
51352
CsD373=
AA
AA
AA
AA
AA
AA
AA
AG
Ars24958364
4756766
03AC
(TG
)Ex
on5
1385
CsP3
84=
AA
AA
AA
AA
AA
AA
AA
AC
Ars27978454
75676592
GA
(CT)
Exon
51396
CnsP
388L
GG
GG
GG
GG
GG
GG
GG
AG
Grs215384328
75676589
GC
(CG
)Ex
on5
1399
CnsA
389G
GG
GG
GG
GG
GG
GG
GG
GC
Grs251728286
75676571
GA
(CT)
Exon
51417
CnsT
395I
Grs247602334
75676567
TC(A
G)
Exon
51421
CsV396=
TT
TT
TT
TT
TT
TT
TT
TC
Trs234216231
75676530
TC(A
G)
Exon
51458
CnsM
409V
TT
TT
TT
TT
TT
TT
TT
TC
Trs212904337
75676526
CT(G
A)
Exon
51462
CnsR
410H
CC
CC
CC
CC
CC
CC
CC
CT
Crs252650779
75676522
TC(A
G)
Exon
51466
CsE4
11=
TT
TT
TT
TT
TT
TT
TT
TC
Trs231273560
75676516
TC(A
G)
Exon
51472
CsQ413=
TT
TT
TT
TT
TT
TT
TT
TC
Trs257886949
756764
13GT
(CA
)Ex
on5
1575
CsR4
48=
GG
GG
GG
GG
GG
GG
GG
GT
Grs241537608
756764
04GC
(CG
)Ex
on5
1584
CnsL
451V
GG
GG
GG
GG
GG
GG
GG
GC
Grs227619071
75676312
TC(A
G)
Exon
51676
CsQ481=
TT
TT
TT
TT
TT
TT
TT
TC
Trs258913831
75676290
AG
(TC)
Exon
51698
CsL4
89=
AA
AA
AA
AA
AA
AA
AA
AG
Ars225593319
75676126
AT
(TA
)Ex
on5
1862
CnsH
543Q
Ars4223233
75676105
GA
(CT)
Exon
51883
CsS550=
GG
GG
GG
GA
GG
GG
GG
GG
Grs4223232
75676032
GT
(CA
)Ex
on5
1956
CnsL
575M
Grs4223231
75675816
CG
UTR
-32172
NC
GG
GG
GG
GG
GG
GG
GG
GC
Grs1346
860
75675682
AT
UTR
-32306
NC
ASequ
ence
varia
tions
inmou
seNr
f2wereob
tained
from
Mou
sePh
enom
eDatabase(http
ph
enom
ejaxorgSNP)
andNCB
ISNPdatabase
(http
wwwncbinlm
nihgovSNP)N
CBIreference
sequ
ence
isMus
Musculusstra
inC5
7BL6J
chromosom
e2GRC
m38p1(NC
0000
687
GI37209910875704641ndash756755192
9123
bp)To
tal968
genetic
mutations
arerepo
rted
inNr
f2gene
and5k
bup
stream
(ge75704
642minus
1)and2k
bdo
wnstre
am(le75675512)sequences
asof
Janu
ary2003A
llSN
Psandallelesa
representedas
geno
miccontig
(reversedsequ
ence
indicated)E
xon175704
641ndash75704364(278
bpT
TS=75704
408)
exon
275679431ndash756791
65(267
bp)exon
375678579ndash75678490(90b
p)exon475677713ndash756775
46(168
bp)andexon
57567718
4ndash75678849
(1666
bp)Proteinam
inoacid
(aa)
resid
uesinNP00
61552
(597
aa)Cs
cod
ing-syno
nymou
sCn
scoding
-non
syno
nymou
sAminoacid
A-alanineD-aspartic
acidE
-glutamicacidF-phenylalanineG
-glycineH
-histidineI-iso
leucineL-leu
cineand
M-m
ethion
ineN-
asparagineP-prolin
eQ-glutamineR-arginineS-serineT-threon
ineandV-valin
elowastErrorsin
databasesfi
xedNC
notcon
firmed
20 Oxidative Medicine and Cellular Longevity
Table 6 NRF2 somatic mutations revealed in various human cancers
Domain Amino acid residues DNA mutation Cancer types (cases) ReferencesLocus Wild type Mutant
DLG motif
24 W (Trp) C (Cys) c72GgtCGgtT NSCLC neck ESC [54 62]K (Lys) c72TgtC ESC [62]
26 Q (Gln) E (Glu) c76CgtG NSCLC ESC [54 62]
27 D (Asp) G (Gly)lowast c80GgtA NSCLC [60]Y (Tyr) c79GgtT ESC [61]
28 I (Ile) T (Thr) c83CgtT NSCLC [54]
29 D (Asp) G (Gly) c86AgtG Head and neck ESC [54 62]H (His) c85GgtC NSCLC laynx [60 61]
30 L (Leu) F (Phe) c88CgtT NSCLC ESC [54 62]
31 G (Gly) A (Ala) c92GgtC NSCLC ESC skin [54 60ndash62]
32 V (Val) T (Thr) c95TgtG NSCLC [54]del c93 95delAGT ESC [61]
33ndash36 S-R-E-V S-R-E-V-S-R-E-Vlowast c97 108dupAGTCGAGCCGTA ESC [61]
34 R (Arg) Q (Gln) c101GgtA NCSCL [54 60 61]P (Pro) c101GgtC NSCLC [63]
ETGF motif
75 Q (Gln) H (His) c225AgtC Head and neck ESC [54 62]
77 D (Asp)
V (Val) c230AgtT NSCLC ESC [54 60 62]G (Gly) c230AgtG ESC [62]A (Ala) c230AgtC NSCLC [61]N (Asn) c229GgtA Larynx [61]
78 E (Glu) K (Lys) c232GgtA NSCLC ESC [54 62]
79 E (Glu)
K (Lys) c235GgtA NSCLC ESC [54 61 62]Q (Gln) c235GgtC NSCLC ESC [54 60ndash62]G (Gly) c236AgtG Larynx [61]E-E c234 236dupAGA ESC [61]
80 T (Thr) K (Lys) c239CgtACgtG NSCLC ESC [54 61 62]
80 T (Thr)P (Phe) c238AgtC ESC [62]I (Ile) c239CgtT Head and neck [54]A (Ala) c238AgtG NSCLC [63]
81 G (Gly) V (Val) c242GgtT ESC [61]D (Asp) c242GgtA NSCLC ESC [60ndash62]
82 E (Glu)
D (Asp) c246AgtT ESC oral cancer cell line [54 62]G (Gly) c245AgtG NSCLC [54]Q (Gln)lowast c244GgtC NSCLC ESC [60 61]V (Val)lowast c245AgtT ESC [61]
83 F (Phe) L (Leu)lowast c247TgtC NSCLC [60]NSCLC non-small cell lung cancer ESC esophageal squamous cancer and lowasterrors in reference fixed Number of cases 82 NSCLC and 10 ESC in [62] 125NSCLC 70 ESC 23 larynx and 17 skin in [61] 103 NSCLC and 12 head and neck in [54] 90 NSCLC in [63] 103 NSCLC in [60]
be involved in squamous lung cancer cell proliferation [64]suggesting that the mutation is functional and overcomesKEAP1 inhibition Singh et al [65] determined in vitrothat RNAi-mediated depletion of NRF2 in lung cancer cellsenhanced ROS production and susceptibility to cell deathby ionizing radiation These studies support the concept thatelevated NRF2 and ARE responsiveness provides cancer cellswith proliferative advantage for malignant transformation
and undue protection from anti-cancer therapy Onco-genic epidermal growth factor receptor (EGFR) signaling isrecently found to be critical in NRF2-mediated proliferationof NSCLC cells [66]
Collectively ldquogain of functionrdquo mutations in NRF2 thatreduce KEAP1 recognition are suggested to be predictivemarkers for poor responsiveness to chemotherapy and radi-ation therapy Although NRF2-mediated cellular defense
Oxidative Medicine and Cellular Longevity 21
processes are essential in the initiation stage enhancedNRF2-ARE activity in advanced stages of cancer developmentmay create a favorable intracellular environment for tumorcell growth and survival [67 68] In this context NRF2may be a potential molecular target for the treatment ofradio-resistance cancers especially those that have ldquoloss offunctionrdquo mutations in EGFR KRAS or KEAP1 as well asldquogain of functionrdquo mutations in NRF2
5 Epigenetic Alterations of NF-E2-RelatedFactor 2
Epigenetic modifications are alterations of molecules inter-acting with genes without changes to the primary DNAsequence They include post-translational modification ofhistones DNA methylation events chromatin conforma-tional changes and alterations to noncoding regulatoryRNAs Epigenetic alterations are stable and often inheritablebut are reversible and may affect expression of the geneDysregulation or defects in epigenetic processes particularlyhypermethylation of tumor suppressor gene promoters (egCpG islands) or histone modifications are thought to beassociated with carcinogenesis Investigators have reportedhypermethylation in CpG islands of KEAP1 which wereassociated with reduced KEAP1 expression in human cancersfrom lung prostate colon and so forth [69ndash72] Similar tosomatic mutations epigenetic changes on KEAP1 impairedthe function of its encoded protein leading to constitutiveNRF2 activation
Supporting the role of ldquopathogenic mutationsrdquo in NRF2expression of Nrf2 and downstream Nqo1 was suppressedin prostate tumors of mice (transgenic adenocarcinomaof mouse prostate TRAMP) Among 15 promoter CpGislands located between minus942 and minus654 (cminus1175 cminus1132and cminus1059 cminus887 gap in cminus1131 cminus1060 see Figure 3)hypermethylation of the first 5 CpG islands (minus942 minus899 andcminus1175 cminus1132) was significantly associated with tumorige-nesis [73] Moreover treatment with inhibitors for DNAmethyltransferase and histone deacetylase restored Nrf2expression in these tumor cells [73] A dietary phytochemicalcurcumin known as a DNA hypomethylation agent restoredepigenetically silent Nrf2 expression through CpG demethy-lation in carcinogen-induced mouse tumor cells [74]
The whole genome epigenetic datasets for 5 species arepublicly accessible at NCBI Epigenomics [75 76]The humanNRF2 epigenome of primary cells (breast penis) and H1stem cell line as well as mouse Nrf2 CpG island methylationdata for sperm blood and cerebellum are currently avail-able (httpwwwncbinlmnihgovepigenomics) Althoughno direct evidence of disease-associated epigenetic modu-lation has been identified in human NRF2 various phyto-chemical NRF2 agonists such as sulforaphane and curcuminhave shown their roles in DNA methylation and histonemodification (see reviews by Lee and colleagues eg [77])Taken together epigenetic modifications of theNRF2KEAP1axle are predicted to cause dysregulation of ARE-mediatedcellular defense leading to deleterious health effects and
phytochemical antioxidants as epigenetic modulators forNRF2 are suggested to be useful in cancer prevention
6 Conclusions
NRF2 is evolutionally conserved with high-sequence homol-ogy in many species However it is a highly mutable geneand numerous genetic variants have been discovered inhuman ethnic groups Importantly certain SNPs or haplo-types have been identified in various diseases as ldquoat-riskrdquoalleles and are related to functional alterations In additionto genetic variations multiple somatic mutations identifiedin the KEAP1 recognition domain of NRF2 in cancer cellshave been found to be oncogenic due to dysregulation ofNRF2 homeostasis by its excess ldquogain of functionrdquo Epigeneticalteration of theNRF2 is under investigation and is predictedto have pathogenic influences as learned from mouse andphytochemical agonist studies Continuous updates of Nrf2allelic variants in inbred mouse strains will provide a usefultool for effective experimental designs formodels of oxidativedisorders to provide insight into the disease mechanisms andintervention strategies
Abbreviations
AD Alzheimerrsquos diseaseALI acute lung injuryARE antioxidant response elementbZIP basic leucine zippercds coding DNA sequencesCI confidence intervalCOPD chronic obstructive pulmonary diseaseESC esophageal squamous cancerFEV1 forced expiratory volume in one second
Gbp giga base pairsGST glutathione S-transferaseGWAS genome-wide association studyHGVS Human Genome Variation SocietyHO-1 heme oxygenase-1119867 119901119910119897119900119903119894 119867119890119897119894119888119900119887119886119888119905119890119903 119901119910119897119900119903119894KEAP1 Kelch-like ECH activating protein 1MPD Mouse Phenome DatabaseMRP multidrug resistance proteinNCBI National Center for Biotechnology InformationNeh NRF2-ECH homologyNfe2l2 nuclear factor (erythroid-derived 2)-like 2Nrf2 NF-E2-related factor 2NSCLC nonsmall cell lung cancerNQO1 NAD(P)Hquinone oxidoreductase 1OR odds ratioPD Parkinsonrsquos diseasePM particulate matterROS reactive oxygen speciesSLE systemic lupus erythematosusSNP single nucleotide polymorphismSOD superoxide dismutaseURT untranslated region
22 Oxidative Medicine and Cellular Longevity
Disclosure
Authorrsquos contribution to the Work was done as part of theAuthorrsquos official duties as a NIH employee and is a Work ofthe United States Government Therefore copyright may notbe established in the United States
Conflict of Interests
The author declares that there is no conflict of interests
Acknowledgments
The research related to this paper was supported by theIntramural Research Program of the National Institutes ofHealth of the National Institute of Environmental HealthSciences (NIEHS) Drs Steven Kleeberger and StephanieLondon at the NIEHS provided excellent critical review ofthis paper The author thanks Mrs Jacqui Marzec for herhelpful comments and English editing
References
[1] J Y Chan M C Cheung P Moi K Chan and Y W KanldquoChromosomal localization of the humanNF-E2 family of bZIPtranscription factors by fluorescence in situ hybridizationrdquoHuman Genetics vol 95 no 3 pp 265ndash269 1995
[2] K Itoh K Igarashi N Hayashi M Nishizawa and MYamamoto ldquoCloning and characterization of a novel erythroidcell-derived CNC family transcription factor heterodimerizingwith the small Maf family proteinsrdquo Molecular and CellularBiology vol 15 no 8 pp 4184ndash4193 1995
[3] W O Osburn and T W Kensler ldquoNRF2 signaling an adaptiveresponse pathway for protection against environmental toxicinsultsrdquoMutation Research vol 659 no 1-2 pp 31ndash39 2008
[4] D Papp K Lenti D Modos D Fazekas Z Dul D Tureiet al ldquoThe NRF2-related interactome and regulome containmultifunctional proteins and fine-tuned autoregulatory loopsrdquoFEBS Letters vol 586 no 13 pp 1795ndash1802 2012
[5] X Wang D J Tomso B N Chorley et al ldquoIdentificationof polymorphic antioxidant response elements in the humangenomerdquo Human Molecular Genetics vol 16 no 10 pp 1188ndash1200 2007
[6] D Malhotra E Portales-Casamar A Singh et al ldquoGlobalmapping of binding sites for NRF2 identifies novel targets incell survival response through ChIP-Seq profiling and networkanalysisrdquo Nucleic Acids Research vol 38 no 17 pp 5718ndash57342010
[7] K Chan R Lu J C Chang and Y W Kan ldquoNRF2 a memberof the NFE2 family of transcription factors is not essential formurine erythropoiesis growth and developmentrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 93 no 24 pp 13943ndash13948 1996
[8] K Itoh T Chiba S Takahashi et al ldquoAn NRF2small Mafheterodimer mediates the induction of phase II detoxifyingenzyme genes through antioxidant response elementsrdquo Bio-chemical and Biophysical Research Communications vol 236no 2 pp 313ndash322 1997
[9] FMartin J M vanDeursen R A Shivdasani CW Jackson AG Troutman and P A Ney ldquoErythroid maturation and globin
gene expression inmicewith combined deficiency ofNF-E2 andNrf-2rdquo Blood vol 91 no 9 pp 3459ndash3466 1998
[10] K Itoh N Wakabayashi Y Katoh et al ldquoKEAP1 repressesnuclear activation of antioxidant responsive elements by NRF2through binding to the amino-terminal Neh2 domainrdquo Genesand Development vol 13 no 1 pp 76ndash86 1999
[11] A Kobayashi M I Kang H Okawa et al ldquoOxidative stresssensor KEAP1 functions as an adaptor for Cul3-based E3 ligaseto regulate proteasomal degradation of NRF2rdquo Molecular andCellular Biology vol 24 no 16 pp 7130ndash7139 2004
[12] K Taguchi H Motohashi and M Yamamoto ldquoMolecularmechanisms of the KEAP1-NRF2 pathway in stress responseand cancer evolutionrdquo Genes to Cells vol 16 no 2 pp 123ndash1402011
[13] K Mukaigasa L T Nguyen L Li H Nakajima M Yamamotoand M Kobayashi ldquoGenetic evidence of an evolutionarilyconserved role for NRF2 in the protection against oxidativestressrdquoMolecular and Cellular Biology vol 32 no 21 pp 4455ndash4461 2012
[14] JMaher andMYamamoto ldquoThe rise of antioxidant signalingmdashthe evolution and hormetic actions of NRF2rdquo Toxicology andApplied Pharmacology vol 244 no 1 pp 4ndash15 2010
[15] S C Lo X Li M T Henzl L J Beamer and M HanninkldquoStructure of the KEAP1NRF2 interface provides mechanisticinsight intoNRF2 signalingrdquoThe EMBO Journal vol 25 no 15pp 3605ndash3617 2006
[16] V A McKusick ldquoMendelian inheritance in man and its onlineversion OMIMrdquo American Journal of Human Genetics vol 80no 4 pp 588ndash604 2007
[17] B Yalcin D J Adams J Flint and T M Keane ldquoNext-generation sequencing of experimental mouse strainsrdquo Mam-malian Genome vol 23 no 9-10 pp 490ndash498 2012
[18] D R Bentley S Balasubramanian H P Swerdlow et alldquoAccurate whole human genome sequencing using reversibleterminator chemistryrdquo Nature vol 456 no 7218 pp 53ndash592008
[19] P S G Chain D V Grafham R S Fulton et al ldquoGenomeproject standards in a new era of sequencingrdquo Science vol 326no 5950 pp 236ndash237 2009
[20] RHWaterstonK Lindblad-Toh E Birney J Rogers J F AbrilP Agarwal et al ldquoInitial sequencing and comparative analysis ofthe mouse genomerdquo Nature vol 420 pp 520ndash562 2002
[21] T M Keane L Goodstadt P Danecek M A White K WongB Yalcin et al ldquoMouse genomic variation and its effect onphenotypes and gene regulationrdquoNature vol 477 pp 289ndash2942011
[22] T Yamamoto K Yoh A Kobayashi et al ldquoIdentification ofpolymorphisms in the promoter region of the human NRF2generdquo Biochemical and Biophysical Research Communicationsvol 321 no 1 pp 72ndash79 2004
[23] J M Marzec J D Christie S P Reddy et al ldquoFunctionalpolymorphisms in the transcription factor NRF2 in humansincrease the risk of acute lung injuryrdquo The FASEB Journal vol21 no 9 pp 2237ndash2246 2007
[24] H Fukushima-Uesaka Y Saito K Maekawa et al ldquoGeneticvariations and haplotype structures of transcriptional factorNRF2 and its cytosolic reservoir protein KEAP1 in JapaneserdquoDrug Metabolism and Pharmacokinetics vol 22 no 3 pp 212ndash219 2007
[25] E J Cordova R Velazquez-Cruz F Centeno V Baca and LOrozco ldquoThe NRF2 gene variant -653GA is associated with
Oxidative Medicine and Cellular Longevity 23
nephritis in childhood-onset systemic lupus erythematosusrdquoLupus vol 19 no 10 pp 1237ndash1242 2010
[26] M von Otter S Landgren S Nilsson et al ldquoAssociation ofNRF2-encoding NFE2L2 haplotypes with Parkinsonrsquos diseaserdquoBMCMedical Genetics vol 11 no 1 article 36 2010
[27] JMHartikainenM TengstromVMKosmaV L Kinnula AMannermaa andY Soini ldquoGenetic polymorphisms and proteinexpression of NRF2 and sulfiredoxin predict survival outcomesin breast cancerrdquo Cancer Research vol 72 pp 5537ndash5546 2012
[28] T Arisawa T Tahara T Shibata et al ldquoThe relationshipbetween Helicobacter pylori infection and promoter polymor-phism of the NRF2 gene in chronic gastritisrdquo InternationalJournal of Molecular Medicine vol 19 no 1 pp 143ndash148 2007
[29] T Arisawa T Tahara T Shibata et al ldquoThe influence ofpromoter polymorphism of nuclear factor-erythroid 2-relatedfactor 2 gene on the aberrant DNA methylation in gastricepitheliumrdquo Oncology Reports vol 19 no 1 pp 211ndash216 2008
[30] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene pro-moter polymorphism and gastric carcinogenesisrdquo Hepato-Gastroenterology vol 55 no 82-83 pp 750ndash754 2008
[31] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene promoterpolymorphism is associated with ulcerative colitis in a Japanesepopulationrdquo Hepato-Gastroenterology vol 55 no 82-83 pp394ndash397 2008
[32] C C Hua L C Chang J C Tseng C M Chu Y C Liu andW B Shieh ldquoFunctional haplotypes in the promoter region oftranscription factor NRF2 in chronic obstructive pulmonarydiseaserdquo Disease Markers vol 28 no 3 pp 185ndash193 2010
[33] S O Shaheen R B Newson S M Ring M J Rose-ZerilliJ W Holloway and A J Henderson ldquoPrenatal and infantacetaminophen exposure antioxidant gene polymorphismsand childhood asthmardquo The Journal of Allergy and ClinicalImmunology vol 126 no 6 pp 1141e7ndash1148e7 2010
[34] H Masuko T Sakamoto Y Kaneko et al ldquoLower FEV1 innon-COPD nonasthmatic subjects association with smokingannual decline in FEV1 total IgE levels and TSLP genotypesrdquoInternational Journal of Chronic Obstructive Pulmonary Diseasevol 6 pp 181ndash189 2011
[35] C P Guan M N Zhou A E Xu et al ldquoThe susceptibilityto vitiligo is associated with NF-E2-related factor 2 (NRF2)gene polymorphisms a study on Chinese Han populationrdquoExperimental Dermatology vol 17 no 12 pp 1059ndash1062 2008
[36] J Bouligand O Cabaret M Canonico et al ldquoEffect of NFE2L2genetic polymorphism on the association between oral estrogentherapy and the risk of venous thromboembolism in post-menopausal womenrdquo Clinical Pharmacology amp Therapeuticsvol 89 no 1 pp 60ndash64 2011
[37] D S OrsquoMahony B J Glavan T D Holden C Fong R A BlackG Rona et al ldquoInflammation and immune-related candidategene associations with acute lung injury susceptibility andseverity a validation studyrdquo PLoS ONE vol 7 no 12 Article IDe51104 2012
[38] I Ungvari E Hadadi V Virag et al ldquoRelationship between airpollutionNFE2L2 gene polymorphisms and childhood asthmain aHungarian populationrdquo Journal of CommunityGenetics vol3 no 1 pp 25ndash33 2012
[39] M SiedlinskiD S Postma JMA Boer et al ldquoLevel and courseof FEV1 in relation to polymorphisms inNFE2L2 andKEAP1 inthe general populationrdquo Respiratory Research vol 10 article 732009
[40] C C Hong C B Ambrosone J Ahn et al ldquoGenetic variabilityin iron-related oxidative stress pathways (NRF2 NQ01 NOS3
and HO-1) iron intake and risk of postmenopausal breastcancerrdquo Cancer Epidemiology Biomarkers and Prevention vol16 no 9 pp 1784ndash1794 2007
[41] C Canova C Dunster F J Kelly C Minelli P L Shah CCaneja et al ldquoPM10-induced hospital admissions for asthmaand chronic obstructive pulmonary disease the modifyingeffect of individual characteristicsrdquo Epidemiology vol 23 no 4pp 607ndash615 2012
[42] A J Henderson R B Newson M Rose-Zerilli S M RingJ W Holloway and S O Shaheen ldquoMaternal NRF2 andgluthathione-S-transferase polymorphisms donotmodify asso-ciations of prenatal tobacco smoke exposure with asthma andlung function in school-aged childrenrdquo Thorax vol 65 no 10pp 897ndash902 2010
[43] C Xing A L Sestak J A Kelly et al ldquoLocalization andreplication of the systemic lupus erythematosus linkage signalat 4p16 interaction with 2p11 12q24 and 19q13 in EuropeanAmericansrdquo Human Genetics vol 120 no 5 pp 623ndash631 2007
[44] T F Wong K Yoshinaga Y Monma K Ito H NiikuraS Nagase et al ldquoAssociation of KEAP1 and NRF2 geneticmutations and polymorphisms with endometrioid endometrialadenocarcinoma survivalrdquo International Journal of Gynecologi-cal Cancer vol 21 no 8 pp 1428ndash1435 2011
[45] M von Otter S Landgren S Nilsson et al ldquoNRF2-encodingNFE2L2 haplotypes influence disease progression but not riskin Alzheimerrsquos disease and age-related cataractrdquoMechanisms ofAgeing and Development vol 131 no 2 pp 105ndash110 2010
[46] S Tsang Z Sun B Luke et al ldquoA comprehensive SNP-basedgenetic analysis of inbred mouse strainsrdquoMammalian Genomevol 16 no 7 pp 476ndash480 2005
[47] C M Wade E J Kulbokas III A W Kirby et al ldquoThe mosaicstructure of variation in the laboratory mouse genomerdquoNaturevol 420 no 6915 pp 574ndash578 2002
[48] T Wiltshire M T Pletcher S Batalov et al ldquoGenome-widesingle-nucleotide polymorphism analysis defines haplotypepatterns in mouserdquo Proceedings of the National Academy ofSciences of the United States of America vol 100 no 6 pp 3380ndash3385 2003
[49] K A Frazer E Eskin H M Kang et al ldquoA sequence-basedvariation map of 827 million SNPs in inbred mouse strainsrdquoNature vol 448 no 7157 pp 1050ndash1053 2007
[50] T P Maddatu S C Grubb C J Bult andM A Bogue ldquoMousephenome database (MPD)rdquo Nucleic Acids Research vol 40 ppD887ndashD894 2007
[51] A Kirby H M Kang C M Wade et al ldquoFine mapping in 94inbred mouse strains using a high-density haplotype resourcerdquoGenetics vol 185 no 3 pp 1081ndash1095 2010
[52] H Y Cho A E Jedlicka S P M Reddy L Y Zhang T WKensler and S R Kleeberger ldquoLinkage analysis of susceptibilityto hyperoxia NRF2 is a candidate generdquo American Journal ofRespiratory Cell and Molecular Biology vol 26 no 1 pp 42ndash512002
[53] H Y Cho A E Jedlicka S P M Reddy et al ldquoRole of NRF2in protection against hyperoxic lung injury in micerdquo AmericanJournal of Respiratory Cell and Molecular Biology vol 26 no 2pp 175ndash182 2002
[54] T Shibata T Ohta K I Tong et al ldquoCancer related mutationsin NRF2 impair its recognition by KEAP1-Cul3 E3 ligase andpromote malignancyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 36 pp13568ndash13573 2008
24 Oxidative Medicine and Cellular Longevity
[55] A Singh V Misra R K Thimmulappa et al ldquoDysfunctionalKEAP1-NRF2 interaction in non-small-cell lung cancerrdquo PLoSMedicine vol 3 no 10 article e420 2006
[56] T Ohta K Iijima M Miyamoto et al ldquoLoss of KEAP1 functionactivates NRF2 and provides advantages for lung cancer cellgrowthrdquo Cancer Research vol 68 no 5 pp 1303ndash1309 2008
[57] B Padmanabhan K I Tong T Ohta et al ldquoStructural basisfor defects of KEAP1 activity provoked by its point mutationsin lung cancerrdquoMolecular Cell vol 21 no 5 pp 689ndash700 2006
[58] T Shibata A Kokubu M Gotoh et al ldquoGenetic alteration ofKEAP1 confers constitutive NRF2 activation and resistance tochemotherapy in gallbladder cancerrdquoGastroenterology vol 135no 4 pp 1358e4ndash1368e4 2008
[59] N J Yoo H R Kim Y R Kim C H An and S H LeeldquoSomatic mutations of the KEAP1 gene in common solidcancersrdquo Histopathology vol 60 no 6 pp 943ndash952 2012
[60] Y Hu Y Ju D Lin Z Wang Y Huang S Zhang et alldquoMutation of the NRF2 gene in non-small cell lung cancerrdquoMolecular Biology Reports vol 39 no 4 pp 4743ndash4747 2012
[61] Y R Kim J EOhM S Kim et al ldquoOncogenicNRF2mutationsin squamous cell carcinomas of oesophagus and skinrdquo TheJournal of Pathology vol 220 no 4 pp 446ndash451 2010
[62] T Shibata A Kokubu S Saito et al ldquoNRF2 mutation confersmalignant potential and resistance to chemoradiation therapyin advanced esophageal squamous cancerrdquo Neoplasia vol 13pp 864ndash873 2011
[63] H Sasaki M Shitara K Yokota Y Hikosaka S MoriyamaM Yano et al ldquoIncreased NRF2 gene (NFE2L2) copy numbercorrelates with mutations in lung squamous cell carcinomasrdquoMolecular Medicine Reports vol 6 no 2 pp 391ndash394 2012
[64] H Sasaki M Shitara K Yokota Y Hikosaka S Moriyama MYano et al ldquoRagD gene expression andNRF2mutations in lungsquamous cell carcinomasrdquo Oncology Letters vol 4 pp 1167ndash1170 2012
[65] A Singh M Bodas N Wakabayashi F Bunz and S BiswalldquoGain of NRF2 function in non-small-cell lung cancer cellsconfers radioresistancerdquo Antioxidants and Redox Signaling vol13 no 11 pp 1627ndash1637 2010
[66] T Yamadori Y Ishii SHommaYMorishimaKKurishimaKItoh et al ldquoMolecular mechanisms for the regulation of NRF2-mediated cell proliferation in non-small-cell lung cancersrdquoOncogene vol 31 pp 4768ndash4777 2012
[67] A K Bauer H Y Cho L Miller-Degraff C Walker K HelmsJ Fostel et al ldquoTargeted deletion of NRF2 reduces urethane-induced lung tumor development in micerdquo PLoS ONE vol 6no 10 Article ID e26590 2011
[68] H Y Cho and S R Kleeberger ldquoNRF2 protects against airwaydisordersrdquo Toxicology and Applied Pharmacology vol 244 no1 pp 43ndash56 2010
[69] R Wang J An F Ji H Jiao H Sun and D Zhou ldquoHyperme-thylation of the KEAP1 gene in human lung cancer cell linesand lung cancer tissuesrdquo Biochemical and Biophysical ResearchCommunications vol 373 no 1 pp 151ndash154 2008
[70] P Zhang A Singh S Yegnasubramanian et al ldquoLoss ofkelch-like ECH-associated protein 1 function in prostate cancercells causes chemoresistance and radioresistance and promotestumor growthrdquoMolecular Cancer Therapeutics vol 9 no 2 pp336ndash346 2010
[71] N Hanada T Takahata Q Zhou et al ldquoMethylation of theKEAP1 gene promoter region in human colorectal cancerrdquoBMCCancer vol 12 article 66 2012
[72] L A Muscarella R Barbano V DrsquoAngelo et al ldquoRegulationof KEAP1 expression by promoter methylation in malignantgliomas and association with patientrsquos outcomerdquo Epigeneticsvol 6 no 3 pp 317ndash325 2011
[73] S Yu T O Khor K L Cheung et al ldquoNRF2 expressionis regulated by epigenetic mechanisms in prostate cancer ofTRAMP micerdquo PLoS ONE vol 5 no 1 Article ID e8579 2010
[74] T O Khor Y Huang T Y Wu L Shu J Lee and A N KongldquoPharmacodynamics of curcumin as DNA hypomethylationagent in restoring the expression of NRF2 via promoter CpGsdemethylationrdquo Biochemical Pharmacology vol 82 no 9 pp1073ndash1078 2011
[75] I M Fingerman L McDaniel X Zhang et al ldquoNCBI epige-nomics a new public resource for exploring epigenomic datasetsrdquo Nucleic Acids Research vol 39 supplemen 1 pp D908ndashD912 2011
[76] I M Fingerman X Zhang W Ratzat N Husain R F Cohenand G D Schuler ldquoNCBI epigenomics whatrsquos new for 2013rdquoNucleic Acids Research vol 41 no 1 pp D221ndashD225 2013
[77] J H Lee T O Khor L Shu Z Y Su F Fuentes andA N Kong ldquoDietary phytochemicals and cancer preventionNRF2 signaling epigenetics and cell death mechanisms inblocking cancer initiation and progressionrdquo Pharmacology ampTherapeutics vol 137 no 2 pp 153ndash171 2012
Submit your manuscripts athttpwwwhindawicom
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
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Behavioural Neurology
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Disease Markers
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BioMed Research International
OncologyJournal of
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Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
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Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
4 Oxidative Medicine and Cellular Longevity
Table1Con
tinued
Species
Rhesus
mon
key
Macacamulatta
Chicken
Gallusgallus
Zebrafish
Daniorerio
Mou
seMus
musculus
Rat
Rattu
snorvegicus
Officialfullname
nucle
arfactor
(erythroid-derived
2)-like
2nu
clear
factorerythroid-derived
2lik
e2Antho
rnam
enu
clear
factor
erythroid2-related
factor
2NF-E2
-related
factor
2NFE
2-related
factor
2Antho
rnam
eHEB
P1mdash
mdashmdash
mdash
Genes
ynon
yms
NFE
2L2NRF
2Mmu966
NFE
2L2NRF
2nfe2l2N
rf2N
rf2a
wufc15g09wufj6
7e03
Nfe2
l2N
rf2A
I194320
Nfe2
l2N
rf2
Chromosom
emap
127
92C
3(4475
cM)
3q23
Genom
esequence
(NCB
IBuild)
NC
0078691
NC
0060
943
NC
0071205
(Zv9)
NC
0000
687
(GRC
m38p1)
NC
0051023
(Rno
r50)
Prim
arySource
mdashCG
NC
4960
4ZF
INZ
DB-GEN
E-030723-2
MGI108420
RGD620360
GeneIDmapping
(orie
ntationlowast
)70
7606
40848940minus40
8846
00(minus)
3960
1415304546minus15320356
3601
491643746minus
1670288
1802
475675519minus757046
41(minus)
8361
969041647minus69069070
(minus)
Ensembleg
eneID
(mapping
)EN
SMMUG00
0000
01861
(40848948minus
40884174)
ENSG
ALG
0000
0009240
(16897956minus
16922637)
ENSD
ARG
0000
0042824
(1643631minus1672715)
ENSM
USG
0000
0015839
(75675513minus
757046
41)
ENSR
NOG00
0000
01548
(58366
693minus
58394118)
RefSeq
IDm
RNA
size
NM
0012576071
(GI383872375)2335b
pNM
2051171
(GI45384113)2555
bpNM
1828891
(GI33504557)2149
bpNM
0109023
(GI76573877)2469
bpNM
0317892
(GI402692377)2305b
pProteinID
(aaMW
sect )NP001244
5361
(606
67703)
NP9904
481
(58265160)
NP8783091
(58665757)
NP0350321
(59766770)
NP1139771
(59766825)
UniProt
IDF7GPD
8Q90834
Q8JIM
1Q60795
O54968
Varia
nts
2transcrip
ts2iso
form
s1transcript1isoform
1transcript1isoform
1transcript
1transcript1isoform
Hom
olog
ygeneprotein
988990
726674
546491
834825
838832
Referto
httpwwwncbinlm
nihgovhom
ologene2412
and
reference
[14]forcross-speciesho
molog
yDetails
ofho
molog
yscores
and
sequ
encescan
bealso
obtained
byblastagainsthu
man
sequ
ence
(http
blastn
cbin
lmnihgov)
Furtherinform
ation
ontranscrip
tvaria
ntsand
protein
isoform
sareview
able
bygene
IDat
NCB
I(http
wwwncbinlm
nihgovrefseqrsg)
and
eEn
semble
(http
useastensembleo
rgin
dexhtml)or
byproteinID
atUniProt(http
wwwun
iproto
rg)
sect MWpredicted
molecularweightfrom
NCB
I(MW
inUniProtvarie
sslightly)Re
fSeqreference
sequ
enceaaam
ino
acidsandlowastgene
incomplem
ento
rientation
Oxidative Medicine and Cellular Longevity 5
Table 2 Protein domains of NF-E2-related factor 2
Domains Amino acid positionslowast Predicted functionsHuman (605 aa) Mouse (597 aa)
Neh216ndash89
DLG motif 17ndash32ETGF motif 77ndash82
16ndash89 KEAP1 repression through DLGETGF motif-DC motif binding fastredox-sensitive proteasomal degradation
Neh4 111ndash134 111ndash134 Translocation and transactivation Phosphorylation or CBP bindingNeh5 182ndash209 172ndash201
Neh6 337ndash394(or 338ndash388) 328ndash385 Degron motif-associated constitutive turnover slow redox-insensitive
(Keap1-independent)
Neh1435ndash568
basic motif 503ndash518leucine zipper 525ndash539
427ndash560basic motif 494ndash509leucine zipper 517ndash531
Dimerization for nuclear translocation DNA binding through basicmotif-leucine zipper
Neh3 569ndash605 561ndash597 CHD6 binding stability or transactivationlowastVaries slightly among publications
homology (Neh) domains configuring the protein sequenceof either species (Table 2) and the potential functions of eachregion particularly the highly conserved KEAP1-bindingNeh2 and DNA- (ARE-) binding Neh1 domains have beenintensively investigated [12 14 15]
3 Genetic Variation of NF-E2-Related Factor 2in Human and Mouse
31 Evolution Genome Sequence and Polymorphism Dis-covery in Human and Mouse While rare and monogenicMendelian diseases are inheritable mutations in a single gene[16] many common diseases are complex traits and thedisease phenotypes are affected by variants inmultiple geneticloci Recent advancements in high-throughput technologyhave enabled sequencing of entire mammalian genomes[17ndash19] and information on DNA sequence and variationhas facilitated the study of complex traits of human dis-orders Genome-wide association studies (GWAS) examinewhether SNPs are associated with important disease traitsand ascertains ldquoat-riskrdquo genotypes that are significantly moreprevalent in the affected group than in the nonaffected groupThe HapMap Project (httphapmapncbinlmnihgov) hasmapped combinations of alleles at specific loci (haplotypes)that is common patterns of sequence variation in severalhuman populations It has supported efficient mapping ofmultiple loci for complex traits in GWAS Candidate geneapproaches based on findings from GWAS of similar dis-orders have also been useful for determining the potentialgenetic mechanisms of diseases
The evolutionary divergence of human and mouse lin-eages occurred for roughly 75million years and their genomesequences have been altered by nearly one substitution (ordeletion or addition) for every twonucleotides [20]Howeverthis slow evolution process resulted in a high degree ofconservation across the two species which allows alignmentof orthologous sequences gt90 of the human and mousegenomes is partitioned into corresponding regions of con-served synteny and at the nucleotide level approximately 40of the human genome is aligned to themouse [20]Due to this
fact biomedical studies of human genes are complemented byexperimental manipulation of corresponding mouse genesand they have aided functional understanding of genesin human health Following the 2003 completion of theHuman Genome Project of approximately 31 giga base pairs(Gbp) the Mouse Genome Project assembled the completegenome sequence of one strain (C57BL6J 2716965481 bp)in 2011 Using this reference strain whole genome sequenc-ing data across 16 additional inbred strains were done(httpwwwsangeracukresourcesmousegenomes [21])Discovery of high-density SNPs in the mouse genome sup-ports evolutionary history of the strain and provides a toolto investigate models of human disease processes that cannotoften be practically achieved through direct human studies
32 GeneticMutations in HumanNRF2 HumanNRF2 codesthree major isoforms of protein (Figure 2) Transcript variant2 (NM 0011454122 2746 bp) has an alternate promoter 51015840-UTR and a downstream start codon compared to variant 1(NM 0061644 2859 bp) It encodes an isoform 2 missing N-terminal 16 aa (NP 001138884 or Q16236-2 589 aa) relativeto isoform 1 (NP 0061552 or Q16236 605 aa) Isoform 3(NP 0011388851 or Q16236-3 582 aa) is encoded by tran-script variant 3 (NM 0011454132 2725 bp) and lacks aninternal segment relative to isoform 2 due to an alternate in-frame splice site in the 31015840 coding region In public databasesmore than 583 sequence mutations are reported in NRF2(34827 bp) and 7000 bp upstream (Table S1 data acquired asof December 2012) (See Table S1 in Supplementary Materialavailable on line at httpdxdoiorg1011552013286524)NRF2 locates on 178130354-178129304 bp of GRCh37p10PrimaryAssembly and Figure 2 shows sequences of proximalpromoter (minus1 to minus500) partial mRNA variant 1 including 51015840-UTR (exon 1 up to TSS) and protein isoform 1 (NP 006155encoded by variant 1 NM 0061644 556ndash2373 bp) Basedon the current assembly and sequence update previouspromoter positions minus686 [22]minus653 [23] are identified asminus214 minus684 [22]minus651 [23] as minus212 and minus650 [22]minus617 [23]asminus178Overall frequency ofNRF2 SNPs and othermutationsis about 1 per 72 bp The genetic mutations include 37 in
6 Oxidative Medicine and Cellular Longevity
Translation start site for variants 2 and 3
1 missed in isoforms 2 and 3pA72T pS99P pQ121H missed in isoform 3
pP406S pE423D pK428R pR437W pR449C
minus400
minus500
minus50minus100
minus200
minus300
minus450
minus350
minus250
minus150
g minus
g
pE152K pV155I pL177V
pV268M
pR517K pH453L
pS365I pS387N pK389R pL309F pI317M pD341V
pK219R pP233S pK237 = fs
51101151201251301351401451501551601
Protein isoform 1 (NP 006155)
g minus66minus65insC
Termination codon (c +2371 +2373)
Translation start site (c +1)
151
101151201251301351401451501551601
2351
frame stop codonc minus156 154 in-old + 1 [23]
old + 1 [22]
g minus1 (178130354)
c minus96delC
c minus438 minus437delTT
minus89 minus87delGCC
transcription start site)+1 (c minus555
p R43Q p R43W
Genomic DNA (reversed contig GRCh37 p10 primary assemblyNM 0061644)minus477
minus214 g minus212gminus188
c 9minus95
g minus178
c minus304
c 8minus13
c 2minus16
TgtA
CgtT
GgtA
GgtA
GgtA
GgtT
GgtCAgtG
AgtCAgtG
Figure 2 DNA (variant 1 partial promoter and exons 1 and 5) and protein (isoform 1) sequence of Human NRF2 SNPs and amino acidresidues for nonsynonymous SNPs are marked
the 51015840 flanking promoter and 59 in exons (Tables 3 and S1)Among exon SNPs 26 are nonsynonymous (Cns) mutationsA triplet repeat variation (rs143406266 GCC
4versus GCC
5
previously published as minus20minus6) in the 51015840-UTR was uniquelyidentified in Asian populations [22 24]
33 SNPs Haplotypes and Association with Disease RiskThe use of gene knockout mice in model systems hasprovided potential insights into the role of NRF2 in thepathogenesis of various human disorders (see Figure 1)Recent epidemiological and association studies have revealedsignificant associations of NRF2 sequence variations withdisease risks which further supportsNRF2 as a susceptibilitygene Most of the phenotype-associated variants are in thepromoter region and presumed to be involved in NRF2 generegulation Table 4summarizes NRF2 SNP andor haplotypealleles that have been associated with oxidant-related diseaserisks Interestingly there is no evidence for exon SNPs as at-risk alleles For convenience and consistency intronic and31015840 distal SNP alleles are presented as chromosome contig(HGVS) alleles while promoter and exon SNPs are presentedas reversed contig alleles throughout the text
Pulmonary Diseases NRF2 SNPs in the promoter and intron1 sequences have been investigated for their potential asso-ciations with risk of pulmonary critical disorders includingacute lung injury (ALI) cigarette smoke-induced chronicobstructive pulmonary disease (COPD) and asthma Aheterozygous CA SNP at minus178 position (rs6721961TgtCor TgtG previously minus617 or minus650) significantly increasedthe risk for developing ALI following major trauma inEuropean and African-American populations (odds ratioOR 644 95 confidence interval CI 134ndash308 allelicfrequency = 119 at 21180) [23] Promoter activity ofthe A allele (AC or AA) determined in vivo and invitro was significantly lower than CC allele at that locus(minus178 in an ARE-like motif) indicating that it is a func-tional SNP for autoregulation [23] The minus178GG werealso nominally associated with ALI-related 28-day mor-tality following systemic inflammatory response syndrome[37] In a Japanese cohort SNP haplotype (rs2001350Trs6726395Ars1962142Ars2364722Ars6721961T) co-tainingthe minus178AA homozygote was associated with an annualdecline of rapid forced expiratory volume in one second(FEV1) in relation to cigarette-smoking status [34] In addi-
tion a promoter and 51015840-UTR SNP haplotype consistingof minus214G allele (52 rs35652124 previously minus686minus653)
Oxidative Medicine and Cellular Longevity 7Ta
ble3Geneticmutations
inprom
oter
andexon
sofh
uman
NRF
2
IDMap
onchromosom
e2lowast
(HGVS
name)
Positionin
Nfe2
l2
(HGVS
name)
Region
s(position
from
mRN
A)
Varia
tioncla
ssand
consequences
(HGVS
name)dagger
Minor
allelefrequ
ency
(MAF)
MAcoun
tsDagger(coh
ort
size)
MAFsources
rs16865105
g178136629AgtC
cminus555minus
6770Tgt
G51015840Flanking
(minus6770)
SNP
C=01928421
1000
Genom
esrs7557529
g178135097CgtT
cminus555minus
5238GgtA
51015840Flanking
(minus5238)
SNP
nars6750320
g178131796CgtT
cminus555minus
1937GgtA
51015840Flanking
(minus1937)
SNP
T=0030266
1000
Genom
esrs18116
2518
g178131774TgtC
cminus555minus
1915AgtG
51015840Flanking
(minus1915)
SNP
C=000
051
1000
Genom
esrs1900
44775
g178131746CgtT
cminus555minus
1887GgtA
51015840Flanking
(minus1887)
SNP
T=000
051
1000
Genom
esrs185117338
g178131704AgtG
cminus555minus
1845Tgt
C51015840Flanking
(minus1845)
SNP
G=000
051
1000
Genom
esrs149947189
g178131697CgtT
cminus555minus
1838GgtA
51015840Flanking
(minus1838)
SNP
T=000
092
1000
Genom
esrs139771244
g178131625AgtG
cminus555minus
1766Tgt
C51015840Flanking
(minus1766)
SNP
G=000
092
1000
Genom
esrs6747203
g17813160
4CgtG
cminus555minus
1745GgtC
51015840Flanking
(minus1745)
SNP
G=000
613
1000
Genom
esrs193101749
g178131504TgtC
cminus555minus
1645AgtG
51015840Flanking
(minus1645)
SNP
C=000
4610
1000
Genom
esrs190630762
g178131495GgtC
cminus555minus
1636Cgt
G51015840Flanking
(minus1636)
SNP
C=000
092
1000
Genom
esrs183764
402
g178131366CgtA
cminus555minus
1507GgtT
51015840Flanking
(minus1507)
SNP
A=000
051
1000
Genom
esrs19122296
4g178131211GgtA
cminus555minus
1352Cgt
T51015840Flanking
(minus1352)
SNP
A=000
092
1000
Genom
esrs187137522
g178131165TgtG
cminus555minus
1306AgtC
51015840Flanking
(minus1306)
SNP
G=000
092
1000
Genom
esrs182620359
g178131158AgtG
cminus555minus
1299Tgt
C51015840Flanking
(minus1299)
SNP
G=000
051
1000
Genom
esrs4893819
(rs61433302)
g178131134CgtT
cminus555minus
1275GgtA
51015840Flanking
(minus1275)
SNP
C=04263931
1000
Genom
es
rs191547130
g178131017CgtT
cminus555minus
1158GgtA
51015840Flanking
(minus1158)
SNP
T=000
051
1000
Genom
esrs188422217
g178131003AgtG
cminus555minus
1144TgtC
51015840Flanking
(minus114
4)SN
PG=000143
1000
Genom
esrs143047764
g178130865AgtG
cminus555minus
1006Tgt
C51015840Flanking
(minus1006)
SNP
G=000
6915
1000
Genom
esrs74432849
g178130766CgtA
cminus555minus
907GgtT
51015840Flanking
(minus907)
SNP
nars116
79252
g178130691CgtG
cminus555minus
832GgtC
51015840Flanking
(minus832)
SNP
nars12993217
g178130516AgtG
cminus555minus
657TgtC
51015840Flanking
(minus657)
SNP
nars115644
826
g17813044
2TgtA
cminus555minus
583AgtT
51015840Flanking
(minus583)
SNP
A=0015133
1000
Genom
esrs140803524
g178130431GgtA
cminus555minus
572CgtT
51015840Flanking
(minus572)
SNP
A=000
4610
1000
Genom
es
rs776844
20g178130427TgtC
cminus555minus
568AgtG
51015840Flanking
(minus568)
SNP
C=0033974
C=0190(84)
1000
Genom
es[24]
rs183651094
g178130336AgtT
cminus555minus
477TgtA
51015840Flanking
(minus477)
SNP
T=000184
1000
Genom
es
rs35652124
sect
(rs57695243)
g178130073TgtC
cminus555minus
214AgtG
51015840Flanking
(minus214)
SNP
C=03512767
T=0429(84)
T=0413181Dagger
C=033827Dagger
C=03517
69
1000
Genom
es[24]
[22]
[23]
[25]
rs6706649
sectg178130071CgtT
cminus555minus
212GgtA
51015840Flanking
(minus212)
SNP
T=0078170
T=004
8(84)
T=004
821Dagger
T=00753Dagger
1000
Genom
es[24]
[22]
[23]
rs1506
48896
g178130047CgtG
cminus555minus
188GgtC
51015840Flanking
(minus188)
SNP
G=000235
G=000
6(84)
1000
Genom
es[24]
rs6721961
sect
(rs117801448)
g178130037TgtC
TgtG
cminus555minus
178AgtC
AgtG
51015840Flanking
(minus178)
SNP
T=0150328
T=0283124Dagger
T=031325Dagger
T=0321(84)
1000
Genom
es[22]
[23]
[24]
8 Oxidative Medicine and Cellular Longevity
Table3Con
tinued
IDMap
onchromosom
e2lowast
(HGVS
name)
Positionin
Nfe2
l2
(HGVS
name)
Region
s(position
from
mRN
A)
Varia
tioncla
ssand
consequences
(HGVS
name)dagger
Minor
allelefrequ
ency
(MAF)
MAcoun
tsDagger(coh
ort
size)
MAFsources
rs20134560
4g178129924178129925insG
cminus555-66minus555-65insC
51015840Flanking
(minus66minus65)
Insertion
G=0017939
1000
Genom
es
rs200432479
g178129741178
129742delAA
cminus438minus437delT
Texon
1UTR
-51015840(118minus119
)Dele
tion
-=000378
-=000
6(84)
1000
Genom
es[24]
rs75485459
g178129608CgtA
cminus304GgtT
Exon
1UTR
-51015840(252)
SNP
nars192086766
g17812946
6CgtT
cminus162GgtA
Exon
1UTR
-51015840(394)
SNP
T=002248
1000
Genom
esmdash
g17812944
2GgtA
cminus138CgtT
Exon
1UTR
-51015840(418)
SNP
A=0012(84)
[24]
rs7166
8246
g17812940
0delG
cminus96delC
Exon
1UTR
-51015840(460)
Dele
tion
nars187291840
g178129399CgtT
cminus95GgtA
Exon
1UTR
-51015840(461)
SNP
T=0054912
01000
Genom
es
rs143406266
g178129391178129393delGGC
cminus89minus87delGCC
Exon
1UTR
-51015840(467minus469)
Dele
tion
-=064
4282Dagger
-=0589(84)
[22]
[24]
rs182428269
g178098918GgtA
c127Cgt
TEx
on2(682)
Cns(pArg43Trp)
A=000
051
1000
Genom
esrs35248500
g178098917CgtT
c128GgtA
Exon
2(683)
Cns(pArg43Gln)
T=000
613
1000
Genom
esrs1135118
g178098831CgtT
c214GgtA
Exon
2(769)
Cns(pAla72Th
r)TDagger
[23]
rs19969166
0g178098829AgtT
c216Tgt
AEx
on2(771)
Cs(pAla72=)
namdash
g178098769GgtA
c276Cgt
TEx
on2(831)
Cs(pIle92=)
TDagger[23]
rs5031039
g178098750AgtG
c295Tgt
CEx
on2(850)
Cns(pSer99P
ro)
G=0Dagger
[23]
rs200239262
g178098017CgtG
c363GgtC
Exon
3(918)
Cns(pGln121H
is)na
rs183034165
g178098008TgtC
c372GgtA
Exon
3(927)
Cs(pAla124=
)T=000
092
T=000
6(84)
1000
Genom
es[24]
rs199970826
g178097996CgtT
c384GgtA
Exon
3(939)
Cs(pPro128=
)T=000
051
1000
Genom
esrs201992337
g178097260CgtT
c454GgtA
Exon
4(100
9)Cn
s(pGlu152Lys)
nars201589693
g178097251CgtT
c463GgtA
Exon
4(1018)
Cns(pV
al155Ile)
T=000
051
1000
Genom
es
rs35577826
g178097185AgtC
c529Tgt
GEx
on4(1084)
Cns(pLeu177Va
l)C=000143
Alt0005Dagger
1000
Genom
es[23]
rs181513314
g178096710CgtT
c621GgtA
Exon
5(1176)
Cn(pLeu207=
)T=000
051
1000
Genom
esrs60132461
g178096675TgtC
c656AgtG
Exon
5(1211)
Cns(pLys219Arg)
C=000184
1000
Genom
es
rs139187151
g178096634GgtA
c697Cgt
TEx
on5(1252)
Cns(pPro233Ser)
A=000
051
A=0012Dagger
(84)
1000
Genom
es[24]
rs35557421
g178096620d
elT
c711delA
Exon
5(1266)
Fram
eshiftdeletio
n(pLys237=fs)
na
rs34154613
g178096529CgtT
c802GgtA
Exon
5(1357)
Cns(pV
al268M
et)
T=000184
1000
Genom
esrs141363120
g178096
406GgtA
c925Cgt
TEx
on5(14
80)
Cns(pLeu309Ph
e)A=000378
1000
Genom
esrs201661476
g178096380AgtC
c951Tgt
GEx
on5(1506)
Cns(pIle
317M
et)
na
Oxidative Medicine and Cellular Longevity 9
Table3Con
tinued
IDMap
onchromosom
e2lowast
(HGVS
name)
Positionin
Nfe2
l2
(HGVS
name)
Region
s(position
from
mRN
A)
Varia
tioncla
ssand
consequences
(HGVS
name)dagger
Minor
allelefrequ
ency
(MAF)
MAcoun
tsDagger(coh
ort
size)
MAFsources
rs199673454
g178096309TgtA
c1022AgtT
Exon
5(1577)
Cns(pAs
p341Va
l)A=000
051
1000
Genom
esrs35007548
g178096299GgtA
c1032CgtT
Exon
5(1587)
Cs(pSer344=
)A=000
092
1000
Genom
esrs200209692
g178096287TgtC
c104
4AgtG
Exon
5(1599)
Cs(pLeu348=
)C=000
051
1000
Genom
es
mdashg178096237CgtA
c1094GgtT
Exon
5(1649)
Cns(pSer365Ile
)A=0125273
A=000
6Dagger(84)
PubM
ed[24]
rs201214197
g178096171CgtT
c1160GgtA
Exon
5(1715)
Cns(pSer387Asn)
T=000
051
1000
Genom
esrs200494292
g178096165TgtC
c1166AgtG
Exon
5(1721)
Cns(pLys389Arg)
nars186171287
g178096115GgtA
c1216CgtT
Exon
5(1771)
Cns(pPro4
06Ser)
A=000
051
1000
Genom
esrs182276775
g178096062CgtA
c1269GgtT
Exon
5(1824)
Cns(pGlu423A
sp)
A=000
051
1000
Genom
esrs201560221
g17809604
8TgtC
c1283AgtG
Exon
5(1838)
Cns(pLys428Arg)
nars189238236
g178096043AgtG
c1288TgtC
Exon
5(1843)
Cs(pLeu430=
)G=000
051
1000
Genom
esrs184287392
g178096022GgtA
c1309CgtT
Exon
5(1864)
Cns(pArg437Trp)
A=000
051
1000
Genom
esrs201871588
g178095986GgtA
c1345CgtT
Exon
5(19
00)
Cns(pAg449Cy
s)na
rs181294188
g178095985TgtC
c1346GgtA
Exon
5(19
01)
Cns(pArg44
9His)
T=000
092
1000
Genom
esrs20169046
6g178095973TgtA
c1358AgtT
Exon
5(19
13)
Cns(pHis4
53Leu)
A=000
051
1000
Genom
esrs105704
4(rs52789869)
g178095781CgtT
c1550GgtA
Exon
5(2105)
Cns(pArg517Lys)
TDagger[23]
rs200750800
g178095603AgtG
c1728TgtC
Exon
5(2283)
Cs(pTyr576=
)na
rs200175942
g178095567AgtG
c1764TgtC
Exon
5(2319)
Cs(pAsp588=
)G=000
051
1000
Genom
esrs77547666
g178095495GgtC
clowast18Cgt
GEx
on5UTR
-31015840(2391)
SNP
C=000
6915
1000
Genom
esrs73031353
g178095425TgtC
clowast88AgtG
Exon
5UTR
-31015840(2461)
SNP
C=000184
1000
Genom
esrs675944
3g178095345TgtC
clowast168AgtG
Exon
5UTR
-31015840(2541)
SNP
C=000
419
1000
Genom
esrs188674558
g178095279CgtA
clowast234GgtT
Exon
5UTR
-31015840(2607)
SNP
A=000
051
1000
Genom
esrs77685897
g178095247AgtG
clowast266TgtC
Exon
5UTR
-31015840(2639)
SNP
G=000
092
1000
Genom
esrs1057092
g178095162TgtG
clowast351AgtC
Exon
5UTR
-31015840(2724)
SNP
nars3197704
g178095162TgtG
clowast351AgtC
Exon
5UTR
-31015840(2724)
SNP
nars184701151
g178095159TgtC
clowast354AgtG
Exon
5UTR
-31015840(2727)
SNP
C=000276
1000
Genom
esrs11543307
g178095153AgtG
clowast360TgtC
Exon
5UTR
-31015840(2733)
SNP
nars111874043
g178095146AgtG
clowast367TgtC
Exon
5UTR
-31015840(2740)
SNP
nars34012004
g178095102AgtC
clowast411TgtG
Exon
5UTR
-31015840(2784)
SNP
C=0071
[24]
10 Oxidative Medicine and Cellular Longevity
Table3Con
tinued
IDMap
onchromosom
e2lowast
(HGVS
name)
Positionin
Nfe2
l2
(HGVS
name)
Region
s(position
from
mRN
A)
Varia
tioncla
ssand
consequences
(HGVS
name)dagger
Minor
allelefrequ
ency
(MAF)
MAcoun
tsDagger(coh
ort
size)
MAFsources
rs201481890
g178095090178095091in
sTclowast
422lowast423insA
Exon
5UTR
-31015840
(2795minus
2796)
Insertion
na
rs3082500
g178095089178095090d
elTT
delTTinsT
clowast423lowast424d
elAAinsA
Exon
5UTR
-31015840
(2796minus
2797)
Dele
tion
insertion
na
rs71792546
(rs71796710)
g178095079d
elT
clowast425delA
Exon
5UTR
-31015840(2798)
Dele
tion
na
rs1057106
g178095078AgtC
AgtT
clowast435TgtA
TgtG
Exon
5UTR
-31015840(2808)
SNP
na
rs34176791
g178095076AgtC
clowast437TgtG
Exon
5UTR
-31015840(2810)
SNP
C=000235
1000
Genom
esrs35911553
g178095045CgtT
clowast46
8GgtA
Exon
5UTR
-31015840(2841)
SNP
T=000
6915
1000
Genom
esSequ
ence
varia
tions
inup
stream
andexon
sofh
uman
NRF
2fro
m655varia
tions
availablein
publicdatabase
asof
Decem
ber2012
(583
activ
esomeSN
Psmergedge14
SNPs
citedin
PubM
ed)lowastNCB
Ireference
sequ
ence
NC
0000
0211
(Hom
osapiens
chromosom
e2G
RCh37p
10prim
arya
ssem
bly)spanning
178095033ndash1781298
59bp
(com
plem
ent34827
bpforexons
andintro
ns)51015840-Flank
ingregions
startat17812
9860
bp(minus1)in
transcrip
tvariant
1HGVS
Hum
anGenom
eVa
riatio
nSo
ciety
Position
sinvaria
nt1(NM
006164
4)2859
bpsectSN
Pscitedin
PubM
edE
xon11781298
59ndash17812
9260
(600
bpT
TS=1781293
04)exon
2178098999ndash
178098733(267
bp)exon
3178098067ndash1780979
78(90b
p)exon41780973
11ndash178097120
(192b
p)and
exon
5178096736ndash
178095033(1704b
p)daggerProteinam
inoacid
(aa)
resid
uesiniso
form
1(N
P00
61552605
aa)Cn
scoding
-non
syno
nymou
sCs
cod
ing-syno
nymou
sDaggerheterozygositydetectednano
tavailable
Oxidative Medicine and Cellular Longevity 11
Table4Hum
anNRF
2SN
Psassociated
with
diseaser
isk
IDMap
onchromosom
e2 (H
GVS
name)
Region
class
Dise
asea
ssociatio
nandreferences
Risk
allelesa
ndstatistics
Ethn
icgrou
p(num
bero
fcase)
rs7557529
g178135097CgtT
minus5238
Ggt
AParkinsonrsquos
disease(2010
[26])
G=0718252Dagger
inhaplotype
OR=090
or040
CI=
060ndash140or
03ndash06
Swedish
Polish
Caucasian
(357)
rs2886162
g178133165AgtG
minus3306
TgtC
Breastcancer
survival(2012[27])
TT=0324
OR=16
87C
I=1105ndash275
FinlandKB
CP(452)
Chronicg
astritisgastric
ulcer(2007
[28])
GDagger
GDaggerin
haplotypefor
ulcer(OR=
252C
I=119ndash
545)
Japanese
(159)
P14methylationin
gastric
cancer
Hpyloriinfectio
n(2008[29])
Gin
haplotype
OR=290C
I=114ndash
736
Japanese
(209)
Gastriccancer
inHpylori-n
egative
cases(2008
[30])
Ain
haplotype
119875=0022
Japanese
(209)
rs35652124
(rs57695243)
g178130073TgtC
minus214Agt
G(previou
slyminus653[23]
orminus686[22])
Ulcerativec
olitis(2008
[31])
AG
(OR=045C
I=022ndash0
93)
G(O
R=257C
I=10
1ndash660)
Japanese
(89)
Lupu
swith
neph
ritisin
female(2010
[25])
GA
OR=18
1CI
=10
4ndash312
Mexican
mestizo(362)
Parkinsonrsquos
disease(2010
[26])
A=0884312Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
COPD
(2010[32])
G=052Dagger
hazard
ratio
=095C
I=091ndash0
99
(Haplotype)
German
(69)
Gastriculcer(2007
[28])
GDaggerin
haplotype
OR=252C
I=119ndash
545
Japanese
(159)
P14methylationin
gastric
cancer
H
pyloriinfection(2008[29])
Gin
haplotype
OR=290C
I=114ndash
736
Japanese
(209)
Gastriccancer
inHpylori-negative
cases(2008
[30])
Gin
haplotype119875=0022
Japanese
(209)
rs6706
649
g178130071CgtT
minus212Ggt
A(previou
slyminus651[23]
orminus684[22])
Ulcerativec
olitis(2008
[31])
AG
(OR=045C
I=022ndash0
93)
G(O
R=257C
I=10
1ndash660)
Japanese
(89)
Maternalacetaminop
henandasthma
(2010[33])
A=02321137Dagger
OR=17
3CI
=12
2245
UKALSPA
C(gt40
00mothersgt
5000
child
ren)
12 Oxidative Medicine and Cellular Longevity
Table4Con
tinued
IDMap
onchromosom
e2 (H
GVS
name)
Region
class
Dise
asea
ssociatio
nandreferences
Risk
allelesa
ndstatistics
Ethn
icgrou
p(num
bero
fcase)
COPD
(2010[32])
G=098Daggerin
haplotype
hazard
ratio
=095C
I=091ndash0
99
German
(69)
Parkinsonrsquos
disease(2010
[26])
G=0972343Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
03ndash06
Swedish
Polish
Caucasian
(357)
Acutelun
ginjury
follo
wingtrauma
(2007[23])
CA=0119Dagger
OR=644
CI=
134ndash
3080
Caucasia
nAfrican-
American
(164
)
Ann
ualF
EV1decline
(2011[34])
A=0082in
haplotype
Japanese
(915)
Vitiligo(2008[35])
CAA
AOR=17
24C
I=13
5ndash221
Chinese(300)
COPD
(2010[32])
G=073Daggerin
haplotype
hazard
ratio
=095C
I=091ndash0
99
German
(69)
rs6721961
(rs117801448)
g178130037TgtC
TgtG
minus178Agt
GAgt
C(previou
slyminus617[23]
orminus650[22])
Parkinsonrsquos
disease(2010
[26])
C=0980346Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
Postm
enop
ausalvenou
sthrombo
embo
lism
(2011[36])
A=033311
OR=25CI
=370ndash8570
Caucasia
n(161)
Breastcancer
survivalandNRF
2proteinexpressio
n(2012[27])
AA
OR=4656CI
=13
5ndash1606
FinlandKB
CP(452)
Acutelun
ginjury-related
mortality
followingsyste
micinflammatory
respon
sesynd
rome(2012
[37])
GG
OR=97
3CI
=12
7ndash7480
Caucasia
n(750)
Infection-indu
cedasthma(
2012
[38])
AC
OR=0437CI
=028ndash0
80
Hun
garia
nCaucasia
nGypsy
(307)
rs143406266
g178129391178
129393delGGC
Exon
1(467ndash4
69G
CC)
COPD
(2010[32])
GCC
4=053Dagger
hazard
ratio
=095C
I=091ndash0
99
Taiwanese(69)
rs2886161
g178127839Tgt
ClowastIntro
n1
Parkinsonrsquos
disease(2010
[26])
T=0878309Dagger
inhaplotype
OR=09or
04CI
=06ndash
14or
03ndash06
Swedish
Polish
Caucasian
(357)
rs2364723
g178126546Ggt
CIntro
n1
Basaland
smoker
FEV
1(200
9[39])
CI=minus6360simminus1780C
=0525Dagger
Also
ashaplotype
Netherla
nd(2542)
rs2364722
g178124787Agt
GIntro
n1
Ann
ualF
EV1decline
(2011[34])
A=0082in
haplotype
Japanese
(915)
rs13001694
g178118990Agt
GIntro
n1
Basaland
smoker
FEV
1(200
9[39])
G=040
11578Dagger
inhaplotype
Netherla
nd(2542)
Oxidative Medicine and Cellular Longevity 13
Table4Con
tinued
IDMap
onchromosom
e2 (H
GVS
name)
Region
class
Dise
asea
ssociatio
nandreferences
Risk
allelesa
ndstatistics
Ethn
icgrou
p(num
bero
fcase)
Breastcancer
[40]
Twith
NQO1N
OS3H
O1risk
allelesOR=15
6CI
=097ndash251
Caucasia
nandothers(505)
rs1806
649
(rs58745895)
g178118152Cgt
TIntro
n1
Basaland
smoker
FEV
1(200
9[39])
T=02631119Daggerin
haplotype
CI=minus8730ndash
(minus17
0)Netherla
nd(2542)
Parkinsonrsquos
disease(2010
[26])
T=0422148Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
Particulatem
attera
ndasthmaCO
PDadmiss
ion(2012[41])
Cwith
lowvitamin
Clevel(OR=
31CI
=15
0ndash630)
UK(209)
rs4243387
(rs6003846
4)g178117765Cgt
TIntro
n1
Basaland
smoker
FEV
1(200
9[39])
T=00914
25Daggerin
haplotype
Netherla
nd(2542)
rs1962142
(rs58448508)
g178113484Agt
GIntro
n1
Ann
ualF
EV1decline
(2011[34])
A=0082in
haplotype
Japanese
(915)
Breastcancer
NRF
2andARE
expressio
n(2012[27])
A(119875=0036)
FinlandKB
CP(452)
rs6726395
(rs57309289)
g178103229Agt
GIntro
n1
Smok
ing-relatedFE
V1decline
and
annu
alFE
V1decline
(2011[34])
G=0884Dagger
A=0082in
haplotype
Japanese
(915)
Basaland
smoker
FEV
1(200
9[39])
G=046
41764Daggerin
haplotype
Netherla
nd(2542)
rs2001350
(rs17515179
rs60883775)
g178100
425Cgt
TIntro
n1
Ann
ualF
EV1decline
(2011[34])
T=0082in
haplotype
Japanese
(915)
Parkinsonrsquos
disease(2010
[26])
T=0986350Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
rs10183914
(rs58731187
rs61374844
)g17809766
6Cgt
TIntro
n3
Parkinsonrsquos
disease(2010
[26])
T=0536188Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
rs2706110
g178092162Tgt
C31015840Flanking
Breastcancer
(2012[27])
TT
OR=2079CI
=118ndash368
FinlandKB
CP(452)
rs2588882
g178087165Ggt
T31015840Flanking
Infection-indu
cedasthma(
2012
[38])
TG
OR=0290CI
=013ndash0
62
Hun
garia
nCaucasia
nGypsy
(307)
minus686in
reference[22]
=minus653in
reference[23]
=currentlyminus214minus684in
reference[22]
=minus651inreference[23]
=currentlyminus212minus651inreference[22]
=minus617in
reference[23]
=currentlyminus178Ch
romosom
econtig(in
tron
31015840flank
ing)
orreversed
(51015840flank
ingprom
oter)a
llelesinbo
ldhave
been
used
inthetextand
TableOR
odds
ratio
CI95confi
denceinterval
14 Oxidative Medicine and Cellular Longevity
minus212GG (98 rs67006649 previously minus684minus651) minus178Callele (73) and GCC
4(53) was predicted to increase
respiratory failure development (hazard ratio = 095 CI091ndash099) in German COPD patients [32] Significant inter-action was also identified between an intronic SNP Gallele (rs6726395 g178103229AgtG 884 frequency) andsmoking status on FEV
1decline relative to the reference
AA allele in the above Japanese cohort [34] Siedlinskiet al [39] reported that the CC genotype of anotherintronic SNP (rs2364723 g178126546GgtC) was associatedwith a lower FEV
1level compared to the wild-type geno-
type (GG) in two Netherland cohorts (CI minus636minus178frequency = 0525 and pooled cohort size = 2542) ThisSNP alone or as a haplotype with 4 more intronic SNPs(rs13001694Grs1806649Trs4243387Trs6726395G)was alsoassociated with high FEV
1levels in individuals that ever
smoked [39] In a Hungarian population of childhoodasthma SNPs at minus178 (CA) and 31015840 flanking (rs2588882TG)loci were inversely associated with infection-induced asthma(OR 0437 CI 028ndash080 OR 0290 CI 013ndash062 resp) andthese SNPs significantly influenced an asthma-environmentalpollution interaction [38]The intronic SNP rs1806649 (CgtT)was associated but not significantly with an increased risk ofhospitalization during high-level particulate matter (PM
10)
periods in asthma or COPD patients (119899 = 209) of the UnitedKingdom (UK) [41] Asthma andCOPDadmission rateswererelated to the increase in environmental PM
10concentration
Importantly effects of interaction between prenatal stressand NRF2 SNPs on descendant pulmonary health wereinvestigated by the Avalon Longitudinal Study in the UKmaternal smoking during pregnancy was not associated withlung function change determined by maximum mild expi-ratory flow (FEF
25ndash75) or with asthma incidence in school-aged children and this relation was not modified by NRF2SNP genotypes [42]However early gestation acetaminophenexposure significantly influenced the risk of asthma andwheezing at the age of 7 years in gt4000 mothers and gt5000children [33] When maternal copies of the minus212A allele werepresent association with asthma (11374891 OR 173 CI122ndash245) and wheezing (11494949 OR 153 95 CI 106ndash220) was significantly increased [33]
Gastrointestinal Disorders While there was no evidence inlung cancer cases studies in Japanese populations suggesteda potential association of NRF2 variations with gastro-intestinal tumorigenesisHelicobacter pylori (H pylori) causesgastritis which can lead to gastric atrophy and cancer Ingastric epithelium from the Japanese cancer cohorts (39gastric cancers 46 controls) H pylori infection was posi-tively correlated with aberrant CpG island methylation oftumor suppressor genes (eg p14) and minus214Gminus212G orminus214Aminus212G NRF2 haplotype was significantly associatedwith increased (OR 290 95 CI 114ndash736) or decreased(OR 033 95 CI 013ndash088) risk of the CpG methyla-tion respectively in the H pylori-infected patients [29]Further study from the same investigators determined thatminus214Aminus212G allele carriers had significantly (119875 = 0022)reduced risk of gastric cancer inH pylori-negative cases [30]The minus214AGminus212AG genotypes were negatively associated
(OR 045 CI 022ndash093) and the minus214Gminus212G genotypeswere positively associated (chronic continuous phenotypeOR 257 CI 101ndash660) with ulcerative colitis (89 patients 141controls) in a Japanese population [31]
Autoimmune Disorders Systemic lupus erythematosus (SLE)is a long-term autoimmune disease more frequently found infemales than in males It affects organs including skin jointskidneys and brain and nephritis is an aggressive character-istic in some patients Genome-wide association studies inhumans identified a suggestive quantitative trait locus nearNRF2 [43] A study of a Mexican Mestizo population (362patients with childhood-onset SLE 379 controls and 212nephritis diagnosed) determined that lupus with nephritiswas significantly (OR 181 CI 104ndash312) associated with theminus214GA SNP in females [25] The same SNPs were notclosely associated with SLE risk in a Japanese cohort [22]Vitiligo is a skin condition in which there is a loss of browncolor (pigment) from areas of skin resulting in irregularwhite patches It is thought to be an autoimmune diseasecaused by loss of cells (melanocytes) that produce brownpigment A study indicated that theminus178A allele increased therisk of vitiligo dose-dependently (OR 1724 95 CI 135ndash221for CA OR 2902 CI 162ndash519 for AA) [35]
Female Disorders It is well known that estrogen metabolites(eg catechols) cause ROS formation suggesting correla-tion of NRF2 and downstream effectors in postmenopausalmammary cancer In a study of a Finish population (KuopioBreast Cancer Project 119899 = 452 patients 370 controls)the minus178AA homozygous genotype (OR 4656 CI = 135ndash1606) and 31015840 flanking rs2706110 (TT OR 2079 CI 118ndash368) genotype were associated with increased risk of breastcancer while the 51015840 flanking minus3306TT homozygous allelewas significantly associated with lower survival (frequency =71219 OR 1687 CI 1105ndash275) [27] suggesting that NRF2genetic polymorphisms affect susceptibility and outcome ofthe patients The minus178A allele carriers together with intronicrs1962142A allele carriers were associated with lowered tissuelevels of NRF2 proteins [27] In postmenopausal women theminus178A allele (OR 179 95CI 370ndash8570) appeared tomodifythe risk of venous thromboembolism caused by oral estrogentherapy (AA or AC frequency = 333) as demonstratedby the French ESTHER study (161 cases 474 controls) [36]An intronic rs1806649CgtT SNP did not associate with breastcancer risk in postmenopausal women [40] However whenthis SNP and other at-risk alleles of ARE-responsive genes(NQO1 HO-1 NOS3) were combined there was a significantgene-dose effect on the breast cancer risk [40] Althoughcoding region SNPs in NRF2 and KEAP1 were identifiedin the Japanese endometrial adenocarcinoma patients noassociation of NRF2 SNPs with the disease was found [44]
Neurodegenerative Diseases Oxidative stress is known to beinvolved in Parkinsonrsquos disease (PD) presumably due toproduction of ROS fromhigh-dopaminemetabolism and lowlevels of antioxidants in the substantia nigra of the brainInvestigators found a protective NRF2 haplotype consistingof four 51015840 flanking SNPs (minus5238Gminus214Aminus212Gminus178C)
Oxidative Medicine and Cellular Longevity 15
and 4 intronic SNPs (rs2886161Ars1806649Ars2001350Ars10183914A) from Swedish (OR 09 CI 060ndash140) and Polish(OR 04 CI 030ndash060) populations (total 165+192 PD cases190+192 controls) [26]The investigators also suggested thatNRF2 haplotype alleles were associated with 2 years earlierage of Alzheimerrsquos disease (AD) onset 4 years earlier age ofposterior subcapsular cataract surgery and 4 years later ageof cortical cataract surgery while they were not significantlyrelated to AD or age-related cataract risk [45]
34 Genetic Mutations in Mouse Nrf2 Tsang et al [46]compiled 673 SNPs in 55 mouse strains and constructedtheir phylogenetic tree to correlate and clarify the originsof strains based on the assembled mouse genome sequenceand SNP data [20 47 48] Recently using the completegenome sequence of C57BL6J (B6) mouse as a referencehigh-density SNP screening in other laboratory strains orin panels of strains has been published (see [17]) Althoughmillions of mouse SNPs (gt10089892 as of December 2012)and haplotype mappings from more than 120 strains havebeen published as valuable references for dissecting thegenetic basis of complex traits [49ndash51] little attention hasbeen paid to polymorphisms of Nrf2 and their correlationwith disease phenotypes
Figure 3 demonstrates the proximal promoter region(minus1 to minus950)51015840-UTR (exon 1 up to TSS) and proteinsequence of mouse Nrf2 based on GRCm38p1 PrimaryAssembly (75704641ndash75675513 bp) mRNA variant1 and protein (NP 035032 encoded by NM 010902234ndash2027 bp) sequences Genetic variations in the Nrf2genome of inbred strains collected from public databasesare listed in Tables S2 and 5 (See Supplementary TableS2) Overall 968 genetic mutations are compiled forNrf2 gene and 5 kb upstream2 kb downstream regions785 SNPs between B6 and another 16 strains wereacquired from the Mouse Phenome Database (MPDhttpphenomejaxorg dbqrtn=snpret1) and additionalSNPs and other mutations were acquired from NCBI dbSNP(httpwwwncbinlmnihgovsnpterm=mus+musculus20nfe2l2) In total 132 mutations are in the promoter (37in proximal 1 kb) 49 in exons (38 in coding region 19 Cns)727 in introns and 60 in the 31015840 flanking region Excludingmutations in the 51015840 and 31015840 flanking sequences murine Nrf2sequences appear to be more highly variable (1 variationper 375 bp) than much of the mouse genome which has anapproximate frequency of one SNP per every 245 bp (httpwwwinformaticsjaxorgmgihomehomepagesstatsallstatsshtmlallstats snp)
Nrf2 was found to be a susceptibility gene from genome-wide linkage analysis in a murine model of hyperoxia-induced ALI [52] A promoter SNP minus103TgtC (previouslypublished as minus336TgtC) in Nrf2 was found and predicted toadd an additional Sp1 binding site in hyperoxia-susceptibleB6 mice but not in resistant C3HHeJ mice [52] Genotypesfrom the SNP and from simple-sequence length polymor-phism markers of the Nrf2 locus (D2Mit248 and D2Mit94)cosegregated in the B6C3F
2mouse cohort [52] and Nrf2
deficient mice were significantly more susceptible to ALI
sub-phenotypes caused by hyperoxia than similarly exposedwild-type mice supporting Nrf2 as a contributor to thephenotypic traits [53] Although no other functional analyseson Nrf2 SNPs or haplotype association studies have beenconducted in inbred mice strains bearing haplotypes suchas multiple Cns in functional domains (eg F71L L451VH543Q and L575M) may be useful to elucidate the role ofNrf2 in differential susceptibility to oxidative diseases
4 Oncogenic Somatic Mutations in HumanNF-E2-Related Factor 2
Somatic mutation is a change in the DNA of somatic cellsthat affects derived cells but is not inherited by offspringEfforts to discover somatic mutations have provided insightinto mutagenesis and cancer development Lung cancerparticularly non-small cell lung cancer (NSCLC) is theleading cause of cancer death worldwide Somatic mutationsof NRF2 and KEAP1 discovered in lung cancer patients havedetermined the oncogenic potential ofNRF2 [54 55] KEAP1somatic mutations were associated with its reduced proteinlevels in lung cancer tissues and cells [56 57] Investigationsof NSCLC in various ethnic populations as well as cancersin gastrointestine breast and prostate have coordinatelydemonstrated that multiple Cns somatic mutations in KEAP1cause dysfunction of the translated protein and in turnconstitutive activation of NRF2 increasing risk of neoplasiaand chemoresistance [12 55 58 59] Somatic mutations ofNRF2 have been detected in various cancer tissues (largelysquamous cell carcinomas) in Asian populations (Table 6)NRF2 mutations were significantly associated with NSCLCcases (squamous cell lung carcinoma adenocarcinoma) ofthe Japanese (107 [54]) the Chinese (23 [60]) andthe Koreans (8 [61]) as well as with lung cancer celllines Smoking history was also correlated with mutationoccurrence in all of the studies [54 60 61] In addition tolung cancers laryngeal squamous carcinoma (13 in [61])esophageal squamous cancer (ESC 22 in [60] 114 in[61]) head and neck cancers (25 in [54]) skin (117 casein [61]) and oral cancer cell lines had somatic changesin NRF2 In contrast to wide-spread KEAP1 mutationsmutations in NRF2 were clustered in DLGETGE motifs ofthe Neh2 domain which are critical in the ldquohinge and latchrdquomodel of KEAP1 binding [12] Similar to KEAP1 somaticmutations it has been postulated that NRF2 mutations incancer cells lead to NRF2 accumulation by suppressing itsubiquitination or KEAP1 binding which eventually confersmalignant potential and resistance to chemotherapy
Most variable sites in NRF2 included aa residues 29 (AspD) 31 (Gly G) 77 (Asp D) and 79 (Glu E) (Table 6)Residue 33 (Ser S) in the Neh2 domain is mutated by eithergenetic or somatic processes (Figure 4) Cns in the EDGFmotif of NRF2 was experimentally determined to impairrecognition of KEAP1 [54] NRF2 mutations were signif-icantly correlated with increased (25-fold) copy number(31 of mutants versus 3 wild types) in Japanese NSCLCcases [63] Aberrant mutation of NRF2 also led to increasedexpression of downstream effectors includingRagD known to
16 Oxidative Medicine and Cellular Longevity
pL575M
pF71L
pH543QpL451V
pA151P pA151V pN159D pQ198delpV105D
pA204T pS224I225S226del pT253S pS278ApD305G
pM409V pR410HpP388L pA389G pT395I
Genomic DNA (reversed contig GRCm38 p1 C57BL6JNM 0109023)
Protein (NP 035032)1
51101151201251301351401451501551
minus400
minus500
minus50
minus100
minus200
minus300
minus450
minus350
minus250
minus150
minus950
minus900
minus850
minus800
minus750
minus700
151
101151201
2001
g minus470gg minusg minus
c minus41c minus
c
c
c c
gg minus 6C
g minus125insTTC g minus118insC
g minus281delC
g minus63delCg minus1 (75704642 c minus234)
Translation start site (c +1)Termination codon (c +2025 +2027)
c minus136 minus134in-frame stop codonc minus8585
g minus41
246
440
minus381
minus11
minus91 minus90
minus332
minus153
minus98
c minus197c minus209 minus202
gminus942 minus899 (cminus1175 minus1132) CpG islands [71]
g 460CgtT
g minus103CCgtT [51]
CgtTCgtG
g minus320CgtT
CgtT
CgtT
CgtTg minus426GgtA
GgtACgtA
CgtA
GgtA
g minus238GgtTTgtC
TgtC
TgtC
TgtCTgtG
g minus229AgtG
CgtG
CgtG
GgtC
gminus826 minus654 (cminus1059 ) CpG islands [71]minus887
Figure 3 DNA (partial promoter and exons 1 and 5) and protein sequence ofmouseNrf2 SNPs and amino acid residues for non-synonymousSNPs are marked Promoter regions bearing 5CpG islands are underlined
151
201251301351401451501551601
Genetic mutation (Table 3 and Figure 2)Somatic mutation (Table 6)
Neh2
Neh3
Neh4Neh5
Neh6
Neh1
Protein isoform 1 (NP 006155)
101151
Figure 4 Genetic and somatic mutation loci in human NRF2 protein
Oxidative Medicine and Cellular Longevity 17
Table5Mou
seup
stream
andexon
varia
tions
ofNfe2
l2locusin17
inbred
strainsR
eference
sequ
ence
isC5
7BL6J
(B6strain
1)geno
me(GI14933824975547698ndash75513576)SNPalleleand
geno
typesareshow
nas
chromosom
econ
tigsequ
enceStrains
2129S1S
vlmJ3AJ
4AKR
J5BA
LBcJ6C3
HH
eJ7C
57BL
6NJ8CA
STEiJ9CB
AJ
10D
BA2J11FVBNJ12LPJ
13N
ODShiLtJ14N
ZOH
ILtJ
15P
WK
PhJ16SPR
ETEiJ
17W
SBEiJ
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs2566
08517
75705565
GA
(CT)
51015840Flankingminus924
NC
GG
GG
GG
GG
GG
GG
GG
AA
Grs47274959
75705562
AT
(TA
)51015840Flankingminus921
NC
AT
TA
TT
AA
TT
AT
AT
TT
Ars216940398
75705554ndash75705555
Tadd(A
add)
51015840Flankingminus913minus914
NC
CGrs239114134
75705540
CT(G
A)
51015840Flankingminus899
NC
CT
TC
TT
CC
TT
CT
CT
CT
Crs46
461765
75705528
TG(A
C)oA
C
51015840Flankingminus887
NC
TG
GT
GG
TT
GG
TG
TG
GG
Trs5144
9853
75705525
CA(G
T)
51015840Flankingminus884
NC
CA
AC
AA
CC
AA
CA
CA
CC
Crs249093111
75705512
CT(G
A)
51015840Flankingminus871
NC
CT
TC
TT
CC
TT
CT
CT
TT
Crs263745200
75705510
CG(G
C)
51015840Flankingminus869
NC
GG
GG
GG
GG
GG
GG
GG
CG
Grs45651867
75705498
TG(A
C)
51015840Flankingminus857
NC
TG
GT
GG
TT
GG
TG
TG
GG
Trs219997531
75705495
GA
(CT)
51015840Flankingminus854
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs251918379
75705451
TA(A
T)
51015840Flankingminus810
NC
TT
TT
TT
TT
TT
TT
TT
TA
Trs27978306
75705449
TA(A
T)
51015840Flankingminus808
NC
TA
AT
AA
TT
AA
TA
TA
TT
Trs216087412
75705429
TG(A
C)lowast
51015840Flankingminus788
NC
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
GTlowast
Tlowast
rs261229914
75705423ndash75705424
AGdel(CT
del)
51015840Flankingminus783minus784
NC
AGrs27978307
75705400
CT(G
A)
51015840Flankingminus759
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs243167395
75705359
GA
(CT)
51015840Flankingminus718
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs27978308
75705307
GA
(CT)
51015840Flankingminus66
6NC
GG
GG
GG
GG
GG
GG
GG
AG
Grs254744
09875705294
AT
(TA
)51015840Flankingminus653
NC
AA
AA
AA
AA
AA
AA
AA
AT
Ars228133419
75705179
GT
(CA
)51015840Flankingminus538
NC
GG
GG
GG
GG
GG
GG
GG
GT
Grs214719520
75705111
GA
(CT)
51015840Flankingminus470
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs2440
87730
75705101
GA
(CT)
51015840Flankingminus46
0NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs227781699
75705081
CG(G
C)
51015840Flankingminus44
0NC
CC
CC
CC
CC
CC
CC
CC
GC
Crs257870353
75705067
CT(G
A)
51015840Flankingminus426
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs24487744
075705022
AG
(TC)
51015840Flankingminus381
NC
AA
AA
AA
AA
AA
AA
AA
AG
Ars234628138
75704973
AC
(TG
)51015840Flankingminus332
NC
AA
AA
AA
AA
AA
AA
AA
AC
A
18 Oxidative Medicine and Cellular Longevity
Table5Con
tinued
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs247275247
75704961
GA
(CT)
51015840Flankingminus320
NC
GG
GG
GG
GG
GG
GG
AG
GG
Grs247519047
75704922
Gdel(Cdel)
51015840Flankingminus281
NC
Grs27978309
(rs51915758)
75704887
GA
C(C
TG
)51015840Flankingminus246
NC
GA
AG
AA
GG
AA
GA
GA
CC
G
rs218139102
75704879
CA(G
T)
51015840Flankingminus238
NC
CC
CC
CC
CC
CC
CC
CC
AC
Crs251990355
75704870
TC(A
G)
51015840Flankingminus229
NC
TT
TT
TT
TT
TT
TT
TT
TC
Trs238746955
75704794
AG
(TC)
51015840Flankingminus153
NC
AA
AA
AA
AA
AA
AA
AA
GA
Ars221664
405
75704786
AG
(TC)
51015840Flankingminus145
NC
AA
AA
AA
AA
AA
AA
AA
AG
Ars217054035
75704766
ndash75704767
GAAadd(TTC
add)
51015840Flankingminus125minus126
NC
GA
rs248182931
75704759ndash75704760
Gadd(C
add)
51015840Flankingminus118
minus119
NC
AGrs217197904
75704744
GA
(CT)
51015840Flankingminus103
(+)S
p1[23]
GA
AG
AA
GG
AA
GA
AA
GG
Ars2644
9364975704704
Gdel(Cdel)
51015840Flankingminus63
NC
Grs2404
81112
757046
82CT(G
A)
51015840Flankingminus41
NC
CC
CC
CC
CC
CC
CC
CC
CT
Crs27978310
75704617
AG
(TC)
UTR
-525
NC
AA
AA
AA
AA
AA
AA
AA
GG
Ars27978311
75704610
CG(G
C)
UTR
-532
NC
GG
GG
GG
GG
GG
GG
GG
GG
Crs27978312
757046
05GA
(CT)
UTR
-537
NC
GA
AG
AA
GG
AA
GA
GA
GG
Grs214784220
757044
99AG
(TC)
UTR
-5143
NC
AA
AA
AA
AA
AA
AA
AA
GG
Ars244146318
757044
98GT
(CA
)UTR
-5144
NC
GG
GG
GG
GG
GG
GG
GG
GT
Grs27978313
757044
97GC
(CG
)UTR
-5145
NC
GC
CG
CC
GC
CC
GC
CC
CC
Crs215431944
757044
93CT(G
A)
UTR
-5149
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs257015697
757044
49GA
(CT)
UTR
-5193
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs252234782
757044
19GT
(CA
)UTR
-5223
NC
Grs1346
0861
75679262
AG
(TC)
Exon
244
6Cn
sF71L
AG
GA
GG
AG
GG
AG
AG
GG
Grs1346
0859
75679202
GA
(CT)lowast
Exon
2506
CsH91=
GA
AG
AA
GG
AA
GA
GA
GG
Grs27978436
75679187
GA
(CT)lowast
Exon
2521
CsT9
6=G
GG
GG
GG
AG
GG
GG
GA
GG
rs226131070
75679178
GA
(CT)
Exon
2530
CsS99=
GG
GG
GG
GG
GG
GG
GG
GA
Grs227683834
75678576
AT
(TA
)Ex
on3
547
CnsV
105D
AA
AA
AA
AA
AA
AA
AA
AT
Ars257321187
75678506
TC(A
G)
Exon
3617
CsP128=
TT
TT
TT
TC
TT
TT
TT
TT
Trs27978444
75678500
TA(A
T)
Exon
3623
CsV130=
TT
TT
TT
TA
TT
TT
TT
AT
Trs227743136
75677686
GA
(CT)
Exon
4662
CsH143=
GG
GG
GG
GG
GG
GG
GG
GA
Grs215327202
75677664
CG(G
C)
Exon
4684
CnsA
151P
CC
CC
CC
CG
CC
CC
CC
CC
Crs247539755
75677663
GA
(CT)
Exon
4685
CnsA
151V
GG
GG
GG
GA
GG
GG
GG
GG
Grs233164
668
75677640
TC(A
G)
Exon
4708
CnsN
159D
TT
TT
TT
TT
TT
TT
TT
TC
T
rs2340
95816
75677162ndash75677160
GCT
del
(AGCdel)
Exon
5826minus
828
Cds-in
delQ
198
GCT
Oxidative Medicine and Cellular Longevity 19
Table5Con
tinued
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs27978452
75677145
CT(G
A)
Exon
5843
CnsA
204T
CC
CC
CC
CC
CC
CC
CC
CC
T
rs225047937
75677086ndash75677078
GAG
ATCG
ATdel
(ATG
CATC
TCdel)
Exon
5902minus
910
Cds-in
del
(S224I225S226)
GAG
ATC
GAT
rs241469868
75676998
TA(G
T)
Exon
5990
CnT2
53S
TT
TT
TT
TT
TT
TT
TT
TA
Trs221602571
75676923
AC
(TG
)Ex
on5
1065
CnsS
278A
AA
AA
AA
AA
AA
AA
AA
AC
Ars252576472
75676876
GA
(CT)
Exon
51112
CsS293=
GG
GG
GG
GG
GG
GG
GG
GA
Grs250802933
75676841
TC(A
G)
Exon
5114
7Cn
sD305G
TT
TT
TT
TT
TT
TT
TT
TC
Trs225425698
75676792
CT(G
A)
Exon
5119
6Cs
P321=
CC
CC
CC
CC
CC
CC
CC
CT
Crs13459064
75676732
CT(G
A)
Exon
51256
CsT3
41=
CT
TC
TT
CT
TT
CT
CT
TC
Crs27978453
75676717
CT(G
A)
Exon
51271
CsA346=
CC
CC
CC
CC
CC
CC
CC
TC
Crs214335034
75676636
AG
(TC)
Exon
51352
CsD373=
AA
AA
AA
AA
AA
AA
AA
AG
Ars24958364
4756766
03AC
(TG
)Ex
on5
1385
CsP3
84=
AA
AA
AA
AA
AA
AA
AA
AC
Ars27978454
75676592
GA
(CT)
Exon
51396
CnsP
388L
GG
GG
GG
GG
GG
GG
GG
AG
Grs215384328
75676589
GC
(CG
)Ex
on5
1399
CnsA
389G
GG
GG
GG
GG
GG
GG
GG
GC
Grs251728286
75676571
GA
(CT)
Exon
51417
CnsT
395I
Grs247602334
75676567
TC(A
G)
Exon
51421
CsV396=
TT
TT
TT
TT
TT
TT
TT
TC
Trs234216231
75676530
TC(A
G)
Exon
51458
CnsM
409V
TT
TT
TT
TT
TT
TT
TT
TC
Trs212904337
75676526
CT(G
A)
Exon
51462
CnsR
410H
CC
CC
CC
CC
CC
CC
CC
CT
Crs252650779
75676522
TC(A
G)
Exon
51466
CsE4
11=
TT
TT
TT
TT
TT
TT
TT
TC
Trs231273560
75676516
TC(A
G)
Exon
51472
CsQ413=
TT
TT
TT
TT
TT
TT
TT
TC
Trs257886949
756764
13GT
(CA
)Ex
on5
1575
CsR4
48=
GG
GG
GG
GG
GG
GG
GG
GT
Grs241537608
756764
04GC
(CG
)Ex
on5
1584
CnsL
451V
GG
GG
GG
GG
GG
GG
GG
GC
Grs227619071
75676312
TC(A
G)
Exon
51676
CsQ481=
TT
TT
TT
TT
TT
TT
TT
TC
Trs258913831
75676290
AG
(TC)
Exon
51698
CsL4
89=
AA
AA
AA
AA
AA
AA
AA
AG
Ars225593319
75676126
AT
(TA
)Ex
on5
1862
CnsH
543Q
Ars4223233
75676105
GA
(CT)
Exon
51883
CsS550=
GG
GG
GG
GA
GG
GG
GG
GG
Grs4223232
75676032
GT
(CA
)Ex
on5
1956
CnsL
575M
Grs4223231
75675816
CG
UTR
-32172
NC
GG
GG
GG
GG
GG
GG
GG
GC
Grs1346
860
75675682
AT
UTR
-32306
NC
ASequ
ence
varia
tions
inmou
seNr
f2wereob
tained
from
Mou
sePh
enom
eDatabase(http
ph
enom
ejaxorgSNP)
andNCB
ISNPdatabase
(http
wwwncbinlm
nihgovSNP)N
CBIreference
sequ
ence
isMus
Musculusstra
inC5
7BL6J
chromosom
e2GRC
m38p1(NC
0000
687
GI37209910875704641ndash756755192
9123
bp)To
tal968
genetic
mutations
arerepo
rted
inNr
f2gene
and5k
bup
stream
(ge75704
642minus
1)and2k
bdo
wnstre
am(le75675512)sequences
asof
Janu
ary2003A
llSN
Psandallelesa
representedas
geno
miccontig
(reversedsequ
ence
indicated)E
xon175704
641ndash75704364(278
bpT
TS=75704
408)
exon
275679431ndash756791
65(267
bp)exon
375678579ndash75678490(90b
p)exon475677713ndash756775
46(168
bp)andexon
57567718
4ndash75678849
(1666
bp)Proteinam
inoacid
(aa)
resid
uesinNP00
61552
(597
aa)Cs
cod
ing-syno
nymou
sCn
scoding
-non
syno
nymou
sAminoacid
A-alanineD-aspartic
acidE
-glutamicacidF-phenylalanineG
-glycineH
-histidineI-iso
leucineL-leu
cineand
M-m
ethion
ineN-
asparagineP-prolin
eQ-glutamineR-arginineS-serineT-threon
ineandV-valin
elowastErrorsin
databasesfi
xedNC
notcon
firmed
20 Oxidative Medicine and Cellular Longevity
Table 6 NRF2 somatic mutations revealed in various human cancers
Domain Amino acid residues DNA mutation Cancer types (cases) ReferencesLocus Wild type Mutant
DLG motif
24 W (Trp) C (Cys) c72GgtCGgtT NSCLC neck ESC [54 62]K (Lys) c72TgtC ESC [62]
26 Q (Gln) E (Glu) c76CgtG NSCLC ESC [54 62]
27 D (Asp) G (Gly)lowast c80GgtA NSCLC [60]Y (Tyr) c79GgtT ESC [61]
28 I (Ile) T (Thr) c83CgtT NSCLC [54]
29 D (Asp) G (Gly) c86AgtG Head and neck ESC [54 62]H (His) c85GgtC NSCLC laynx [60 61]
30 L (Leu) F (Phe) c88CgtT NSCLC ESC [54 62]
31 G (Gly) A (Ala) c92GgtC NSCLC ESC skin [54 60ndash62]
32 V (Val) T (Thr) c95TgtG NSCLC [54]del c93 95delAGT ESC [61]
33ndash36 S-R-E-V S-R-E-V-S-R-E-Vlowast c97 108dupAGTCGAGCCGTA ESC [61]
34 R (Arg) Q (Gln) c101GgtA NCSCL [54 60 61]P (Pro) c101GgtC NSCLC [63]
ETGF motif
75 Q (Gln) H (His) c225AgtC Head and neck ESC [54 62]
77 D (Asp)
V (Val) c230AgtT NSCLC ESC [54 60 62]G (Gly) c230AgtG ESC [62]A (Ala) c230AgtC NSCLC [61]N (Asn) c229GgtA Larynx [61]
78 E (Glu) K (Lys) c232GgtA NSCLC ESC [54 62]
79 E (Glu)
K (Lys) c235GgtA NSCLC ESC [54 61 62]Q (Gln) c235GgtC NSCLC ESC [54 60ndash62]G (Gly) c236AgtG Larynx [61]E-E c234 236dupAGA ESC [61]
80 T (Thr) K (Lys) c239CgtACgtG NSCLC ESC [54 61 62]
80 T (Thr)P (Phe) c238AgtC ESC [62]I (Ile) c239CgtT Head and neck [54]A (Ala) c238AgtG NSCLC [63]
81 G (Gly) V (Val) c242GgtT ESC [61]D (Asp) c242GgtA NSCLC ESC [60ndash62]
82 E (Glu)
D (Asp) c246AgtT ESC oral cancer cell line [54 62]G (Gly) c245AgtG NSCLC [54]Q (Gln)lowast c244GgtC NSCLC ESC [60 61]V (Val)lowast c245AgtT ESC [61]
83 F (Phe) L (Leu)lowast c247TgtC NSCLC [60]NSCLC non-small cell lung cancer ESC esophageal squamous cancer and lowasterrors in reference fixed Number of cases 82 NSCLC and 10 ESC in [62] 125NSCLC 70 ESC 23 larynx and 17 skin in [61] 103 NSCLC and 12 head and neck in [54] 90 NSCLC in [63] 103 NSCLC in [60]
be involved in squamous lung cancer cell proliferation [64]suggesting that the mutation is functional and overcomesKEAP1 inhibition Singh et al [65] determined in vitrothat RNAi-mediated depletion of NRF2 in lung cancer cellsenhanced ROS production and susceptibility to cell deathby ionizing radiation These studies support the concept thatelevated NRF2 and ARE responsiveness provides cancer cellswith proliferative advantage for malignant transformation
and undue protection from anti-cancer therapy Onco-genic epidermal growth factor receptor (EGFR) signaling isrecently found to be critical in NRF2-mediated proliferationof NSCLC cells [66]
Collectively ldquogain of functionrdquo mutations in NRF2 thatreduce KEAP1 recognition are suggested to be predictivemarkers for poor responsiveness to chemotherapy and radi-ation therapy Although NRF2-mediated cellular defense
Oxidative Medicine and Cellular Longevity 21
processes are essential in the initiation stage enhancedNRF2-ARE activity in advanced stages of cancer developmentmay create a favorable intracellular environment for tumorcell growth and survival [67 68] In this context NRF2may be a potential molecular target for the treatment ofradio-resistance cancers especially those that have ldquoloss offunctionrdquo mutations in EGFR KRAS or KEAP1 as well asldquogain of functionrdquo mutations in NRF2
5 Epigenetic Alterations of NF-E2-RelatedFactor 2
Epigenetic modifications are alterations of molecules inter-acting with genes without changes to the primary DNAsequence They include post-translational modification ofhistones DNA methylation events chromatin conforma-tional changes and alterations to noncoding regulatoryRNAs Epigenetic alterations are stable and often inheritablebut are reversible and may affect expression of the geneDysregulation or defects in epigenetic processes particularlyhypermethylation of tumor suppressor gene promoters (egCpG islands) or histone modifications are thought to beassociated with carcinogenesis Investigators have reportedhypermethylation in CpG islands of KEAP1 which wereassociated with reduced KEAP1 expression in human cancersfrom lung prostate colon and so forth [69ndash72] Similar tosomatic mutations epigenetic changes on KEAP1 impairedthe function of its encoded protein leading to constitutiveNRF2 activation
Supporting the role of ldquopathogenic mutationsrdquo in NRF2expression of Nrf2 and downstream Nqo1 was suppressedin prostate tumors of mice (transgenic adenocarcinomaof mouse prostate TRAMP) Among 15 promoter CpGislands located between minus942 and minus654 (cminus1175 cminus1132and cminus1059 cminus887 gap in cminus1131 cminus1060 see Figure 3)hypermethylation of the first 5 CpG islands (minus942 minus899 andcminus1175 cminus1132) was significantly associated with tumorige-nesis [73] Moreover treatment with inhibitors for DNAmethyltransferase and histone deacetylase restored Nrf2expression in these tumor cells [73] A dietary phytochemicalcurcumin known as a DNA hypomethylation agent restoredepigenetically silent Nrf2 expression through CpG demethy-lation in carcinogen-induced mouse tumor cells [74]
The whole genome epigenetic datasets for 5 species arepublicly accessible at NCBI Epigenomics [75 76]The humanNRF2 epigenome of primary cells (breast penis) and H1stem cell line as well as mouse Nrf2 CpG island methylationdata for sperm blood and cerebellum are currently avail-able (httpwwwncbinlmnihgovepigenomics) Althoughno direct evidence of disease-associated epigenetic modu-lation has been identified in human NRF2 various phyto-chemical NRF2 agonists such as sulforaphane and curcuminhave shown their roles in DNA methylation and histonemodification (see reviews by Lee and colleagues eg [77])Taken together epigenetic modifications of theNRF2KEAP1axle are predicted to cause dysregulation of ARE-mediatedcellular defense leading to deleterious health effects and
phytochemical antioxidants as epigenetic modulators forNRF2 are suggested to be useful in cancer prevention
6 Conclusions
NRF2 is evolutionally conserved with high-sequence homol-ogy in many species However it is a highly mutable geneand numerous genetic variants have been discovered inhuman ethnic groups Importantly certain SNPs or haplo-types have been identified in various diseases as ldquoat-riskrdquoalleles and are related to functional alterations In additionto genetic variations multiple somatic mutations identifiedin the KEAP1 recognition domain of NRF2 in cancer cellshave been found to be oncogenic due to dysregulation ofNRF2 homeostasis by its excess ldquogain of functionrdquo Epigeneticalteration of theNRF2 is under investigation and is predictedto have pathogenic influences as learned from mouse andphytochemical agonist studies Continuous updates of Nrf2allelic variants in inbred mouse strains will provide a usefultool for effective experimental designs formodels of oxidativedisorders to provide insight into the disease mechanisms andintervention strategies
Abbreviations
AD Alzheimerrsquos diseaseALI acute lung injuryARE antioxidant response elementbZIP basic leucine zippercds coding DNA sequencesCI confidence intervalCOPD chronic obstructive pulmonary diseaseESC esophageal squamous cancerFEV1 forced expiratory volume in one second
Gbp giga base pairsGST glutathione S-transferaseGWAS genome-wide association studyHGVS Human Genome Variation SocietyHO-1 heme oxygenase-1119867 119901119910119897119900119903119894 119867119890119897119894119888119900119887119886119888119905119890119903 119901119910119897119900119903119894KEAP1 Kelch-like ECH activating protein 1MPD Mouse Phenome DatabaseMRP multidrug resistance proteinNCBI National Center for Biotechnology InformationNeh NRF2-ECH homologyNfe2l2 nuclear factor (erythroid-derived 2)-like 2Nrf2 NF-E2-related factor 2NSCLC nonsmall cell lung cancerNQO1 NAD(P)Hquinone oxidoreductase 1OR odds ratioPD Parkinsonrsquos diseasePM particulate matterROS reactive oxygen speciesSLE systemic lupus erythematosusSNP single nucleotide polymorphismSOD superoxide dismutaseURT untranslated region
22 Oxidative Medicine and Cellular Longevity
Disclosure
Authorrsquos contribution to the Work was done as part of theAuthorrsquos official duties as a NIH employee and is a Work ofthe United States Government Therefore copyright may notbe established in the United States
Conflict of Interests
The author declares that there is no conflict of interests
Acknowledgments
The research related to this paper was supported by theIntramural Research Program of the National Institutes ofHealth of the National Institute of Environmental HealthSciences (NIEHS) Drs Steven Kleeberger and StephanieLondon at the NIEHS provided excellent critical review ofthis paper The author thanks Mrs Jacqui Marzec for herhelpful comments and English editing
References
[1] J Y Chan M C Cheung P Moi K Chan and Y W KanldquoChromosomal localization of the humanNF-E2 family of bZIPtranscription factors by fluorescence in situ hybridizationrdquoHuman Genetics vol 95 no 3 pp 265ndash269 1995
[2] K Itoh K Igarashi N Hayashi M Nishizawa and MYamamoto ldquoCloning and characterization of a novel erythroidcell-derived CNC family transcription factor heterodimerizingwith the small Maf family proteinsrdquo Molecular and CellularBiology vol 15 no 8 pp 4184ndash4193 1995
[3] W O Osburn and T W Kensler ldquoNRF2 signaling an adaptiveresponse pathway for protection against environmental toxicinsultsrdquoMutation Research vol 659 no 1-2 pp 31ndash39 2008
[4] D Papp K Lenti D Modos D Fazekas Z Dul D Tureiet al ldquoThe NRF2-related interactome and regulome containmultifunctional proteins and fine-tuned autoregulatory loopsrdquoFEBS Letters vol 586 no 13 pp 1795ndash1802 2012
[5] X Wang D J Tomso B N Chorley et al ldquoIdentificationof polymorphic antioxidant response elements in the humangenomerdquo Human Molecular Genetics vol 16 no 10 pp 1188ndash1200 2007
[6] D Malhotra E Portales-Casamar A Singh et al ldquoGlobalmapping of binding sites for NRF2 identifies novel targets incell survival response through ChIP-Seq profiling and networkanalysisrdquo Nucleic Acids Research vol 38 no 17 pp 5718ndash57342010
[7] K Chan R Lu J C Chang and Y W Kan ldquoNRF2 a memberof the NFE2 family of transcription factors is not essential formurine erythropoiesis growth and developmentrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 93 no 24 pp 13943ndash13948 1996
[8] K Itoh T Chiba S Takahashi et al ldquoAn NRF2small Mafheterodimer mediates the induction of phase II detoxifyingenzyme genes through antioxidant response elementsrdquo Bio-chemical and Biophysical Research Communications vol 236no 2 pp 313ndash322 1997
[9] FMartin J M vanDeursen R A Shivdasani CW Jackson AG Troutman and P A Ney ldquoErythroid maturation and globin
gene expression inmicewith combined deficiency ofNF-E2 andNrf-2rdquo Blood vol 91 no 9 pp 3459ndash3466 1998
[10] K Itoh N Wakabayashi Y Katoh et al ldquoKEAP1 repressesnuclear activation of antioxidant responsive elements by NRF2through binding to the amino-terminal Neh2 domainrdquo Genesand Development vol 13 no 1 pp 76ndash86 1999
[11] A Kobayashi M I Kang H Okawa et al ldquoOxidative stresssensor KEAP1 functions as an adaptor for Cul3-based E3 ligaseto regulate proteasomal degradation of NRF2rdquo Molecular andCellular Biology vol 24 no 16 pp 7130ndash7139 2004
[12] K Taguchi H Motohashi and M Yamamoto ldquoMolecularmechanisms of the KEAP1-NRF2 pathway in stress responseand cancer evolutionrdquo Genes to Cells vol 16 no 2 pp 123ndash1402011
[13] K Mukaigasa L T Nguyen L Li H Nakajima M Yamamotoand M Kobayashi ldquoGenetic evidence of an evolutionarilyconserved role for NRF2 in the protection against oxidativestressrdquoMolecular and Cellular Biology vol 32 no 21 pp 4455ndash4461 2012
[14] JMaher andMYamamoto ldquoThe rise of antioxidant signalingmdashthe evolution and hormetic actions of NRF2rdquo Toxicology andApplied Pharmacology vol 244 no 1 pp 4ndash15 2010
[15] S C Lo X Li M T Henzl L J Beamer and M HanninkldquoStructure of the KEAP1NRF2 interface provides mechanisticinsight intoNRF2 signalingrdquoThe EMBO Journal vol 25 no 15pp 3605ndash3617 2006
[16] V A McKusick ldquoMendelian inheritance in man and its onlineversion OMIMrdquo American Journal of Human Genetics vol 80no 4 pp 588ndash604 2007
[17] B Yalcin D J Adams J Flint and T M Keane ldquoNext-generation sequencing of experimental mouse strainsrdquo Mam-malian Genome vol 23 no 9-10 pp 490ndash498 2012
[18] D R Bentley S Balasubramanian H P Swerdlow et alldquoAccurate whole human genome sequencing using reversibleterminator chemistryrdquo Nature vol 456 no 7218 pp 53ndash592008
[19] P S G Chain D V Grafham R S Fulton et al ldquoGenomeproject standards in a new era of sequencingrdquo Science vol 326no 5950 pp 236ndash237 2009
[20] RHWaterstonK Lindblad-Toh E Birney J Rogers J F AbrilP Agarwal et al ldquoInitial sequencing and comparative analysis ofthe mouse genomerdquo Nature vol 420 pp 520ndash562 2002
[21] T M Keane L Goodstadt P Danecek M A White K WongB Yalcin et al ldquoMouse genomic variation and its effect onphenotypes and gene regulationrdquoNature vol 477 pp 289ndash2942011
[22] T Yamamoto K Yoh A Kobayashi et al ldquoIdentification ofpolymorphisms in the promoter region of the human NRF2generdquo Biochemical and Biophysical Research Communicationsvol 321 no 1 pp 72ndash79 2004
[23] J M Marzec J D Christie S P Reddy et al ldquoFunctionalpolymorphisms in the transcription factor NRF2 in humansincrease the risk of acute lung injuryrdquo The FASEB Journal vol21 no 9 pp 2237ndash2246 2007
[24] H Fukushima-Uesaka Y Saito K Maekawa et al ldquoGeneticvariations and haplotype structures of transcriptional factorNRF2 and its cytosolic reservoir protein KEAP1 in JapaneserdquoDrug Metabolism and Pharmacokinetics vol 22 no 3 pp 212ndash219 2007
[25] E J Cordova R Velazquez-Cruz F Centeno V Baca and LOrozco ldquoThe NRF2 gene variant -653GA is associated with
Oxidative Medicine and Cellular Longevity 23
nephritis in childhood-onset systemic lupus erythematosusrdquoLupus vol 19 no 10 pp 1237ndash1242 2010
[26] M von Otter S Landgren S Nilsson et al ldquoAssociation ofNRF2-encoding NFE2L2 haplotypes with Parkinsonrsquos diseaserdquoBMCMedical Genetics vol 11 no 1 article 36 2010
[27] JMHartikainenM TengstromVMKosmaV L Kinnula AMannermaa andY Soini ldquoGenetic polymorphisms and proteinexpression of NRF2 and sulfiredoxin predict survival outcomesin breast cancerrdquo Cancer Research vol 72 pp 5537ndash5546 2012
[28] T Arisawa T Tahara T Shibata et al ldquoThe relationshipbetween Helicobacter pylori infection and promoter polymor-phism of the NRF2 gene in chronic gastritisrdquo InternationalJournal of Molecular Medicine vol 19 no 1 pp 143ndash148 2007
[29] T Arisawa T Tahara T Shibata et al ldquoThe influence ofpromoter polymorphism of nuclear factor-erythroid 2-relatedfactor 2 gene on the aberrant DNA methylation in gastricepitheliumrdquo Oncology Reports vol 19 no 1 pp 211ndash216 2008
[30] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene pro-moter polymorphism and gastric carcinogenesisrdquo Hepato-Gastroenterology vol 55 no 82-83 pp 750ndash754 2008
[31] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene promoterpolymorphism is associated with ulcerative colitis in a Japanesepopulationrdquo Hepato-Gastroenterology vol 55 no 82-83 pp394ndash397 2008
[32] C C Hua L C Chang J C Tseng C M Chu Y C Liu andW B Shieh ldquoFunctional haplotypes in the promoter region oftranscription factor NRF2 in chronic obstructive pulmonarydiseaserdquo Disease Markers vol 28 no 3 pp 185ndash193 2010
[33] S O Shaheen R B Newson S M Ring M J Rose-ZerilliJ W Holloway and A J Henderson ldquoPrenatal and infantacetaminophen exposure antioxidant gene polymorphismsand childhood asthmardquo The Journal of Allergy and ClinicalImmunology vol 126 no 6 pp 1141e7ndash1148e7 2010
[34] H Masuko T Sakamoto Y Kaneko et al ldquoLower FEV1 innon-COPD nonasthmatic subjects association with smokingannual decline in FEV1 total IgE levels and TSLP genotypesrdquoInternational Journal of Chronic Obstructive Pulmonary Diseasevol 6 pp 181ndash189 2011
[35] C P Guan M N Zhou A E Xu et al ldquoThe susceptibilityto vitiligo is associated with NF-E2-related factor 2 (NRF2)gene polymorphisms a study on Chinese Han populationrdquoExperimental Dermatology vol 17 no 12 pp 1059ndash1062 2008
[36] J Bouligand O Cabaret M Canonico et al ldquoEffect of NFE2L2genetic polymorphism on the association between oral estrogentherapy and the risk of venous thromboembolism in post-menopausal womenrdquo Clinical Pharmacology amp Therapeuticsvol 89 no 1 pp 60ndash64 2011
[37] D S OrsquoMahony B J Glavan T D Holden C Fong R A BlackG Rona et al ldquoInflammation and immune-related candidategene associations with acute lung injury susceptibility andseverity a validation studyrdquo PLoS ONE vol 7 no 12 Article IDe51104 2012
[38] I Ungvari E Hadadi V Virag et al ldquoRelationship between airpollutionNFE2L2 gene polymorphisms and childhood asthmain aHungarian populationrdquo Journal of CommunityGenetics vol3 no 1 pp 25ndash33 2012
[39] M SiedlinskiD S Postma JMA Boer et al ldquoLevel and courseof FEV1 in relation to polymorphisms inNFE2L2 andKEAP1 inthe general populationrdquo Respiratory Research vol 10 article 732009
[40] C C Hong C B Ambrosone J Ahn et al ldquoGenetic variabilityin iron-related oxidative stress pathways (NRF2 NQ01 NOS3
and HO-1) iron intake and risk of postmenopausal breastcancerrdquo Cancer Epidemiology Biomarkers and Prevention vol16 no 9 pp 1784ndash1794 2007
[41] C Canova C Dunster F J Kelly C Minelli P L Shah CCaneja et al ldquoPM10-induced hospital admissions for asthmaand chronic obstructive pulmonary disease the modifyingeffect of individual characteristicsrdquo Epidemiology vol 23 no 4pp 607ndash615 2012
[42] A J Henderson R B Newson M Rose-Zerilli S M RingJ W Holloway and S O Shaheen ldquoMaternal NRF2 andgluthathione-S-transferase polymorphisms donotmodify asso-ciations of prenatal tobacco smoke exposure with asthma andlung function in school-aged childrenrdquo Thorax vol 65 no 10pp 897ndash902 2010
[43] C Xing A L Sestak J A Kelly et al ldquoLocalization andreplication of the systemic lupus erythematosus linkage signalat 4p16 interaction with 2p11 12q24 and 19q13 in EuropeanAmericansrdquo Human Genetics vol 120 no 5 pp 623ndash631 2007
[44] T F Wong K Yoshinaga Y Monma K Ito H NiikuraS Nagase et al ldquoAssociation of KEAP1 and NRF2 geneticmutations and polymorphisms with endometrioid endometrialadenocarcinoma survivalrdquo International Journal of Gynecologi-cal Cancer vol 21 no 8 pp 1428ndash1435 2011
[45] M von Otter S Landgren S Nilsson et al ldquoNRF2-encodingNFE2L2 haplotypes influence disease progression but not riskin Alzheimerrsquos disease and age-related cataractrdquoMechanisms ofAgeing and Development vol 131 no 2 pp 105ndash110 2010
[46] S Tsang Z Sun B Luke et al ldquoA comprehensive SNP-basedgenetic analysis of inbred mouse strainsrdquoMammalian Genomevol 16 no 7 pp 476ndash480 2005
[47] C M Wade E J Kulbokas III A W Kirby et al ldquoThe mosaicstructure of variation in the laboratory mouse genomerdquoNaturevol 420 no 6915 pp 574ndash578 2002
[48] T Wiltshire M T Pletcher S Batalov et al ldquoGenome-widesingle-nucleotide polymorphism analysis defines haplotypepatterns in mouserdquo Proceedings of the National Academy ofSciences of the United States of America vol 100 no 6 pp 3380ndash3385 2003
[49] K A Frazer E Eskin H M Kang et al ldquoA sequence-basedvariation map of 827 million SNPs in inbred mouse strainsrdquoNature vol 448 no 7157 pp 1050ndash1053 2007
[50] T P Maddatu S C Grubb C J Bult andM A Bogue ldquoMousephenome database (MPD)rdquo Nucleic Acids Research vol 40 ppD887ndashD894 2007
[51] A Kirby H M Kang C M Wade et al ldquoFine mapping in 94inbred mouse strains using a high-density haplotype resourcerdquoGenetics vol 185 no 3 pp 1081ndash1095 2010
[52] H Y Cho A E Jedlicka S P M Reddy L Y Zhang T WKensler and S R Kleeberger ldquoLinkage analysis of susceptibilityto hyperoxia NRF2 is a candidate generdquo American Journal ofRespiratory Cell and Molecular Biology vol 26 no 1 pp 42ndash512002
[53] H Y Cho A E Jedlicka S P M Reddy et al ldquoRole of NRF2in protection against hyperoxic lung injury in micerdquo AmericanJournal of Respiratory Cell and Molecular Biology vol 26 no 2pp 175ndash182 2002
[54] T Shibata T Ohta K I Tong et al ldquoCancer related mutationsin NRF2 impair its recognition by KEAP1-Cul3 E3 ligase andpromote malignancyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 36 pp13568ndash13573 2008
24 Oxidative Medicine and Cellular Longevity
[55] A Singh V Misra R K Thimmulappa et al ldquoDysfunctionalKEAP1-NRF2 interaction in non-small-cell lung cancerrdquo PLoSMedicine vol 3 no 10 article e420 2006
[56] T Ohta K Iijima M Miyamoto et al ldquoLoss of KEAP1 functionactivates NRF2 and provides advantages for lung cancer cellgrowthrdquo Cancer Research vol 68 no 5 pp 1303ndash1309 2008
[57] B Padmanabhan K I Tong T Ohta et al ldquoStructural basisfor defects of KEAP1 activity provoked by its point mutationsin lung cancerrdquoMolecular Cell vol 21 no 5 pp 689ndash700 2006
[58] T Shibata A Kokubu M Gotoh et al ldquoGenetic alteration ofKEAP1 confers constitutive NRF2 activation and resistance tochemotherapy in gallbladder cancerrdquoGastroenterology vol 135no 4 pp 1358e4ndash1368e4 2008
[59] N J Yoo H R Kim Y R Kim C H An and S H LeeldquoSomatic mutations of the KEAP1 gene in common solidcancersrdquo Histopathology vol 60 no 6 pp 943ndash952 2012
[60] Y Hu Y Ju D Lin Z Wang Y Huang S Zhang et alldquoMutation of the NRF2 gene in non-small cell lung cancerrdquoMolecular Biology Reports vol 39 no 4 pp 4743ndash4747 2012
[61] Y R Kim J EOhM S Kim et al ldquoOncogenicNRF2mutationsin squamous cell carcinomas of oesophagus and skinrdquo TheJournal of Pathology vol 220 no 4 pp 446ndash451 2010
[62] T Shibata A Kokubu S Saito et al ldquoNRF2 mutation confersmalignant potential and resistance to chemoradiation therapyin advanced esophageal squamous cancerrdquo Neoplasia vol 13pp 864ndash873 2011
[63] H Sasaki M Shitara K Yokota Y Hikosaka S MoriyamaM Yano et al ldquoIncreased NRF2 gene (NFE2L2) copy numbercorrelates with mutations in lung squamous cell carcinomasrdquoMolecular Medicine Reports vol 6 no 2 pp 391ndash394 2012
[64] H Sasaki M Shitara K Yokota Y Hikosaka S Moriyama MYano et al ldquoRagD gene expression andNRF2mutations in lungsquamous cell carcinomasrdquo Oncology Letters vol 4 pp 1167ndash1170 2012
[65] A Singh M Bodas N Wakabayashi F Bunz and S BiswalldquoGain of NRF2 function in non-small-cell lung cancer cellsconfers radioresistancerdquo Antioxidants and Redox Signaling vol13 no 11 pp 1627ndash1637 2010
[66] T Yamadori Y Ishii SHommaYMorishimaKKurishimaKItoh et al ldquoMolecular mechanisms for the regulation of NRF2-mediated cell proliferation in non-small-cell lung cancersrdquoOncogene vol 31 pp 4768ndash4777 2012
[67] A K Bauer H Y Cho L Miller-Degraff C Walker K HelmsJ Fostel et al ldquoTargeted deletion of NRF2 reduces urethane-induced lung tumor development in micerdquo PLoS ONE vol 6no 10 Article ID e26590 2011
[68] H Y Cho and S R Kleeberger ldquoNRF2 protects against airwaydisordersrdquo Toxicology and Applied Pharmacology vol 244 no1 pp 43ndash56 2010
[69] R Wang J An F Ji H Jiao H Sun and D Zhou ldquoHyperme-thylation of the KEAP1 gene in human lung cancer cell linesand lung cancer tissuesrdquo Biochemical and Biophysical ResearchCommunications vol 373 no 1 pp 151ndash154 2008
[70] P Zhang A Singh S Yegnasubramanian et al ldquoLoss ofkelch-like ECH-associated protein 1 function in prostate cancercells causes chemoresistance and radioresistance and promotestumor growthrdquoMolecular Cancer Therapeutics vol 9 no 2 pp336ndash346 2010
[71] N Hanada T Takahata Q Zhou et al ldquoMethylation of theKEAP1 gene promoter region in human colorectal cancerrdquoBMCCancer vol 12 article 66 2012
[72] L A Muscarella R Barbano V DrsquoAngelo et al ldquoRegulationof KEAP1 expression by promoter methylation in malignantgliomas and association with patientrsquos outcomerdquo Epigeneticsvol 6 no 3 pp 317ndash325 2011
[73] S Yu T O Khor K L Cheung et al ldquoNRF2 expressionis regulated by epigenetic mechanisms in prostate cancer ofTRAMP micerdquo PLoS ONE vol 5 no 1 Article ID e8579 2010
[74] T O Khor Y Huang T Y Wu L Shu J Lee and A N KongldquoPharmacodynamics of curcumin as DNA hypomethylationagent in restoring the expression of NRF2 via promoter CpGsdemethylationrdquo Biochemical Pharmacology vol 82 no 9 pp1073ndash1078 2011
[75] I M Fingerman L McDaniel X Zhang et al ldquoNCBI epige-nomics a new public resource for exploring epigenomic datasetsrdquo Nucleic Acids Research vol 39 supplemen 1 pp D908ndashD912 2011
[76] I M Fingerman X Zhang W Ratzat N Husain R F Cohenand G D Schuler ldquoNCBI epigenomics whatrsquos new for 2013rdquoNucleic Acids Research vol 41 no 1 pp D221ndashD225 2013
[77] J H Lee T O Khor L Shu Z Y Su F Fuentes andA N Kong ldquoDietary phytochemicals and cancer preventionNRF2 signaling epigenetics and cell death mechanisms inblocking cancer initiation and progressionrdquo Pharmacology ampTherapeutics vol 137 no 2 pp 153ndash171 2012
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Disease Markers
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OncologyJournal of
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Oxidative Medicine and Cellular Longevity
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Evidence-Based Complementary and Alternative Medicine
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Oxidative Medicine and Cellular Longevity 5
Table 2 Protein domains of NF-E2-related factor 2
Domains Amino acid positionslowast Predicted functionsHuman (605 aa) Mouse (597 aa)
Neh216ndash89
DLG motif 17ndash32ETGF motif 77ndash82
16ndash89 KEAP1 repression through DLGETGF motif-DC motif binding fastredox-sensitive proteasomal degradation
Neh4 111ndash134 111ndash134 Translocation and transactivation Phosphorylation or CBP bindingNeh5 182ndash209 172ndash201
Neh6 337ndash394(or 338ndash388) 328ndash385 Degron motif-associated constitutive turnover slow redox-insensitive
(Keap1-independent)
Neh1435ndash568
basic motif 503ndash518leucine zipper 525ndash539
427ndash560basic motif 494ndash509leucine zipper 517ndash531
Dimerization for nuclear translocation DNA binding through basicmotif-leucine zipper
Neh3 569ndash605 561ndash597 CHD6 binding stability or transactivationlowastVaries slightly among publications
homology (Neh) domains configuring the protein sequenceof either species (Table 2) and the potential functions of eachregion particularly the highly conserved KEAP1-bindingNeh2 and DNA- (ARE-) binding Neh1 domains have beenintensively investigated [12 14 15]
3 Genetic Variation of NF-E2-Related Factor 2in Human and Mouse
31 Evolution Genome Sequence and Polymorphism Dis-covery in Human and Mouse While rare and monogenicMendelian diseases are inheritable mutations in a single gene[16] many common diseases are complex traits and thedisease phenotypes are affected by variants inmultiple geneticloci Recent advancements in high-throughput technologyhave enabled sequencing of entire mammalian genomes[17ndash19] and information on DNA sequence and variationhas facilitated the study of complex traits of human dis-orders Genome-wide association studies (GWAS) examinewhether SNPs are associated with important disease traitsand ascertains ldquoat-riskrdquo genotypes that are significantly moreprevalent in the affected group than in the nonaffected groupThe HapMap Project (httphapmapncbinlmnihgov) hasmapped combinations of alleles at specific loci (haplotypes)that is common patterns of sequence variation in severalhuman populations It has supported efficient mapping ofmultiple loci for complex traits in GWAS Candidate geneapproaches based on findings from GWAS of similar dis-orders have also been useful for determining the potentialgenetic mechanisms of diseases
The evolutionary divergence of human and mouse lin-eages occurred for roughly 75million years and their genomesequences have been altered by nearly one substitution (ordeletion or addition) for every twonucleotides [20]Howeverthis slow evolution process resulted in a high degree ofconservation across the two species which allows alignmentof orthologous sequences gt90 of the human and mousegenomes is partitioned into corresponding regions of con-served synteny and at the nucleotide level approximately 40of the human genome is aligned to themouse [20]Due to this
fact biomedical studies of human genes are complemented byexperimental manipulation of corresponding mouse genesand they have aided functional understanding of genesin human health Following the 2003 completion of theHuman Genome Project of approximately 31 giga base pairs(Gbp) the Mouse Genome Project assembled the completegenome sequence of one strain (C57BL6J 2716965481 bp)in 2011 Using this reference strain whole genome sequenc-ing data across 16 additional inbred strains were done(httpwwwsangeracukresourcesmousegenomes [21])Discovery of high-density SNPs in the mouse genome sup-ports evolutionary history of the strain and provides a toolto investigate models of human disease processes that cannotoften be practically achieved through direct human studies
32 GeneticMutations in HumanNRF2 HumanNRF2 codesthree major isoforms of protein (Figure 2) Transcript variant2 (NM 0011454122 2746 bp) has an alternate promoter 51015840-UTR and a downstream start codon compared to variant 1(NM 0061644 2859 bp) It encodes an isoform 2 missing N-terminal 16 aa (NP 001138884 or Q16236-2 589 aa) relativeto isoform 1 (NP 0061552 or Q16236 605 aa) Isoform 3(NP 0011388851 or Q16236-3 582 aa) is encoded by tran-script variant 3 (NM 0011454132 2725 bp) and lacks aninternal segment relative to isoform 2 due to an alternate in-frame splice site in the 31015840 coding region In public databasesmore than 583 sequence mutations are reported in NRF2(34827 bp) and 7000 bp upstream (Table S1 data acquired asof December 2012) (See Table S1 in Supplementary Materialavailable on line at httpdxdoiorg1011552013286524)NRF2 locates on 178130354-178129304 bp of GRCh37p10PrimaryAssembly and Figure 2 shows sequences of proximalpromoter (minus1 to minus500) partial mRNA variant 1 including 51015840-UTR (exon 1 up to TSS) and protein isoform 1 (NP 006155encoded by variant 1 NM 0061644 556ndash2373 bp) Basedon the current assembly and sequence update previouspromoter positions minus686 [22]minus653 [23] are identified asminus214 minus684 [22]minus651 [23] as minus212 and minus650 [22]minus617 [23]asminus178Overall frequency ofNRF2 SNPs and othermutationsis about 1 per 72 bp The genetic mutations include 37 in
6 Oxidative Medicine and Cellular Longevity
Translation start site for variants 2 and 3
1 missed in isoforms 2 and 3pA72T pS99P pQ121H missed in isoform 3
pP406S pE423D pK428R pR437W pR449C
minus400
minus500
minus50minus100
minus200
minus300
minus450
minus350
minus250
minus150
g minus
g
pE152K pV155I pL177V
pV268M
pR517K pH453L
pS365I pS387N pK389R pL309F pI317M pD341V
pK219R pP233S pK237 = fs
51101151201251301351401451501551601
Protein isoform 1 (NP 006155)
g minus66minus65insC
Termination codon (c +2371 +2373)
Translation start site (c +1)
151
101151201251301351401451501551601
2351
frame stop codonc minus156 154 in-old + 1 [23]
old + 1 [22]
g minus1 (178130354)
c minus96delC
c minus438 minus437delTT
minus89 minus87delGCC
transcription start site)+1 (c minus555
p R43Q p R43W
Genomic DNA (reversed contig GRCh37 p10 primary assemblyNM 0061644)minus477
minus214 g minus212gminus188
c 9minus95
g minus178
c minus304
c 8minus13
c 2minus16
TgtA
CgtT
GgtA
GgtA
GgtA
GgtT
GgtCAgtG
AgtCAgtG
Figure 2 DNA (variant 1 partial promoter and exons 1 and 5) and protein (isoform 1) sequence of Human NRF2 SNPs and amino acidresidues for nonsynonymous SNPs are marked
the 51015840 flanking promoter and 59 in exons (Tables 3 and S1)Among exon SNPs 26 are nonsynonymous (Cns) mutationsA triplet repeat variation (rs143406266 GCC
4versus GCC
5
previously published as minus20minus6) in the 51015840-UTR was uniquelyidentified in Asian populations [22 24]
33 SNPs Haplotypes and Association with Disease RiskThe use of gene knockout mice in model systems hasprovided potential insights into the role of NRF2 in thepathogenesis of various human disorders (see Figure 1)Recent epidemiological and association studies have revealedsignificant associations of NRF2 sequence variations withdisease risks which further supportsNRF2 as a susceptibilitygene Most of the phenotype-associated variants are in thepromoter region and presumed to be involved in NRF2 generegulation Table 4summarizes NRF2 SNP andor haplotypealleles that have been associated with oxidant-related diseaserisks Interestingly there is no evidence for exon SNPs as at-risk alleles For convenience and consistency intronic and31015840 distal SNP alleles are presented as chromosome contig(HGVS) alleles while promoter and exon SNPs are presentedas reversed contig alleles throughout the text
Pulmonary Diseases NRF2 SNPs in the promoter and intron1 sequences have been investigated for their potential asso-ciations with risk of pulmonary critical disorders includingacute lung injury (ALI) cigarette smoke-induced chronicobstructive pulmonary disease (COPD) and asthma Aheterozygous CA SNP at minus178 position (rs6721961TgtCor TgtG previously minus617 or minus650) significantly increasedthe risk for developing ALI following major trauma inEuropean and African-American populations (odds ratioOR 644 95 confidence interval CI 134ndash308 allelicfrequency = 119 at 21180) [23] Promoter activity ofthe A allele (AC or AA) determined in vivo and invitro was significantly lower than CC allele at that locus(minus178 in an ARE-like motif) indicating that it is a func-tional SNP for autoregulation [23] The minus178GG werealso nominally associated with ALI-related 28-day mor-tality following systemic inflammatory response syndrome[37] In a Japanese cohort SNP haplotype (rs2001350Trs6726395Ars1962142Ars2364722Ars6721961T) co-tainingthe minus178AA homozygote was associated with an annualdecline of rapid forced expiratory volume in one second(FEV1) in relation to cigarette-smoking status [34] In addi-
tion a promoter and 51015840-UTR SNP haplotype consistingof minus214G allele (52 rs35652124 previously minus686minus653)
Oxidative Medicine and Cellular Longevity 7Ta
ble3Geneticmutations
inprom
oter
andexon
sofh
uman
NRF
2
IDMap
onchromosom
e2lowast
(HGVS
name)
Positionin
Nfe2
l2
(HGVS
name)
Region
s(position
from
mRN
A)
Varia
tioncla
ssand
consequences
(HGVS
name)dagger
Minor
allelefrequ
ency
(MAF)
MAcoun
tsDagger(coh
ort
size)
MAFsources
rs16865105
g178136629AgtC
cminus555minus
6770Tgt
G51015840Flanking
(minus6770)
SNP
C=01928421
1000
Genom
esrs7557529
g178135097CgtT
cminus555minus
5238GgtA
51015840Flanking
(minus5238)
SNP
nars6750320
g178131796CgtT
cminus555minus
1937GgtA
51015840Flanking
(minus1937)
SNP
T=0030266
1000
Genom
esrs18116
2518
g178131774TgtC
cminus555minus
1915AgtG
51015840Flanking
(minus1915)
SNP
C=000
051
1000
Genom
esrs1900
44775
g178131746CgtT
cminus555minus
1887GgtA
51015840Flanking
(minus1887)
SNP
T=000
051
1000
Genom
esrs185117338
g178131704AgtG
cminus555minus
1845Tgt
C51015840Flanking
(minus1845)
SNP
G=000
051
1000
Genom
esrs149947189
g178131697CgtT
cminus555minus
1838GgtA
51015840Flanking
(minus1838)
SNP
T=000
092
1000
Genom
esrs139771244
g178131625AgtG
cminus555minus
1766Tgt
C51015840Flanking
(minus1766)
SNP
G=000
092
1000
Genom
esrs6747203
g17813160
4CgtG
cminus555minus
1745GgtC
51015840Flanking
(minus1745)
SNP
G=000
613
1000
Genom
esrs193101749
g178131504TgtC
cminus555minus
1645AgtG
51015840Flanking
(minus1645)
SNP
C=000
4610
1000
Genom
esrs190630762
g178131495GgtC
cminus555minus
1636Cgt
G51015840Flanking
(minus1636)
SNP
C=000
092
1000
Genom
esrs183764
402
g178131366CgtA
cminus555minus
1507GgtT
51015840Flanking
(minus1507)
SNP
A=000
051
1000
Genom
esrs19122296
4g178131211GgtA
cminus555minus
1352Cgt
T51015840Flanking
(minus1352)
SNP
A=000
092
1000
Genom
esrs187137522
g178131165TgtG
cminus555minus
1306AgtC
51015840Flanking
(minus1306)
SNP
G=000
092
1000
Genom
esrs182620359
g178131158AgtG
cminus555minus
1299Tgt
C51015840Flanking
(minus1299)
SNP
G=000
051
1000
Genom
esrs4893819
(rs61433302)
g178131134CgtT
cminus555minus
1275GgtA
51015840Flanking
(minus1275)
SNP
C=04263931
1000
Genom
es
rs191547130
g178131017CgtT
cminus555minus
1158GgtA
51015840Flanking
(minus1158)
SNP
T=000
051
1000
Genom
esrs188422217
g178131003AgtG
cminus555minus
1144TgtC
51015840Flanking
(minus114
4)SN
PG=000143
1000
Genom
esrs143047764
g178130865AgtG
cminus555minus
1006Tgt
C51015840Flanking
(minus1006)
SNP
G=000
6915
1000
Genom
esrs74432849
g178130766CgtA
cminus555minus
907GgtT
51015840Flanking
(minus907)
SNP
nars116
79252
g178130691CgtG
cminus555minus
832GgtC
51015840Flanking
(minus832)
SNP
nars12993217
g178130516AgtG
cminus555minus
657TgtC
51015840Flanking
(minus657)
SNP
nars115644
826
g17813044
2TgtA
cminus555minus
583AgtT
51015840Flanking
(minus583)
SNP
A=0015133
1000
Genom
esrs140803524
g178130431GgtA
cminus555minus
572CgtT
51015840Flanking
(minus572)
SNP
A=000
4610
1000
Genom
es
rs776844
20g178130427TgtC
cminus555minus
568AgtG
51015840Flanking
(minus568)
SNP
C=0033974
C=0190(84)
1000
Genom
es[24]
rs183651094
g178130336AgtT
cminus555minus
477TgtA
51015840Flanking
(minus477)
SNP
T=000184
1000
Genom
es
rs35652124
sect
(rs57695243)
g178130073TgtC
cminus555minus
214AgtG
51015840Flanking
(minus214)
SNP
C=03512767
T=0429(84)
T=0413181Dagger
C=033827Dagger
C=03517
69
1000
Genom
es[24]
[22]
[23]
[25]
rs6706649
sectg178130071CgtT
cminus555minus
212GgtA
51015840Flanking
(minus212)
SNP
T=0078170
T=004
8(84)
T=004
821Dagger
T=00753Dagger
1000
Genom
es[24]
[22]
[23]
rs1506
48896
g178130047CgtG
cminus555minus
188GgtC
51015840Flanking
(minus188)
SNP
G=000235
G=000
6(84)
1000
Genom
es[24]
rs6721961
sect
(rs117801448)
g178130037TgtC
TgtG
cminus555minus
178AgtC
AgtG
51015840Flanking
(minus178)
SNP
T=0150328
T=0283124Dagger
T=031325Dagger
T=0321(84)
1000
Genom
es[22]
[23]
[24]
8 Oxidative Medicine and Cellular Longevity
Table3Con
tinued
IDMap
onchromosom
e2lowast
(HGVS
name)
Positionin
Nfe2
l2
(HGVS
name)
Region
s(position
from
mRN
A)
Varia
tioncla
ssand
consequences
(HGVS
name)dagger
Minor
allelefrequ
ency
(MAF)
MAcoun
tsDagger(coh
ort
size)
MAFsources
rs20134560
4g178129924178129925insG
cminus555-66minus555-65insC
51015840Flanking
(minus66minus65)
Insertion
G=0017939
1000
Genom
es
rs200432479
g178129741178
129742delAA
cminus438minus437delT
Texon
1UTR
-51015840(118minus119
)Dele
tion
-=000378
-=000
6(84)
1000
Genom
es[24]
rs75485459
g178129608CgtA
cminus304GgtT
Exon
1UTR
-51015840(252)
SNP
nars192086766
g17812946
6CgtT
cminus162GgtA
Exon
1UTR
-51015840(394)
SNP
T=002248
1000
Genom
esmdash
g17812944
2GgtA
cminus138CgtT
Exon
1UTR
-51015840(418)
SNP
A=0012(84)
[24]
rs7166
8246
g17812940
0delG
cminus96delC
Exon
1UTR
-51015840(460)
Dele
tion
nars187291840
g178129399CgtT
cminus95GgtA
Exon
1UTR
-51015840(461)
SNP
T=0054912
01000
Genom
es
rs143406266
g178129391178129393delGGC
cminus89minus87delGCC
Exon
1UTR
-51015840(467minus469)
Dele
tion
-=064
4282Dagger
-=0589(84)
[22]
[24]
rs182428269
g178098918GgtA
c127Cgt
TEx
on2(682)
Cns(pArg43Trp)
A=000
051
1000
Genom
esrs35248500
g178098917CgtT
c128GgtA
Exon
2(683)
Cns(pArg43Gln)
T=000
613
1000
Genom
esrs1135118
g178098831CgtT
c214GgtA
Exon
2(769)
Cns(pAla72Th
r)TDagger
[23]
rs19969166
0g178098829AgtT
c216Tgt
AEx
on2(771)
Cs(pAla72=)
namdash
g178098769GgtA
c276Cgt
TEx
on2(831)
Cs(pIle92=)
TDagger[23]
rs5031039
g178098750AgtG
c295Tgt
CEx
on2(850)
Cns(pSer99P
ro)
G=0Dagger
[23]
rs200239262
g178098017CgtG
c363GgtC
Exon
3(918)
Cns(pGln121H
is)na
rs183034165
g178098008TgtC
c372GgtA
Exon
3(927)
Cs(pAla124=
)T=000
092
T=000
6(84)
1000
Genom
es[24]
rs199970826
g178097996CgtT
c384GgtA
Exon
3(939)
Cs(pPro128=
)T=000
051
1000
Genom
esrs201992337
g178097260CgtT
c454GgtA
Exon
4(100
9)Cn
s(pGlu152Lys)
nars201589693
g178097251CgtT
c463GgtA
Exon
4(1018)
Cns(pV
al155Ile)
T=000
051
1000
Genom
es
rs35577826
g178097185AgtC
c529Tgt
GEx
on4(1084)
Cns(pLeu177Va
l)C=000143
Alt0005Dagger
1000
Genom
es[23]
rs181513314
g178096710CgtT
c621GgtA
Exon
5(1176)
Cn(pLeu207=
)T=000
051
1000
Genom
esrs60132461
g178096675TgtC
c656AgtG
Exon
5(1211)
Cns(pLys219Arg)
C=000184
1000
Genom
es
rs139187151
g178096634GgtA
c697Cgt
TEx
on5(1252)
Cns(pPro233Ser)
A=000
051
A=0012Dagger
(84)
1000
Genom
es[24]
rs35557421
g178096620d
elT
c711delA
Exon
5(1266)
Fram
eshiftdeletio
n(pLys237=fs)
na
rs34154613
g178096529CgtT
c802GgtA
Exon
5(1357)
Cns(pV
al268M
et)
T=000184
1000
Genom
esrs141363120
g178096
406GgtA
c925Cgt
TEx
on5(14
80)
Cns(pLeu309Ph
e)A=000378
1000
Genom
esrs201661476
g178096380AgtC
c951Tgt
GEx
on5(1506)
Cns(pIle
317M
et)
na
Oxidative Medicine and Cellular Longevity 9
Table3Con
tinued
IDMap
onchromosom
e2lowast
(HGVS
name)
Positionin
Nfe2
l2
(HGVS
name)
Region
s(position
from
mRN
A)
Varia
tioncla
ssand
consequences
(HGVS
name)dagger
Minor
allelefrequ
ency
(MAF)
MAcoun
tsDagger(coh
ort
size)
MAFsources
rs199673454
g178096309TgtA
c1022AgtT
Exon
5(1577)
Cns(pAs
p341Va
l)A=000
051
1000
Genom
esrs35007548
g178096299GgtA
c1032CgtT
Exon
5(1587)
Cs(pSer344=
)A=000
092
1000
Genom
esrs200209692
g178096287TgtC
c104
4AgtG
Exon
5(1599)
Cs(pLeu348=
)C=000
051
1000
Genom
es
mdashg178096237CgtA
c1094GgtT
Exon
5(1649)
Cns(pSer365Ile
)A=0125273
A=000
6Dagger(84)
PubM
ed[24]
rs201214197
g178096171CgtT
c1160GgtA
Exon
5(1715)
Cns(pSer387Asn)
T=000
051
1000
Genom
esrs200494292
g178096165TgtC
c1166AgtG
Exon
5(1721)
Cns(pLys389Arg)
nars186171287
g178096115GgtA
c1216CgtT
Exon
5(1771)
Cns(pPro4
06Ser)
A=000
051
1000
Genom
esrs182276775
g178096062CgtA
c1269GgtT
Exon
5(1824)
Cns(pGlu423A
sp)
A=000
051
1000
Genom
esrs201560221
g17809604
8TgtC
c1283AgtG
Exon
5(1838)
Cns(pLys428Arg)
nars189238236
g178096043AgtG
c1288TgtC
Exon
5(1843)
Cs(pLeu430=
)G=000
051
1000
Genom
esrs184287392
g178096022GgtA
c1309CgtT
Exon
5(1864)
Cns(pArg437Trp)
A=000
051
1000
Genom
esrs201871588
g178095986GgtA
c1345CgtT
Exon
5(19
00)
Cns(pAg449Cy
s)na
rs181294188
g178095985TgtC
c1346GgtA
Exon
5(19
01)
Cns(pArg44
9His)
T=000
092
1000
Genom
esrs20169046
6g178095973TgtA
c1358AgtT
Exon
5(19
13)
Cns(pHis4
53Leu)
A=000
051
1000
Genom
esrs105704
4(rs52789869)
g178095781CgtT
c1550GgtA
Exon
5(2105)
Cns(pArg517Lys)
TDagger[23]
rs200750800
g178095603AgtG
c1728TgtC
Exon
5(2283)
Cs(pTyr576=
)na
rs200175942
g178095567AgtG
c1764TgtC
Exon
5(2319)
Cs(pAsp588=
)G=000
051
1000
Genom
esrs77547666
g178095495GgtC
clowast18Cgt
GEx
on5UTR
-31015840(2391)
SNP
C=000
6915
1000
Genom
esrs73031353
g178095425TgtC
clowast88AgtG
Exon
5UTR
-31015840(2461)
SNP
C=000184
1000
Genom
esrs675944
3g178095345TgtC
clowast168AgtG
Exon
5UTR
-31015840(2541)
SNP
C=000
419
1000
Genom
esrs188674558
g178095279CgtA
clowast234GgtT
Exon
5UTR
-31015840(2607)
SNP
A=000
051
1000
Genom
esrs77685897
g178095247AgtG
clowast266TgtC
Exon
5UTR
-31015840(2639)
SNP
G=000
092
1000
Genom
esrs1057092
g178095162TgtG
clowast351AgtC
Exon
5UTR
-31015840(2724)
SNP
nars3197704
g178095162TgtG
clowast351AgtC
Exon
5UTR
-31015840(2724)
SNP
nars184701151
g178095159TgtC
clowast354AgtG
Exon
5UTR
-31015840(2727)
SNP
C=000276
1000
Genom
esrs11543307
g178095153AgtG
clowast360TgtC
Exon
5UTR
-31015840(2733)
SNP
nars111874043
g178095146AgtG
clowast367TgtC
Exon
5UTR
-31015840(2740)
SNP
nars34012004
g178095102AgtC
clowast411TgtG
Exon
5UTR
-31015840(2784)
SNP
C=0071
[24]
10 Oxidative Medicine and Cellular Longevity
Table3Con
tinued
IDMap
onchromosom
e2lowast
(HGVS
name)
Positionin
Nfe2
l2
(HGVS
name)
Region
s(position
from
mRN
A)
Varia
tioncla
ssand
consequences
(HGVS
name)dagger
Minor
allelefrequ
ency
(MAF)
MAcoun
tsDagger(coh
ort
size)
MAFsources
rs201481890
g178095090178095091in
sTclowast
422lowast423insA
Exon
5UTR
-31015840
(2795minus
2796)
Insertion
na
rs3082500
g178095089178095090d
elTT
delTTinsT
clowast423lowast424d
elAAinsA
Exon
5UTR
-31015840
(2796minus
2797)
Dele
tion
insertion
na
rs71792546
(rs71796710)
g178095079d
elT
clowast425delA
Exon
5UTR
-31015840(2798)
Dele
tion
na
rs1057106
g178095078AgtC
AgtT
clowast435TgtA
TgtG
Exon
5UTR
-31015840(2808)
SNP
na
rs34176791
g178095076AgtC
clowast437TgtG
Exon
5UTR
-31015840(2810)
SNP
C=000235
1000
Genom
esrs35911553
g178095045CgtT
clowast46
8GgtA
Exon
5UTR
-31015840(2841)
SNP
T=000
6915
1000
Genom
esSequ
ence
varia
tions
inup
stream
andexon
sofh
uman
NRF
2fro
m655varia
tions
availablein
publicdatabase
asof
Decem
ber2012
(583
activ
esomeSN
Psmergedge14
SNPs
citedin
PubM
ed)lowastNCB
Ireference
sequ
ence
NC
0000
0211
(Hom
osapiens
chromosom
e2G
RCh37p
10prim
arya
ssem
bly)spanning
178095033ndash1781298
59bp
(com
plem
ent34827
bpforexons
andintro
ns)51015840-Flank
ingregions
startat17812
9860
bp(minus1)in
transcrip
tvariant
1HGVS
Hum
anGenom
eVa
riatio
nSo
ciety
Position
sinvaria
nt1(NM
006164
4)2859
bpsectSN
Pscitedin
PubM
edE
xon11781298
59ndash17812
9260
(600
bpT
TS=1781293
04)exon
2178098999ndash
178098733(267
bp)exon
3178098067ndash1780979
78(90b
p)exon41780973
11ndash178097120
(192b
p)and
exon
5178096736ndash
178095033(1704b
p)daggerProteinam
inoacid
(aa)
resid
uesiniso
form
1(N
P00
61552605
aa)Cn
scoding
-non
syno
nymou
sCs
cod
ing-syno
nymou
sDaggerheterozygositydetectednano
tavailable
Oxidative Medicine and Cellular Longevity 11
Table4Hum
anNRF
2SN
Psassociated
with
diseaser
isk
IDMap
onchromosom
e2 (H
GVS
name)
Region
class
Dise
asea
ssociatio
nandreferences
Risk
allelesa
ndstatistics
Ethn
icgrou
p(num
bero
fcase)
rs7557529
g178135097CgtT
minus5238
Ggt
AParkinsonrsquos
disease(2010
[26])
G=0718252Dagger
inhaplotype
OR=090
or040
CI=
060ndash140or
03ndash06
Swedish
Polish
Caucasian
(357)
rs2886162
g178133165AgtG
minus3306
TgtC
Breastcancer
survival(2012[27])
TT=0324
OR=16
87C
I=1105ndash275
FinlandKB
CP(452)
Chronicg
astritisgastric
ulcer(2007
[28])
GDagger
GDaggerin
haplotypefor
ulcer(OR=
252C
I=119ndash
545)
Japanese
(159)
P14methylationin
gastric
cancer
Hpyloriinfectio
n(2008[29])
Gin
haplotype
OR=290C
I=114ndash
736
Japanese
(209)
Gastriccancer
inHpylori-n
egative
cases(2008
[30])
Ain
haplotype
119875=0022
Japanese
(209)
rs35652124
(rs57695243)
g178130073TgtC
minus214Agt
G(previou
slyminus653[23]
orminus686[22])
Ulcerativec
olitis(2008
[31])
AG
(OR=045C
I=022ndash0
93)
G(O
R=257C
I=10
1ndash660)
Japanese
(89)
Lupu
swith
neph
ritisin
female(2010
[25])
GA
OR=18
1CI
=10
4ndash312
Mexican
mestizo(362)
Parkinsonrsquos
disease(2010
[26])
A=0884312Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
COPD
(2010[32])
G=052Dagger
hazard
ratio
=095C
I=091ndash0
99
(Haplotype)
German
(69)
Gastriculcer(2007
[28])
GDaggerin
haplotype
OR=252C
I=119ndash
545
Japanese
(159)
P14methylationin
gastric
cancer
H
pyloriinfection(2008[29])
Gin
haplotype
OR=290C
I=114ndash
736
Japanese
(209)
Gastriccancer
inHpylori-negative
cases(2008
[30])
Gin
haplotype119875=0022
Japanese
(209)
rs6706
649
g178130071CgtT
minus212Ggt
A(previou
slyminus651[23]
orminus684[22])
Ulcerativec
olitis(2008
[31])
AG
(OR=045C
I=022ndash0
93)
G(O
R=257C
I=10
1ndash660)
Japanese
(89)
Maternalacetaminop
henandasthma
(2010[33])
A=02321137Dagger
OR=17
3CI
=12
2245
UKALSPA
C(gt40
00mothersgt
5000
child
ren)
12 Oxidative Medicine and Cellular Longevity
Table4Con
tinued
IDMap
onchromosom
e2 (H
GVS
name)
Region
class
Dise
asea
ssociatio
nandreferences
Risk
allelesa
ndstatistics
Ethn
icgrou
p(num
bero
fcase)
COPD
(2010[32])
G=098Daggerin
haplotype
hazard
ratio
=095C
I=091ndash0
99
German
(69)
Parkinsonrsquos
disease(2010
[26])
G=0972343Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
03ndash06
Swedish
Polish
Caucasian
(357)
Acutelun
ginjury
follo
wingtrauma
(2007[23])
CA=0119Dagger
OR=644
CI=
134ndash
3080
Caucasia
nAfrican-
American
(164
)
Ann
ualF
EV1decline
(2011[34])
A=0082in
haplotype
Japanese
(915)
Vitiligo(2008[35])
CAA
AOR=17
24C
I=13
5ndash221
Chinese(300)
COPD
(2010[32])
G=073Daggerin
haplotype
hazard
ratio
=095C
I=091ndash0
99
German
(69)
rs6721961
(rs117801448)
g178130037TgtC
TgtG
minus178Agt
GAgt
C(previou
slyminus617[23]
orminus650[22])
Parkinsonrsquos
disease(2010
[26])
C=0980346Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
Postm
enop
ausalvenou
sthrombo
embo
lism
(2011[36])
A=033311
OR=25CI
=370ndash8570
Caucasia
n(161)
Breastcancer
survivalandNRF
2proteinexpressio
n(2012[27])
AA
OR=4656CI
=13
5ndash1606
FinlandKB
CP(452)
Acutelun
ginjury-related
mortality
followingsyste
micinflammatory
respon
sesynd
rome(2012
[37])
GG
OR=97
3CI
=12
7ndash7480
Caucasia
n(750)
Infection-indu
cedasthma(
2012
[38])
AC
OR=0437CI
=028ndash0
80
Hun
garia
nCaucasia
nGypsy
(307)
rs143406266
g178129391178
129393delGGC
Exon
1(467ndash4
69G
CC)
COPD
(2010[32])
GCC
4=053Dagger
hazard
ratio
=095C
I=091ndash0
99
Taiwanese(69)
rs2886161
g178127839Tgt
ClowastIntro
n1
Parkinsonrsquos
disease(2010
[26])
T=0878309Dagger
inhaplotype
OR=09or
04CI
=06ndash
14or
03ndash06
Swedish
Polish
Caucasian
(357)
rs2364723
g178126546Ggt
CIntro
n1
Basaland
smoker
FEV
1(200
9[39])
CI=minus6360simminus1780C
=0525Dagger
Also
ashaplotype
Netherla
nd(2542)
rs2364722
g178124787Agt
GIntro
n1
Ann
ualF
EV1decline
(2011[34])
A=0082in
haplotype
Japanese
(915)
rs13001694
g178118990Agt
GIntro
n1
Basaland
smoker
FEV
1(200
9[39])
G=040
11578Dagger
inhaplotype
Netherla
nd(2542)
Oxidative Medicine and Cellular Longevity 13
Table4Con
tinued
IDMap
onchromosom
e2 (H
GVS
name)
Region
class
Dise
asea
ssociatio
nandreferences
Risk
allelesa
ndstatistics
Ethn
icgrou
p(num
bero
fcase)
Breastcancer
[40]
Twith
NQO1N
OS3H
O1risk
allelesOR=15
6CI
=097ndash251
Caucasia
nandothers(505)
rs1806
649
(rs58745895)
g178118152Cgt
TIntro
n1
Basaland
smoker
FEV
1(200
9[39])
T=02631119Daggerin
haplotype
CI=minus8730ndash
(minus17
0)Netherla
nd(2542)
Parkinsonrsquos
disease(2010
[26])
T=0422148Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
Particulatem
attera
ndasthmaCO
PDadmiss
ion(2012[41])
Cwith
lowvitamin
Clevel(OR=
31CI
=15
0ndash630)
UK(209)
rs4243387
(rs6003846
4)g178117765Cgt
TIntro
n1
Basaland
smoker
FEV
1(200
9[39])
T=00914
25Daggerin
haplotype
Netherla
nd(2542)
rs1962142
(rs58448508)
g178113484Agt
GIntro
n1
Ann
ualF
EV1decline
(2011[34])
A=0082in
haplotype
Japanese
(915)
Breastcancer
NRF
2andARE
expressio
n(2012[27])
A(119875=0036)
FinlandKB
CP(452)
rs6726395
(rs57309289)
g178103229Agt
GIntro
n1
Smok
ing-relatedFE
V1decline
and
annu
alFE
V1decline
(2011[34])
G=0884Dagger
A=0082in
haplotype
Japanese
(915)
Basaland
smoker
FEV
1(200
9[39])
G=046
41764Daggerin
haplotype
Netherla
nd(2542)
rs2001350
(rs17515179
rs60883775)
g178100
425Cgt
TIntro
n1
Ann
ualF
EV1decline
(2011[34])
T=0082in
haplotype
Japanese
(915)
Parkinsonrsquos
disease(2010
[26])
T=0986350Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
rs10183914
(rs58731187
rs61374844
)g17809766
6Cgt
TIntro
n3
Parkinsonrsquos
disease(2010
[26])
T=0536188Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
rs2706110
g178092162Tgt
C31015840Flanking
Breastcancer
(2012[27])
TT
OR=2079CI
=118ndash368
FinlandKB
CP(452)
rs2588882
g178087165Ggt
T31015840Flanking
Infection-indu
cedasthma(
2012
[38])
TG
OR=0290CI
=013ndash0
62
Hun
garia
nCaucasia
nGypsy
(307)
minus686in
reference[22]
=minus653in
reference[23]
=currentlyminus214minus684in
reference[22]
=minus651inreference[23]
=currentlyminus212minus651inreference[22]
=minus617in
reference[23]
=currentlyminus178Ch
romosom
econtig(in
tron
31015840flank
ing)
orreversed
(51015840flank
ingprom
oter)a
llelesinbo
ldhave
been
used
inthetextand
TableOR
odds
ratio
CI95confi
denceinterval
14 Oxidative Medicine and Cellular Longevity
minus212GG (98 rs67006649 previously minus684minus651) minus178Callele (73) and GCC
4(53) was predicted to increase
respiratory failure development (hazard ratio = 095 CI091ndash099) in German COPD patients [32] Significant inter-action was also identified between an intronic SNP Gallele (rs6726395 g178103229AgtG 884 frequency) andsmoking status on FEV
1decline relative to the reference
AA allele in the above Japanese cohort [34] Siedlinskiet al [39] reported that the CC genotype of anotherintronic SNP (rs2364723 g178126546GgtC) was associatedwith a lower FEV
1level compared to the wild-type geno-
type (GG) in two Netherland cohorts (CI minus636minus178frequency = 0525 and pooled cohort size = 2542) ThisSNP alone or as a haplotype with 4 more intronic SNPs(rs13001694Grs1806649Trs4243387Trs6726395G)was alsoassociated with high FEV
1levels in individuals that ever
smoked [39] In a Hungarian population of childhoodasthma SNPs at minus178 (CA) and 31015840 flanking (rs2588882TG)loci were inversely associated with infection-induced asthma(OR 0437 CI 028ndash080 OR 0290 CI 013ndash062 resp) andthese SNPs significantly influenced an asthma-environmentalpollution interaction [38]The intronic SNP rs1806649 (CgtT)was associated but not significantly with an increased risk ofhospitalization during high-level particulate matter (PM
10)
periods in asthma or COPD patients (119899 = 209) of the UnitedKingdom (UK) [41] Asthma andCOPDadmission rateswererelated to the increase in environmental PM
10concentration
Importantly effects of interaction between prenatal stressand NRF2 SNPs on descendant pulmonary health wereinvestigated by the Avalon Longitudinal Study in the UKmaternal smoking during pregnancy was not associated withlung function change determined by maximum mild expi-ratory flow (FEF
25ndash75) or with asthma incidence in school-aged children and this relation was not modified by NRF2SNP genotypes [42]However early gestation acetaminophenexposure significantly influenced the risk of asthma andwheezing at the age of 7 years in gt4000 mothers and gt5000children [33] When maternal copies of the minus212A allele werepresent association with asthma (11374891 OR 173 CI122ndash245) and wheezing (11494949 OR 153 95 CI 106ndash220) was significantly increased [33]
Gastrointestinal Disorders While there was no evidence inlung cancer cases studies in Japanese populations suggesteda potential association of NRF2 variations with gastro-intestinal tumorigenesisHelicobacter pylori (H pylori) causesgastritis which can lead to gastric atrophy and cancer Ingastric epithelium from the Japanese cancer cohorts (39gastric cancers 46 controls) H pylori infection was posi-tively correlated with aberrant CpG island methylation oftumor suppressor genes (eg p14) and minus214Gminus212G orminus214Aminus212G NRF2 haplotype was significantly associatedwith increased (OR 290 95 CI 114ndash736) or decreased(OR 033 95 CI 013ndash088) risk of the CpG methyla-tion respectively in the H pylori-infected patients [29]Further study from the same investigators determined thatminus214Aminus212G allele carriers had significantly (119875 = 0022)reduced risk of gastric cancer inH pylori-negative cases [30]The minus214AGminus212AG genotypes were negatively associated
(OR 045 CI 022ndash093) and the minus214Gminus212G genotypeswere positively associated (chronic continuous phenotypeOR 257 CI 101ndash660) with ulcerative colitis (89 patients 141controls) in a Japanese population [31]
Autoimmune Disorders Systemic lupus erythematosus (SLE)is a long-term autoimmune disease more frequently found infemales than in males It affects organs including skin jointskidneys and brain and nephritis is an aggressive character-istic in some patients Genome-wide association studies inhumans identified a suggestive quantitative trait locus nearNRF2 [43] A study of a Mexican Mestizo population (362patients with childhood-onset SLE 379 controls and 212nephritis diagnosed) determined that lupus with nephritiswas significantly (OR 181 CI 104ndash312) associated with theminus214GA SNP in females [25] The same SNPs were notclosely associated with SLE risk in a Japanese cohort [22]Vitiligo is a skin condition in which there is a loss of browncolor (pigment) from areas of skin resulting in irregularwhite patches It is thought to be an autoimmune diseasecaused by loss of cells (melanocytes) that produce brownpigment A study indicated that theminus178A allele increased therisk of vitiligo dose-dependently (OR 1724 95 CI 135ndash221for CA OR 2902 CI 162ndash519 for AA) [35]
Female Disorders It is well known that estrogen metabolites(eg catechols) cause ROS formation suggesting correla-tion of NRF2 and downstream effectors in postmenopausalmammary cancer In a study of a Finish population (KuopioBreast Cancer Project 119899 = 452 patients 370 controls)the minus178AA homozygous genotype (OR 4656 CI = 135ndash1606) and 31015840 flanking rs2706110 (TT OR 2079 CI 118ndash368) genotype were associated with increased risk of breastcancer while the 51015840 flanking minus3306TT homozygous allelewas significantly associated with lower survival (frequency =71219 OR 1687 CI 1105ndash275) [27] suggesting that NRF2genetic polymorphisms affect susceptibility and outcome ofthe patients The minus178A allele carriers together with intronicrs1962142A allele carriers were associated with lowered tissuelevels of NRF2 proteins [27] In postmenopausal women theminus178A allele (OR 179 95CI 370ndash8570) appeared tomodifythe risk of venous thromboembolism caused by oral estrogentherapy (AA or AC frequency = 333) as demonstratedby the French ESTHER study (161 cases 474 controls) [36]An intronic rs1806649CgtT SNP did not associate with breastcancer risk in postmenopausal women [40] However whenthis SNP and other at-risk alleles of ARE-responsive genes(NQO1 HO-1 NOS3) were combined there was a significantgene-dose effect on the breast cancer risk [40] Althoughcoding region SNPs in NRF2 and KEAP1 were identifiedin the Japanese endometrial adenocarcinoma patients noassociation of NRF2 SNPs with the disease was found [44]
Neurodegenerative Diseases Oxidative stress is known to beinvolved in Parkinsonrsquos disease (PD) presumably due toproduction of ROS fromhigh-dopaminemetabolism and lowlevels of antioxidants in the substantia nigra of the brainInvestigators found a protective NRF2 haplotype consistingof four 51015840 flanking SNPs (minus5238Gminus214Aminus212Gminus178C)
Oxidative Medicine and Cellular Longevity 15
and 4 intronic SNPs (rs2886161Ars1806649Ars2001350Ars10183914A) from Swedish (OR 09 CI 060ndash140) and Polish(OR 04 CI 030ndash060) populations (total 165+192 PD cases190+192 controls) [26]The investigators also suggested thatNRF2 haplotype alleles were associated with 2 years earlierage of Alzheimerrsquos disease (AD) onset 4 years earlier age ofposterior subcapsular cataract surgery and 4 years later ageof cortical cataract surgery while they were not significantlyrelated to AD or age-related cataract risk [45]
34 Genetic Mutations in Mouse Nrf2 Tsang et al [46]compiled 673 SNPs in 55 mouse strains and constructedtheir phylogenetic tree to correlate and clarify the originsof strains based on the assembled mouse genome sequenceand SNP data [20 47 48] Recently using the completegenome sequence of C57BL6J (B6) mouse as a referencehigh-density SNP screening in other laboratory strains orin panels of strains has been published (see [17]) Althoughmillions of mouse SNPs (gt10089892 as of December 2012)and haplotype mappings from more than 120 strains havebeen published as valuable references for dissecting thegenetic basis of complex traits [49ndash51] little attention hasbeen paid to polymorphisms of Nrf2 and their correlationwith disease phenotypes
Figure 3 demonstrates the proximal promoter region(minus1 to minus950)51015840-UTR (exon 1 up to TSS) and proteinsequence of mouse Nrf2 based on GRCm38p1 PrimaryAssembly (75704641ndash75675513 bp) mRNA variant1 and protein (NP 035032 encoded by NM 010902234ndash2027 bp) sequences Genetic variations in the Nrf2genome of inbred strains collected from public databasesare listed in Tables S2 and 5 (See Supplementary TableS2) Overall 968 genetic mutations are compiled forNrf2 gene and 5 kb upstream2 kb downstream regions785 SNPs between B6 and another 16 strains wereacquired from the Mouse Phenome Database (MPDhttpphenomejaxorg dbqrtn=snpret1) and additionalSNPs and other mutations were acquired from NCBI dbSNP(httpwwwncbinlmnihgovsnpterm=mus+musculus20nfe2l2) In total 132 mutations are in the promoter (37in proximal 1 kb) 49 in exons (38 in coding region 19 Cns)727 in introns and 60 in the 31015840 flanking region Excludingmutations in the 51015840 and 31015840 flanking sequences murine Nrf2sequences appear to be more highly variable (1 variationper 375 bp) than much of the mouse genome which has anapproximate frequency of one SNP per every 245 bp (httpwwwinformaticsjaxorgmgihomehomepagesstatsallstatsshtmlallstats snp)
Nrf2 was found to be a susceptibility gene from genome-wide linkage analysis in a murine model of hyperoxia-induced ALI [52] A promoter SNP minus103TgtC (previouslypublished as minus336TgtC) in Nrf2 was found and predicted toadd an additional Sp1 binding site in hyperoxia-susceptibleB6 mice but not in resistant C3HHeJ mice [52] Genotypesfrom the SNP and from simple-sequence length polymor-phism markers of the Nrf2 locus (D2Mit248 and D2Mit94)cosegregated in the B6C3F
2mouse cohort [52] and Nrf2
deficient mice were significantly more susceptible to ALI
sub-phenotypes caused by hyperoxia than similarly exposedwild-type mice supporting Nrf2 as a contributor to thephenotypic traits [53] Although no other functional analyseson Nrf2 SNPs or haplotype association studies have beenconducted in inbred mice strains bearing haplotypes suchas multiple Cns in functional domains (eg F71L L451VH543Q and L575M) may be useful to elucidate the role ofNrf2 in differential susceptibility to oxidative diseases
4 Oncogenic Somatic Mutations in HumanNF-E2-Related Factor 2
Somatic mutation is a change in the DNA of somatic cellsthat affects derived cells but is not inherited by offspringEfforts to discover somatic mutations have provided insightinto mutagenesis and cancer development Lung cancerparticularly non-small cell lung cancer (NSCLC) is theleading cause of cancer death worldwide Somatic mutationsof NRF2 and KEAP1 discovered in lung cancer patients havedetermined the oncogenic potential ofNRF2 [54 55] KEAP1somatic mutations were associated with its reduced proteinlevels in lung cancer tissues and cells [56 57] Investigationsof NSCLC in various ethnic populations as well as cancersin gastrointestine breast and prostate have coordinatelydemonstrated that multiple Cns somatic mutations in KEAP1cause dysfunction of the translated protein and in turnconstitutive activation of NRF2 increasing risk of neoplasiaand chemoresistance [12 55 58 59] Somatic mutations ofNRF2 have been detected in various cancer tissues (largelysquamous cell carcinomas) in Asian populations (Table 6)NRF2 mutations were significantly associated with NSCLCcases (squamous cell lung carcinoma adenocarcinoma) ofthe Japanese (107 [54]) the Chinese (23 [60]) andthe Koreans (8 [61]) as well as with lung cancer celllines Smoking history was also correlated with mutationoccurrence in all of the studies [54 60 61] In addition tolung cancers laryngeal squamous carcinoma (13 in [61])esophageal squamous cancer (ESC 22 in [60] 114 in[61]) head and neck cancers (25 in [54]) skin (117 casein [61]) and oral cancer cell lines had somatic changesin NRF2 In contrast to wide-spread KEAP1 mutationsmutations in NRF2 were clustered in DLGETGE motifs ofthe Neh2 domain which are critical in the ldquohinge and latchrdquomodel of KEAP1 binding [12] Similar to KEAP1 somaticmutations it has been postulated that NRF2 mutations incancer cells lead to NRF2 accumulation by suppressing itsubiquitination or KEAP1 binding which eventually confersmalignant potential and resistance to chemotherapy
Most variable sites in NRF2 included aa residues 29 (AspD) 31 (Gly G) 77 (Asp D) and 79 (Glu E) (Table 6)Residue 33 (Ser S) in the Neh2 domain is mutated by eithergenetic or somatic processes (Figure 4) Cns in the EDGFmotif of NRF2 was experimentally determined to impairrecognition of KEAP1 [54] NRF2 mutations were signif-icantly correlated with increased (25-fold) copy number(31 of mutants versus 3 wild types) in Japanese NSCLCcases [63] Aberrant mutation of NRF2 also led to increasedexpression of downstream effectors includingRagD known to
16 Oxidative Medicine and Cellular Longevity
pL575M
pF71L
pH543QpL451V
pA151P pA151V pN159D pQ198delpV105D
pA204T pS224I225S226del pT253S pS278ApD305G
pM409V pR410HpP388L pA389G pT395I
Genomic DNA (reversed contig GRCm38 p1 C57BL6JNM 0109023)
Protein (NP 035032)1
51101151201251301351401451501551
minus400
minus500
minus50
minus100
minus200
minus300
minus450
minus350
minus250
minus150
minus950
minus900
minus850
minus800
minus750
minus700
151
101151201
2001
g minus470gg minusg minus
c minus41c minus
c
c
c c
gg minus 6C
g minus125insTTC g minus118insC
g minus281delC
g minus63delCg minus1 (75704642 c minus234)
Translation start site (c +1)Termination codon (c +2025 +2027)
c minus136 minus134in-frame stop codonc minus8585
g minus41
246
440
minus381
minus11
minus91 minus90
minus332
minus153
minus98
c minus197c minus209 minus202
gminus942 minus899 (cminus1175 minus1132) CpG islands [71]
g 460CgtT
g minus103CCgtT [51]
CgtTCgtG
g minus320CgtT
CgtT
CgtT
CgtTg minus426GgtA
GgtACgtA
CgtA
GgtA
g minus238GgtTTgtC
TgtC
TgtC
TgtCTgtG
g minus229AgtG
CgtG
CgtG
GgtC
gminus826 minus654 (cminus1059 ) CpG islands [71]minus887
Figure 3 DNA (partial promoter and exons 1 and 5) and protein sequence ofmouseNrf2 SNPs and amino acid residues for non-synonymousSNPs are marked Promoter regions bearing 5CpG islands are underlined
151
201251301351401451501551601
Genetic mutation (Table 3 and Figure 2)Somatic mutation (Table 6)
Neh2
Neh3
Neh4Neh5
Neh6
Neh1
Protein isoform 1 (NP 006155)
101151
Figure 4 Genetic and somatic mutation loci in human NRF2 protein
Oxidative Medicine and Cellular Longevity 17
Table5Mou
seup
stream
andexon
varia
tions
ofNfe2
l2locusin17
inbred
strainsR
eference
sequ
ence
isC5
7BL6J
(B6strain
1)geno
me(GI14933824975547698ndash75513576)SNPalleleand
geno
typesareshow
nas
chromosom
econ
tigsequ
enceStrains
2129S1S
vlmJ3AJ
4AKR
J5BA
LBcJ6C3
HH
eJ7C
57BL
6NJ8CA
STEiJ9CB
AJ
10D
BA2J11FVBNJ12LPJ
13N
ODShiLtJ14N
ZOH
ILtJ
15P
WK
PhJ16SPR
ETEiJ
17W
SBEiJ
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs2566
08517
75705565
GA
(CT)
51015840Flankingminus924
NC
GG
GG
GG
GG
GG
GG
GG
AA
Grs47274959
75705562
AT
(TA
)51015840Flankingminus921
NC
AT
TA
TT
AA
TT
AT
AT
TT
Ars216940398
75705554ndash75705555
Tadd(A
add)
51015840Flankingminus913minus914
NC
CGrs239114134
75705540
CT(G
A)
51015840Flankingminus899
NC
CT
TC
TT
CC
TT
CT
CT
CT
Crs46
461765
75705528
TG(A
C)oA
C
51015840Flankingminus887
NC
TG
GT
GG
TT
GG
TG
TG
GG
Trs5144
9853
75705525
CA(G
T)
51015840Flankingminus884
NC
CA
AC
AA
CC
AA
CA
CA
CC
Crs249093111
75705512
CT(G
A)
51015840Flankingminus871
NC
CT
TC
TT
CC
TT
CT
CT
TT
Crs263745200
75705510
CG(G
C)
51015840Flankingminus869
NC
GG
GG
GG
GG
GG
GG
GG
CG
Grs45651867
75705498
TG(A
C)
51015840Flankingminus857
NC
TG
GT
GG
TT
GG
TG
TG
GG
Trs219997531
75705495
GA
(CT)
51015840Flankingminus854
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs251918379
75705451
TA(A
T)
51015840Flankingminus810
NC
TT
TT
TT
TT
TT
TT
TT
TA
Trs27978306
75705449
TA(A
T)
51015840Flankingminus808
NC
TA
AT
AA
TT
AA
TA
TA
TT
Trs216087412
75705429
TG(A
C)lowast
51015840Flankingminus788
NC
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
GTlowast
Tlowast
rs261229914
75705423ndash75705424
AGdel(CT
del)
51015840Flankingminus783minus784
NC
AGrs27978307
75705400
CT(G
A)
51015840Flankingminus759
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs243167395
75705359
GA
(CT)
51015840Flankingminus718
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs27978308
75705307
GA
(CT)
51015840Flankingminus66
6NC
GG
GG
GG
GG
GG
GG
GG
AG
Grs254744
09875705294
AT
(TA
)51015840Flankingminus653
NC
AA
AA
AA
AA
AA
AA
AA
AT
Ars228133419
75705179
GT
(CA
)51015840Flankingminus538
NC
GG
GG
GG
GG
GG
GG
GG
GT
Grs214719520
75705111
GA
(CT)
51015840Flankingminus470
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs2440
87730
75705101
GA
(CT)
51015840Flankingminus46
0NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs227781699
75705081
CG(G
C)
51015840Flankingminus44
0NC
CC
CC
CC
CC
CC
CC
CC
GC
Crs257870353
75705067
CT(G
A)
51015840Flankingminus426
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs24487744
075705022
AG
(TC)
51015840Flankingminus381
NC
AA
AA
AA
AA
AA
AA
AA
AG
Ars234628138
75704973
AC
(TG
)51015840Flankingminus332
NC
AA
AA
AA
AA
AA
AA
AA
AC
A
18 Oxidative Medicine and Cellular Longevity
Table5Con
tinued
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs247275247
75704961
GA
(CT)
51015840Flankingminus320
NC
GG
GG
GG
GG
GG
GG
AG
GG
Grs247519047
75704922
Gdel(Cdel)
51015840Flankingminus281
NC
Grs27978309
(rs51915758)
75704887
GA
C(C
TG
)51015840Flankingminus246
NC
GA
AG
AA
GG
AA
GA
GA
CC
G
rs218139102
75704879
CA(G
T)
51015840Flankingminus238
NC
CC
CC
CC
CC
CC
CC
CC
AC
Crs251990355
75704870
TC(A
G)
51015840Flankingminus229
NC
TT
TT
TT
TT
TT
TT
TT
TC
Trs238746955
75704794
AG
(TC)
51015840Flankingminus153
NC
AA
AA
AA
AA
AA
AA
AA
GA
Ars221664
405
75704786
AG
(TC)
51015840Flankingminus145
NC
AA
AA
AA
AA
AA
AA
AA
AG
Ars217054035
75704766
ndash75704767
GAAadd(TTC
add)
51015840Flankingminus125minus126
NC
GA
rs248182931
75704759ndash75704760
Gadd(C
add)
51015840Flankingminus118
minus119
NC
AGrs217197904
75704744
GA
(CT)
51015840Flankingminus103
(+)S
p1[23]
GA
AG
AA
GG
AA
GA
AA
GG
Ars2644
9364975704704
Gdel(Cdel)
51015840Flankingminus63
NC
Grs2404
81112
757046
82CT(G
A)
51015840Flankingminus41
NC
CC
CC
CC
CC
CC
CC
CC
CT
Crs27978310
75704617
AG
(TC)
UTR
-525
NC
AA
AA
AA
AA
AA
AA
AA
GG
Ars27978311
75704610
CG(G
C)
UTR
-532
NC
GG
GG
GG
GG
GG
GG
GG
GG
Crs27978312
757046
05GA
(CT)
UTR
-537
NC
GA
AG
AA
GG
AA
GA
GA
GG
Grs214784220
757044
99AG
(TC)
UTR
-5143
NC
AA
AA
AA
AA
AA
AA
AA
GG
Ars244146318
757044
98GT
(CA
)UTR
-5144
NC
GG
GG
GG
GG
GG
GG
GG
GT
Grs27978313
757044
97GC
(CG
)UTR
-5145
NC
GC
CG
CC
GC
CC
GC
CC
CC
Crs215431944
757044
93CT(G
A)
UTR
-5149
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs257015697
757044
49GA
(CT)
UTR
-5193
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs252234782
757044
19GT
(CA
)UTR
-5223
NC
Grs1346
0861
75679262
AG
(TC)
Exon
244
6Cn
sF71L
AG
GA
GG
AG
GG
AG
AG
GG
Grs1346
0859
75679202
GA
(CT)lowast
Exon
2506
CsH91=
GA
AG
AA
GG
AA
GA
GA
GG
Grs27978436
75679187
GA
(CT)lowast
Exon
2521
CsT9
6=G
GG
GG
GG
AG
GG
GG
GA
GG
rs226131070
75679178
GA
(CT)
Exon
2530
CsS99=
GG
GG
GG
GG
GG
GG
GG
GA
Grs227683834
75678576
AT
(TA
)Ex
on3
547
CnsV
105D
AA
AA
AA
AA
AA
AA
AA
AT
Ars257321187
75678506
TC(A
G)
Exon
3617
CsP128=
TT
TT
TT
TC
TT
TT
TT
TT
Trs27978444
75678500
TA(A
T)
Exon
3623
CsV130=
TT
TT
TT
TA
TT
TT
TT
AT
Trs227743136
75677686
GA
(CT)
Exon
4662
CsH143=
GG
GG
GG
GG
GG
GG
GG
GA
Grs215327202
75677664
CG(G
C)
Exon
4684
CnsA
151P
CC
CC
CC
CG
CC
CC
CC
CC
Crs247539755
75677663
GA
(CT)
Exon
4685
CnsA
151V
GG
GG
GG
GA
GG
GG
GG
GG
Grs233164
668
75677640
TC(A
G)
Exon
4708
CnsN
159D
TT
TT
TT
TT
TT
TT
TT
TC
T
rs2340
95816
75677162ndash75677160
GCT
del
(AGCdel)
Exon
5826minus
828
Cds-in
delQ
198
GCT
Oxidative Medicine and Cellular Longevity 19
Table5Con
tinued
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs27978452
75677145
CT(G
A)
Exon
5843
CnsA
204T
CC
CC
CC
CC
CC
CC
CC
CC
T
rs225047937
75677086ndash75677078
GAG
ATCG
ATdel
(ATG
CATC
TCdel)
Exon
5902minus
910
Cds-in
del
(S224I225S226)
GAG
ATC
GAT
rs241469868
75676998
TA(G
T)
Exon
5990
CnT2
53S
TT
TT
TT
TT
TT
TT
TT
TA
Trs221602571
75676923
AC
(TG
)Ex
on5
1065
CnsS
278A
AA
AA
AA
AA
AA
AA
AA
AC
Ars252576472
75676876
GA
(CT)
Exon
51112
CsS293=
GG
GG
GG
GG
GG
GG
GG
GA
Grs250802933
75676841
TC(A
G)
Exon
5114
7Cn
sD305G
TT
TT
TT
TT
TT
TT
TT
TC
Trs225425698
75676792
CT(G
A)
Exon
5119
6Cs
P321=
CC
CC
CC
CC
CC
CC
CC
CT
Crs13459064
75676732
CT(G
A)
Exon
51256
CsT3
41=
CT
TC
TT
CT
TT
CT
CT
TC
Crs27978453
75676717
CT(G
A)
Exon
51271
CsA346=
CC
CC
CC
CC
CC
CC
CC
TC
Crs214335034
75676636
AG
(TC)
Exon
51352
CsD373=
AA
AA
AA
AA
AA
AA
AA
AG
Ars24958364
4756766
03AC
(TG
)Ex
on5
1385
CsP3
84=
AA
AA
AA
AA
AA
AA
AA
AC
Ars27978454
75676592
GA
(CT)
Exon
51396
CnsP
388L
GG
GG
GG
GG
GG
GG
GG
AG
Grs215384328
75676589
GC
(CG
)Ex
on5
1399
CnsA
389G
GG
GG
GG
GG
GG
GG
GG
GC
Grs251728286
75676571
GA
(CT)
Exon
51417
CnsT
395I
Grs247602334
75676567
TC(A
G)
Exon
51421
CsV396=
TT
TT
TT
TT
TT
TT
TT
TC
Trs234216231
75676530
TC(A
G)
Exon
51458
CnsM
409V
TT
TT
TT
TT
TT
TT
TT
TC
Trs212904337
75676526
CT(G
A)
Exon
51462
CnsR
410H
CC
CC
CC
CC
CC
CC
CC
CT
Crs252650779
75676522
TC(A
G)
Exon
51466
CsE4
11=
TT
TT
TT
TT
TT
TT
TT
TC
Trs231273560
75676516
TC(A
G)
Exon
51472
CsQ413=
TT
TT
TT
TT
TT
TT
TT
TC
Trs257886949
756764
13GT
(CA
)Ex
on5
1575
CsR4
48=
GG
GG
GG
GG
GG
GG
GG
GT
Grs241537608
756764
04GC
(CG
)Ex
on5
1584
CnsL
451V
GG
GG
GG
GG
GG
GG
GG
GC
Grs227619071
75676312
TC(A
G)
Exon
51676
CsQ481=
TT
TT
TT
TT
TT
TT
TT
TC
Trs258913831
75676290
AG
(TC)
Exon
51698
CsL4
89=
AA
AA
AA
AA
AA
AA
AA
AG
Ars225593319
75676126
AT
(TA
)Ex
on5
1862
CnsH
543Q
Ars4223233
75676105
GA
(CT)
Exon
51883
CsS550=
GG
GG
GG
GA
GG
GG
GG
GG
Grs4223232
75676032
GT
(CA
)Ex
on5
1956
CnsL
575M
Grs4223231
75675816
CG
UTR
-32172
NC
GG
GG
GG
GG
GG
GG
GG
GC
Grs1346
860
75675682
AT
UTR
-32306
NC
ASequ
ence
varia
tions
inmou
seNr
f2wereob
tained
from
Mou
sePh
enom
eDatabase(http
ph
enom
ejaxorgSNP)
andNCB
ISNPdatabase
(http
wwwncbinlm
nihgovSNP)N
CBIreference
sequ
ence
isMus
Musculusstra
inC5
7BL6J
chromosom
e2GRC
m38p1(NC
0000
687
GI37209910875704641ndash756755192
9123
bp)To
tal968
genetic
mutations
arerepo
rted
inNr
f2gene
and5k
bup
stream
(ge75704
642minus
1)and2k
bdo
wnstre
am(le75675512)sequences
asof
Janu
ary2003A
llSN
Psandallelesa
representedas
geno
miccontig
(reversedsequ
ence
indicated)E
xon175704
641ndash75704364(278
bpT
TS=75704
408)
exon
275679431ndash756791
65(267
bp)exon
375678579ndash75678490(90b
p)exon475677713ndash756775
46(168
bp)andexon
57567718
4ndash75678849
(1666
bp)Proteinam
inoacid
(aa)
resid
uesinNP00
61552
(597
aa)Cs
cod
ing-syno
nymou
sCn
scoding
-non
syno
nymou
sAminoacid
A-alanineD-aspartic
acidE
-glutamicacidF-phenylalanineG
-glycineH
-histidineI-iso
leucineL-leu
cineand
M-m
ethion
ineN-
asparagineP-prolin
eQ-glutamineR-arginineS-serineT-threon
ineandV-valin
elowastErrorsin
databasesfi
xedNC
notcon
firmed
20 Oxidative Medicine and Cellular Longevity
Table 6 NRF2 somatic mutations revealed in various human cancers
Domain Amino acid residues DNA mutation Cancer types (cases) ReferencesLocus Wild type Mutant
DLG motif
24 W (Trp) C (Cys) c72GgtCGgtT NSCLC neck ESC [54 62]K (Lys) c72TgtC ESC [62]
26 Q (Gln) E (Glu) c76CgtG NSCLC ESC [54 62]
27 D (Asp) G (Gly)lowast c80GgtA NSCLC [60]Y (Tyr) c79GgtT ESC [61]
28 I (Ile) T (Thr) c83CgtT NSCLC [54]
29 D (Asp) G (Gly) c86AgtG Head and neck ESC [54 62]H (His) c85GgtC NSCLC laynx [60 61]
30 L (Leu) F (Phe) c88CgtT NSCLC ESC [54 62]
31 G (Gly) A (Ala) c92GgtC NSCLC ESC skin [54 60ndash62]
32 V (Val) T (Thr) c95TgtG NSCLC [54]del c93 95delAGT ESC [61]
33ndash36 S-R-E-V S-R-E-V-S-R-E-Vlowast c97 108dupAGTCGAGCCGTA ESC [61]
34 R (Arg) Q (Gln) c101GgtA NCSCL [54 60 61]P (Pro) c101GgtC NSCLC [63]
ETGF motif
75 Q (Gln) H (His) c225AgtC Head and neck ESC [54 62]
77 D (Asp)
V (Val) c230AgtT NSCLC ESC [54 60 62]G (Gly) c230AgtG ESC [62]A (Ala) c230AgtC NSCLC [61]N (Asn) c229GgtA Larynx [61]
78 E (Glu) K (Lys) c232GgtA NSCLC ESC [54 62]
79 E (Glu)
K (Lys) c235GgtA NSCLC ESC [54 61 62]Q (Gln) c235GgtC NSCLC ESC [54 60ndash62]G (Gly) c236AgtG Larynx [61]E-E c234 236dupAGA ESC [61]
80 T (Thr) K (Lys) c239CgtACgtG NSCLC ESC [54 61 62]
80 T (Thr)P (Phe) c238AgtC ESC [62]I (Ile) c239CgtT Head and neck [54]A (Ala) c238AgtG NSCLC [63]
81 G (Gly) V (Val) c242GgtT ESC [61]D (Asp) c242GgtA NSCLC ESC [60ndash62]
82 E (Glu)
D (Asp) c246AgtT ESC oral cancer cell line [54 62]G (Gly) c245AgtG NSCLC [54]Q (Gln)lowast c244GgtC NSCLC ESC [60 61]V (Val)lowast c245AgtT ESC [61]
83 F (Phe) L (Leu)lowast c247TgtC NSCLC [60]NSCLC non-small cell lung cancer ESC esophageal squamous cancer and lowasterrors in reference fixed Number of cases 82 NSCLC and 10 ESC in [62] 125NSCLC 70 ESC 23 larynx and 17 skin in [61] 103 NSCLC and 12 head and neck in [54] 90 NSCLC in [63] 103 NSCLC in [60]
be involved in squamous lung cancer cell proliferation [64]suggesting that the mutation is functional and overcomesKEAP1 inhibition Singh et al [65] determined in vitrothat RNAi-mediated depletion of NRF2 in lung cancer cellsenhanced ROS production and susceptibility to cell deathby ionizing radiation These studies support the concept thatelevated NRF2 and ARE responsiveness provides cancer cellswith proliferative advantage for malignant transformation
and undue protection from anti-cancer therapy Onco-genic epidermal growth factor receptor (EGFR) signaling isrecently found to be critical in NRF2-mediated proliferationof NSCLC cells [66]
Collectively ldquogain of functionrdquo mutations in NRF2 thatreduce KEAP1 recognition are suggested to be predictivemarkers for poor responsiveness to chemotherapy and radi-ation therapy Although NRF2-mediated cellular defense
Oxidative Medicine and Cellular Longevity 21
processes are essential in the initiation stage enhancedNRF2-ARE activity in advanced stages of cancer developmentmay create a favorable intracellular environment for tumorcell growth and survival [67 68] In this context NRF2may be a potential molecular target for the treatment ofradio-resistance cancers especially those that have ldquoloss offunctionrdquo mutations in EGFR KRAS or KEAP1 as well asldquogain of functionrdquo mutations in NRF2
5 Epigenetic Alterations of NF-E2-RelatedFactor 2
Epigenetic modifications are alterations of molecules inter-acting with genes without changes to the primary DNAsequence They include post-translational modification ofhistones DNA methylation events chromatin conforma-tional changes and alterations to noncoding regulatoryRNAs Epigenetic alterations are stable and often inheritablebut are reversible and may affect expression of the geneDysregulation or defects in epigenetic processes particularlyhypermethylation of tumor suppressor gene promoters (egCpG islands) or histone modifications are thought to beassociated with carcinogenesis Investigators have reportedhypermethylation in CpG islands of KEAP1 which wereassociated with reduced KEAP1 expression in human cancersfrom lung prostate colon and so forth [69ndash72] Similar tosomatic mutations epigenetic changes on KEAP1 impairedthe function of its encoded protein leading to constitutiveNRF2 activation
Supporting the role of ldquopathogenic mutationsrdquo in NRF2expression of Nrf2 and downstream Nqo1 was suppressedin prostate tumors of mice (transgenic adenocarcinomaof mouse prostate TRAMP) Among 15 promoter CpGislands located between minus942 and minus654 (cminus1175 cminus1132and cminus1059 cminus887 gap in cminus1131 cminus1060 see Figure 3)hypermethylation of the first 5 CpG islands (minus942 minus899 andcminus1175 cminus1132) was significantly associated with tumorige-nesis [73] Moreover treatment with inhibitors for DNAmethyltransferase and histone deacetylase restored Nrf2expression in these tumor cells [73] A dietary phytochemicalcurcumin known as a DNA hypomethylation agent restoredepigenetically silent Nrf2 expression through CpG demethy-lation in carcinogen-induced mouse tumor cells [74]
The whole genome epigenetic datasets for 5 species arepublicly accessible at NCBI Epigenomics [75 76]The humanNRF2 epigenome of primary cells (breast penis) and H1stem cell line as well as mouse Nrf2 CpG island methylationdata for sperm blood and cerebellum are currently avail-able (httpwwwncbinlmnihgovepigenomics) Althoughno direct evidence of disease-associated epigenetic modu-lation has been identified in human NRF2 various phyto-chemical NRF2 agonists such as sulforaphane and curcuminhave shown their roles in DNA methylation and histonemodification (see reviews by Lee and colleagues eg [77])Taken together epigenetic modifications of theNRF2KEAP1axle are predicted to cause dysregulation of ARE-mediatedcellular defense leading to deleterious health effects and
phytochemical antioxidants as epigenetic modulators forNRF2 are suggested to be useful in cancer prevention
6 Conclusions
NRF2 is evolutionally conserved with high-sequence homol-ogy in many species However it is a highly mutable geneand numerous genetic variants have been discovered inhuman ethnic groups Importantly certain SNPs or haplo-types have been identified in various diseases as ldquoat-riskrdquoalleles and are related to functional alterations In additionto genetic variations multiple somatic mutations identifiedin the KEAP1 recognition domain of NRF2 in cancer cellshave been found to be oncogenic due to dysregulation ofNRF2 homeostasis by its excess ldquogain of functionrdquo Epigeneticalteration of theNRF2 is under investigation and is predictedto have pathogenic influences as learned from mouse andphytochemical agonist studies Continuous updates of Nrf2allelic variants in inbred mouse strains will provide a usefultool for effective experimental designs formodels of oxidativedisorders to provide insight into the disease mechanisms andintervention strategies
Abbreviations
AD Alzheimerrsquos diseaseALI acute lung injuryARE antioxidant response elementbZIP basic leucine zippercds coding DNA sequencesCI confidence intervalCOPD chronic obstructive pulmonary diseaseESC esophageal squamous cancerFEV1 forced expiratory volume in one second
Gbp giga base pairsGST glutathione S-transferaseGWAS genome-wide association studyHGVS Human Genome Variation SocietyHO-1 heme oxygenase-1119867 119901119910119897119900119903119894 119867119890119897119894119888119900119887119886119888119905119890119903 119901119910119897119900119903119894KEAP1 Kelch-like ECH activating protein 1MPD Mouse Phenome DatabaseMRP multidrug resistance proteinNCBI National Center for Biotechnology InformationNeh NRF2-ECH homologyNfe2l2 nuclear factor (erythroid-derived 2)-like 2Nrf2 NF-E2-related factor 2NSCLC nonsmall cell lung cancerNQO1 NAD(P)Hquinone oxidoreductase 1OR odds ratioPD Parkinsonrsquos diseasePM particulate matterROS reactive oxygen speciesSLE systemic lupus erythematosusSNP single nucleotide polymorphismSOD superoxide dismutaseURT untranslated region
22 Oxidative Medicine and Cellular Longevity
Disclosure
Authorrsquos contribution to the Work was done as part of theAuthorrsquos official duties as a NIH employee and is a Work ofthe United States Government Therefore copyright may notbe established in the United States
Conflict of Interests
The author declares that there is no conflict of interests
Acknowledgments
The research related to this paper was supported by theIntramural Research Program of the National Institutes ofHealth of the National Institute of Environmental HealthSciences (NIEHS) Drs Steven Kleeberger and StephanieLondon at the NIEHS provided excellent critical review ofthis paper The author thanks Mrs Jacqui Marzec for herhelpful comments and English editing
References
[1] J Y Chan M C Cheung P Moi K Chan and Y W KanldquoChromosomal localization of the humanNF-E2 family of bZIPtranscription factors by fluorescence in situ hybridizationrdquoHuman Genetics vol 95 no 3 pp 265ndash269 1995
[2] K Itoh K Igarashi N Hayashi M Nishizawa and MYamamoto ldquoCloning and characterization of a novel erythroidcell-derived CNC family transcription factor heterodimerizingwith the small Maf family proteinsrdquo Molecular and CellularBiology vol 15 no 8 pp 4184ndash4193 1995
[3] W O Osburn and T W Kensler ldquoNRF2 signaling an adaptiveresponse pathway for protection against environmental toxicinsultsrdquoMutation Research vol 659 no 1-2 pp 31ndash39 2008
[4] D Papp K Lenti D Modos D Fazekas Z Dul D Tureiet al ldquoThe NRF2-related interactome and regulome containmultifunctional proteins and fine-tuned autoregulatory loopsrdquoFEBS Letters vol 586 no 13 pp 1795ndash1802 2012
[5] X Wang D J Tomso B N Chorley et al ldquoIdentificationof polymorphic antioxidant response elements in the humangenomerdquo Human Molecular Genetics vol 16 no 10 pp 1188ndash1200 2007
[6] D Malhotra E Portales-Casamar A Singh et al ldquoGlobalmapping of binding sites for NRF2 identifies novel targets incell survival response through ChIP-Seq profiling and networkanalysisrdquo Nucleic Acids Research vol 38 no 17 pp 5718ndash57342010
[7] K Chan R Lu J C Chang and Y W Kan ldquoNRF2 a memberof the NFE2 family of transcription factors is not essential formurine erythropoiesis growth and developmentrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 93 no 24 pp 13943ndash13948 1996
[8] K Itoh T Chiba S Takahashi et al ldquoAn NRF2small Mafheterodimer mediates the induction of phase II detoxifyingenzyme genes through antioxidant response elementsrdquo Bio-chemical and Biophysical Research Communications vol 236no 2 pp 313ndash322 1997
[9] FMartin J M vanDeursen R A Shivdasani CW Jackson AG Troutman and P A Ney ldquoErythroid maturation and globin
gene expression inmicewith combined deficiency ofNF-E2 andNrf-2rdquo Blood vol 91 no 9 pp 3459ndash3466 1998
[10] K Itoh N Wakabayashi Y Katoh et al ldquoKEAP1 repressesnuclear activation of antioxidant responsive elements by NRF2through binding to the amino-terminal Neh2 domainrdquo Genesand Development vol 13 no 1 pp 76ndash86 1999
[11] A Kobayashi M I Kang H Okawa et al ldquoOxidative stresssensor KEAP1 functions as an adaptor for Cul3-based E3 ligaseto regulate proteasomal degradation of NRF2rdquo Molecular andCellular Biology vol 24 no 16 pp 7130ndash7139 2004
[12] K Taguchi H Motohashi and M Yamamoto ldquoMolecularmechanisms of the KEAP1-NRF2 pathway in stress responseand cancer evolutionrdquo Genes to Cells vol 16 no 2 pp 123ndash1402011
[13] K Mukaigasa L T Nguyen L Li H Nakajima M Yamamotoand M Kobayashi ldquoGenetic evidence of an evolutionarilyconserved role for NRF2 in the protection against oxidativestressrdquoMolecular and Cellular Biology vol 32 no 21 pp 4455ndash4461 2012
[14] JMaher andMYamamoto ldquoThe rise of antioxidant signalingmdashthe evolution and hormetic actions of NRF2rdquo Toxicology andApplied Pharmacology vol 244 no 1 pp 4ndash15 2010
[15] S C Lo X Li M T Henzl L J Beamer and M HanninkldquoStructure of the KEAP1NRF2 interface provides mechanisticinsight intoNRF2 signalingrdquoThe EMBO Journal vol 25 no 15pp 3605ndash3617 2006
[16] V A McKusick ldquoMendelian inheritance in man and its onlineversion OMIMrdquo American Journal of Human Genetics vol 80no 4 pp 588ndash604 2007
[17] B Yalcin D J Adams J Flint and T M Keane ldquoNext-generation sequencing of experimental mouse strainsrdquo Mam-malian Genome vol 23 no 9-10 pp 490ndash498 2012
[18] D R Bentley S Balasubramanian H P Swerdlow et alldquoAccurate whole human genome sequencing using reversibleterminator chemistryrdquo Nature vol 456 no 7218 pp 53ndash592008
[19] P S G Chain D V Grafham R S Fulton et al ldquoGenomeproject standards in a new era of sequencingrdquo Science vol 326no 5950 pp 236ndash237 2009
[20] RHWaterstonK Lindblad-Toh E Birney J Rogers J F AbrilP Agarwal et al ldquoInitial sequencing and comparative analysis ofthe mouse genomerdquo Nature vol 420 pp 520ndash562 2002
[21] T M Keane L Goodstadt P Danecek M A White K WongB Yalcin et al ldquoMouse genomic variation and its effect onphenotypes and gene regulationrdquoNature vol 477 pp 289ndash2942011
[22] T Yamamoto K Yoh A Kobayashi et al ldquoIdentification ofpolymorphisms in the promoter region of the human NRF2generdquo Biochemical and Biophysical Research Communicationsvol 321 no 1 pp 72ndash79 2004
[23] J M Marzec J D Christie S P Reddy et al ldquoFunctionalpolymorphisms in the transcription factor NRF2 in humansincrease the risk of acute lung injuryrdquo The FASEB Journal vol21 no 9 pp 2237ndash2246 2007
[24] H Fukushima-Uesaka Y Saito K Maekawa et al ldquoGeneticvariations and haplotype structures of transcriptional factorNRF2 and its cytosolic reservoir protein KEAP1 in JapaneserdquoDrug Metabolism and Pharmacokinetics vol 22 no 3 pp 212ndash219 2007
[25] E J Cordova R Velazquez-Cruz F Centeno V Baca and LOrozco ldquoThe NRF2 gene variant -653GA is associated with
Oxidative Medicine and Cellular Longevity 23
nephritis in childhood-onset systemic lupus erythematosusrdquoLupus vol 19 no 10 pp 1237ndash1242 2010
[26] M von Otter S Landgren S Nilsson et al ldquoAssociation ofNRF2-encoding NFE2L2 haplotypes with Parkinsonrsquos diseaserdquoBMCMedical Genetics vol 11 no 1 article 36 2010
[27] JMHartikainenM TengstromVMKosmaV L Kinnula AMannermaa andY Soini ldquoGenetic polymorphisms and proteinexpression of NRF2 and sulfiredoxin predict survival outcomesin breast cancerrdquo Cancer Research vol 72 pp 5537ndash5546 2012
[28] T Arisawa T Tahara T Shibata et al ldquoThe relationshipbetween Helicobacter pylori infection and promoter polymor-phism of the NRF2 gene in chronic gastritisrdquo InternationalJournal of Molecular Medicine vol 19 no 1 pp 143ndash148 2007
[29] T Arisawa T Tahara T Shibata et al ldquoThe influence ofpromoter polymorphism of nuclear factor-erythroid 2-relatedfactor 2 gene on the aberrant DNA methylation in gastricepitheliumrdquo Oncology Reports vol 19 no 1 pp 211ndash216 2008
[30] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene pro-moter polymorphism and gastric carcinogenesisrdquo Hepato-Gastroenterology vol 55 no 82-83 pp 750ndash754 2008
[31] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene promoterpolymorphism is associated with ulcerative colitis in a Japanesepopulationrdquo Hepato-Gastroenterology vol 55 no 82-83 pp394ndash397 2008
[32] C C Hua L C Chang J C Tseng C M Chu Y C Liu andW B Shieh ldquoFunctional haplotypes in the promoter region oftranscription factor NRF2 in chronic obstructive pulmonarydiseaserdquo Disease Markers vol 28 no 3 pp 185ndash193 2010
[33] S O Shaheen R B Newson S M Ring M J Rose-ZerilliJ W Holloway and A J Henderson ldquoPrenatal and infantacetaminophen exposure antioxidant gene polymorphismsand childhood asthmardquo The Journal of Allergy and ClinicalImmunology vol 126 no 6 pp 1141e7ndash1148e7 2010
[34] H Masuko T Sakamoto Y Kaneko et al ldquoLower FEV1 innon-COPD nonasthmatic subjects association with smokingannual decline in FEV1 total IgE levels and TSLP genotypesrdquoInternational Journal of Chronic Obstructive Pulmonary Diseasevol 6 pp 181ndash189 2011
[35] C P Guan M N Zhou A E Xu et al ldquoThe susceptibilityto vitiligo is associated with NF-E2-related factor 2 (NRF2)gene polymorphisms a study on Chinese Han populationrdquoExperimental Dermatology vol 17 no 12 pp 1059ndash1062 2008
[36] J Bouligand O Cabaret M Canonico et al ldquoEffect of NFE2L2genetic polymorphism on the association between oral estrogentherapy and the risk of venous thromboembolism in post-menopausal womenrdquo Clinical Pharmacology amp Therapeuticsvol 89 no 1 pp 60ndash64 2011
[37] D S OrsquoMahony B J Glavan T D Holden C Fong R A BlackG Rona et al ldquoInflammation and immune-related candidategene associations with acute lung injury susceptibility andseverity a validation studyrdquo PLoS ONE vol 7 no 12 Article IDe51104 2012
[38] I Ungvari E Hadadi V Virag et al ldquoRelationship between airpollutionNFE2L2 gene polymorphisms and childhood asthmain aHungarian populationrdquo Journal of CommunityGenetics vol3 no 1 pp 25ndash33 2012
[39] M SiedlinskiD S Postma JMA Boer et al ldquoLevel and courseof FEV1 in relation to polymorphisms inNFE2L2 andKEAP1 inthe general populationrdquo Respiratory Research vol 10 article 732009
[40] C C Hong C B Ambrosone J Ahn et al ldquoGenetic variabilityin iron-related oxidative stress pathways (NRF2 NQ01 NOS3
and HO-1) iron intake and risk of postmenopausal breastcancerrdquo Cancer Epidemiology Biomarkers and Prevention vol16 no 9 pp 1784ndash1794 2007
[41] C Canova C Dunster F J Kelly C Minelli P L Shah CCaneja et al ldquoPM10-induced hospital admissions for asthmaand chronic obstructive pulmonary disease the modifyingeffect of individual characteristicsrdquo Epidemiology vol 23 no 4pp 607ndash615 2012
[42] A J Henderson R B Newson M Rose-Zerilli S M RingJ W Holloway and S O Shaheen ldquoMaternal NRF2 andgluthathione-S-transferase polymorphisms donotmodify asso-ciations of prenatal tobacco smoke exposure with asthma andlung function in school-aged childrenrdquo Thorax vol 65 no 10pp 897ndash902 2010
[43] C Xing A L Sestak J A Kelly et al ldquoLocalization andreplication of the systemic lupus erythematosus linkage signalat 4p16 interaction with 2p11 12q24 and 19q13 in EuropeanAmericansrdquo Human Genetics vol 120 no 5 pp 623ndash631 2007
[44] T F Wong K Yoshinaga Y Monma K Ito H NiikuraS Nagase et al ldquoAssociation of KEAP1 and NRF2 geneticmutations and polymorphisms with endometrioid endometrialadenocarcinoma survivalrdquo International Journal of Gynecologi-cal Cancer vol 21 no 8 pp 1428ndash1435 2011
[45] M von Otter S Landgren S Nilsson et al ldquoNRF2-encodingNFE2L2 haplotypes influence disease progression but not riskin Alzheimerrsquos disease and age-related cataractrdquoMechanisms ofAgeing and Development vol 131 no 2 pp 105ndash110 2010
[46] S Tsang Z Sun B Luke et al ldquoA comprehensive SNP-basedgenetic analysis of inbred mouse strainsrdquoMammalian Genomevol 16 no 7 pp 476ndash480 2005
[47] C M Wade E J Kulbokas III A W Kirby et al ldquoThe mosaicstructure of variation in the laboratory mouse genomerdquoNaturevol 420 no 6915 pp 574ndash578 2002
[48] T Wiltshire M T Pletcher S Batalov et al ldquoGenome-widesingle-nucleotide polymorphism analysis defines haplotypepatterns in mouserdquo Proceedings of the National Academy ofSciences of the United States of America vol 100 no 6 pp 3380ndash3385 2003
[49] K A Frazer E Eskin H M Kang et al ldquoA sequence-basedvariation map of 827 million SNPs in inbred mouse strainsrdquoNature vol 448 no 7157 pp 1050ndash1053 2007
[50] T P Maddatu S C Grubb C J Bult andM A Bogue ldquoMousephenome database (MPD)rdquo Nucleic Acids Research vol 40 ppD887ndashD894 2007
[51] A Kirby H M Kang C M Wade et al ldquoFine mapping in 94inbred mouse strains using a high-density haplotype resourcerdquoGenetics vol 185 no 3 pp 1081ndash1095 2010
[52] H Y Cho A E Jedlicka S P M Reddy L Y Zhang T WKensler and S R Kleeberger ldquoLinkage analysis of susceptibilityto hyperoxia NRF2 is a candidate generdquo American Journal ofRespiratory Cell and Molecular Biology vol 26 no 1 pp 42ndash512002
[53] H Y Cho A E Jedlicka S P M Reddy et al ldquoRole of NRF2in protection against hyperoxic lung injury in micerdquo AmericanJournal of Respiratory Cell and Molecular Biology vol 26 no 2pp 175ndash182 2002
[54] T Shibata T Ohta K I Tong et al ldquoCancer related mutationsin NRF2 impair its recognition by KEAP1-Cul3 E3 ligase andpromote malignancyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 36 pp13568ndash13573 2008
24 Oxidative Medicine and Cellular Longevity
[55] A Singh V Misra R K Thimmulappa et al ldquoDysfunctionalKEAP1-NRF2 interaction in non-small-cell lung cancerrdquo PLoSMedicine vol 3 no 10 article e420 2006
[56] T Ohta K Iijima M Miyamoto et al ldquoLoss of KEAP1 functionactivates NRF2 and provides advantages for lung cancer cellgrowthrdquo Cancer Research vol 68 no 5 pp 1303ndash1309 2008
[57] B Padmanabhan K I Tong T Ohta et al ldquoStructural basisfor defects of KEAP1 activity provoked by its point mutationsin lung cancerrdquoMolecular Cell vol 21 no 5 pp 689ndash700 2006
[58] T Shibata A Kokubu M Gotoh et al ldquoGenetic alteration ofKEAP1 confers constitutive NRF2 activation and resistance tochemotherapy in gallbladder cancerrdquoGastroenterology vol 135no 4 pp 1358e4ndash1368e4 2008
[59] N J Yoo H R Kim Y R Kim C H An and S H LeeldquoSomatic mutations of the KEAP1 gene in common solidcancersrdquo Histopathology vol 60 no 6 pp 943ndash952 2012
[60] Y Hu Y Ju D Lin Z Wang Y Huang S Zhang et alldquoMutation of the NRF2 gene in non-small cell lung cancerrdquoMolecular Biology Reports vol 39 no 4 pp 4743ndash4747 2012
[61] Y R Kim J EOhM S Kim et al ldquoOncogenicNRF2mutationsin squamous cell carcinomas of oesophagus and skinrdquo TheJournal of Pathology vol 220 no 4 pp 446ndash451 2010
[62] T Shibata A Kokubu S Saito et al ldquoNRF2 mutation confersmalignant potential and resistance to chemoradiation therapyin advanced esophageal squamous cancerrdquo Neoplasia vol 13pp 864ndash873 2011
[63] H Sasaki M Shitara K Yokota Y Hikosaka S MoriyamaM Yano et al ldquoIncreased NRF2 gene (NFE2L2) copy numbercorrelates with mutations in lung squamous cell carcinomasrdquoMolecular Medicine Reports vol 6 no 2 pp 391ndash394 2012
[64] H Sasaki M Shitara K Yokota Y Hikosaka S Moriyama MYano et al ldquoRagD gene expression andNRF2mutations in lungsquamous cell carcinomasrdquo Oncology Letters vol 4 pp 1167ndash1170 2012
[65] A Singh M Bodas N Wakabayashi F Bunz and S BiswalldquoGain of NRF2 function in non-small-cell lung cancer cellsconfers radioresistancerdquo Antioxidants and Redox Signaling vol13 no 11 pp 1627ndash1637 2010
[66] T Yamadori Y Ishii SHommaYMorishimaKKurishimaKItoh et al ldquoMolecular mechanisms for the regulation of NRF2-mediated cell proliferation in non-small-cell lung cancersrdquoOncogene vol 31 pp 4768ndash4777 2012
[67] A K Bauer H Y Cho L Miller-Degraff C Walker K HelmsJ Fostel et al ldquoTargeted deletion of NRF2 reduces urethane-induced lung tumor development in micerdquo PLoS ONE vol 6no 10 Article ID e26590 2011
[68] H Y Cho and S R Kleeberger ldquoNRF2 protects against airwaydisordersrdquo Toxicology and Applied Pharmacology vol 244 no1 pp 43ndash56 2010
[69] R Wang J An F Ji H Jiao H Sun and D Zhou ldquoHyperme-thylation of the KEAP1 gene in human lung cancer cell linesand lung cancer tissuesrdquo Biochemical and Biophysical ResearchCommunications vol 373 no 1 pp 151ndash154 2008
[70] P Zhang A Singh S Yegnasubramanian et al ldquoLoss ofkelch-like ECH-associated protein 1 function in prostate cancercells causes chemoresistance and radioresistance and promotestumor growthrdquoMolecular Cancer Therapeutics vol 9 no 2 pp336ndash346 2010
[71] N Hanada T Takahata Q Zhou et al ldquoMethylation of theKEAP1 gene promoter region in human colorectal cancerrdquoBMCCancer vol 12 article 66 2012
[72] L A Muscarella R Barbano V DrsquoAngelo et al ldquoRegulationof KEAP1 expression by promoter methylation in malignantgliomas and association with patientrsquos outcomerdquo Epigeneticsvol 6 no 3 pp 317ndash325 2011
[73] S Yu T O Khor K L Cheung et al ldquoNRF2 expressionis regulated by epigenetic mechanisms in prostate cancer ofTRAMP micerdquo PLoS ONE vol 5 no 1 Article ID e8579 2010
[74] T O Khor Y Huang T Y Wu L Shu J Lee and A N KongldquoPharmacodynamics of curcumin as DNA hypomethylationagent in restoring the expression of NRF2 via promoter CpGsdemethylationrdquo Biochemical Pharmacology vol 82 no 9 pp1073ndash1078 2011
[75] I M Fingerman L McDaniel X Zhang et al ldquoNCBI epige-nomics a new public resource for exploring epigenomic datasetsrdquo Nucleic Acids Research vol 39 supplemen 1 pp D908ndashD912 2011
[76] I M Fingerman X Zhang W Ratzat N Husain R F Cohenand G D Schuler ldquoNCBI epigenomics whatrsquos new for 2013rdquoNucleic Acids Research vol 41 no 1 pp D221ndashD225 2013
[77] J H Lee T O Khor L Shu Z Y Su F Fuentes andA N Kong ldquoDietary phytochemicals and cancer preventionNRF2 signaling epigenetics and cell death mechanisms inblocking cancer initiation and progressionrdquo Pharmacology ampTherapeutics vol 137 no 2 pp 153ndash171 2012
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
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Diabetes ResearchJournal of
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Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
6 Oxidative Medicine and Cellular Longevity
Translation start site for variants 2 and 3
1 missed in isoforms 2 and 3pA72T pS99P pQ121H missed in isoform 3
pP406S pE423D pK428R pR437W pR449C
minus400
minus500
minus50minus100
minus200
minus300
minus450
minus350
minus250
minus150
g minus
g
pE152K pV155I pL177V
pV268M
pR517K pH453L
pS365I pS387N pK389R pL309F pI317M pD341V
pK219R pP233S pK237 = fs
51101151201251301351401451501551601
Protein isoform 1 (NP 006155)
g minus66minus65insC
Termination codon (c +2371 +2373)
Translation start site (c +1)
151
101151201251301351401451501551601
2351
frame stop codonc minus156 154 in-old + 1 [23]
old + 1 [22]
g minus1 (178130354)
c minus96delC
c minus438 minus437delTT
minus89 minus87delGCC
transcription start site)+1 (c minus555
p R43Q p R43W
Genomic DNA (reversed contig GRCh37 p10 primary assemblyNM 0061644)minus477
minus214 g minus212gminus188
c 9minus95
g minus178
c minus304
c 8minus13
c 2minus16
TgtA
CgtT
GgtA
GgtA
GgtA
GgtT
GgtCAgtG
AgtCAgtG
Figure 2 DNA (variant 1 partial promoter and exons 1 and 5) and protein (isoform 1) sequence of Human NRF2 SNPs and amino acidresidues for nonsynonymous SNPs are marked
the 51015840 flanking promoter and 59 in exons (Tables 3 and S1)Among exon SNPs 26 are nonsynonymous (Cns) mutationsA triplet repeat variation (rs143406266 GCC
4versus GCC
5
previously published as minus20minus6) in the 51015840-UTR was uniquelyidentified in Asian populations [22 24]
33 SNPs Haplotypes and Association with Disease RiskThe use of gene knockout mice in model systems hasprovided potential insights into the role of NRF2 in thepathogenesis of various human disorders (see Figure 1)Recent epidemiological and association studies have revealedsignificant associations of NRF2 sequence variations withdisease risks which further supportsNRF2 as a susceptibilitygene Most of the phenotype-associated variants are in thepromoter region and presumed to be involved in NRF2 generegulation Table 4summarizes NRF2 SNP andor haplotypealleles that have been associated with oxidant-related diseaserisks Interestingly there is no evidence for exon SNPs as at-risk alleles For convenience and consistency intronic and31015840 distal SNP alleles are presented as chromosome contig(HGVS) alleles while promoter and exon SNPs are presentedas reversed contig alleles throughout the text
Pulmonary Diseases NRF2 SNPs in the promoter and intron1 sequences have been investigated for their potential asso-ciations with risk of pulmonary critical disorders includingacute lung injury (ALI) cigarette smoke-induced chronicobstructive pulmonary disease (COPD) and asthma Aheterozygous CA SNP at minus178 position (rs6721961TgtCor TgtG previously minus617 or minus650) significantly increasedthe risk for developing ALI following major trauma inEuropean and African-American populations (odds ratioOR 644 95 confidence interval CI 134ndash308 allelicfrequency = 119 at 21180) [23] Promoter activity ofthe A allele (AC or AA) determined in vivo and invitro was significantly lower than CC allele at that locus(minus178 in an ARE-like motif) indicating that it is a func-tional SNP for autoregulation [23] The minus178GG werealso nominally associated with ALI-related 28-day mor-tality following systemic inflammatory response syndrome[37] In a Japanese cohort SNP haplotype (rs2001350Trs6726395Ars1962142Ars2364722Ars6721961T) co-tainingthe minus178AA homozygote was associated with an annualdecline of rapid forced expiratory volume in one second(FEV1) in relation to cigarette-smoking status [34] In addi-
tion a promoter and 51015840-UTR SNP haplotype consistingof minus214G allele (52 rs35652124 previously minus686minus653)
Oxidative Medicine and Cellular Longevity 7Ta
ble3Geneticmutations
inprom
oter
andexon
sofh
uman
NRF
2
IDMap
onchromosom
e2lowast
(HGVS
name)
Positionin
Nfe2
l2
(HGVS
name)
Region
s(position
from
mRN
A)
Varia
tioncla
ssand
consequences
(HGVS
name)dagger
Minor
allelefrequ
ency
(MAF)
MAcoun
tsDagger(coh
ort
size)
MAFsources
rs16865105
g178136629AgtC
cminus555minus
6770Tgt
G51015840Flanking
(minus6770)
SNP
C=01928421
1000
Genom
esrs7557529
g178135097CgtT
cminus555minus
5238GgtA
51015840Flanking
(minus5238)
SNP
nars6750320
g178131796CgtT
cminus555minus
1937GgtA
51015840Flanking
(minus1937)
SNP
T=0030266
1000
Genom
esrs18116
2518
g178131774TgtC
cminus555minus
1915AgtG
51015840Flanking
(minus1915)
SNP
C=000
051
1000
Genom
esrs1900
44775
g178131746CgtT
cminus555minus
1887GgtA
51015840Flanking
(minus1887)
SNP
T=000
051
1000
Genom
esrs185117338
g178131704AgtG
cminus555minus
1845Tgt
C51015840Flanking
(minus1845)
SNP
G=000
051
1000
Genom
esrs149947189
g178131697CgtT
cminus555minus
1838GgtA
51015840Flanking
(minus1838)
SNP
T=000
092
1000
Genom
esrs139771244
g178131625AgtG
cminus555minus
1766Tgt
C51015840Flanking
(minus1766)
SNP
G=000
092
1000
Genom
esrs6747203
g17813160
4CgtG
cminus555minus
1745GgtC
51015840Flanking
(minus1745)
SNP
G=000
613
1000
Genom
esrs193101749
g178131504TgtC
cminus555minus
1645AgtG
51015840Flanking
(minus1645)
SNP
C=000
4610
1000
Genom
esrs190630762
g178131495GgtC
cminus555minus
1636Cgt
G51015840Flanking
(minus1636)
SNP
C=000
092
1000
Genom
esrs183764
402
g178131366CgtA
cminus555minus
1507GgtT
51015840Flanking
(minus1507)
SNP
A=000
051
1000
Genom
esrs19122296
4g178131211GgtA
cminus555minus
1352Cgt
T51015840Flanking
(minus1352)
SNP
A=000
092
1000
Genom
esrs187137522
g178131165TgtG
cminus555minus
1306AgtC
51015840Flanking
(minus1306)
SNP
G=000
092
1000
Genom
esrs182620359
g178131158AgtG
cminus555minus
1299Tgt
C51015840Flanking
(minus1299)
SNP
G=000
051
1000
Genom
esrs4893819
(rs61433302)
g178131134CgtT
cminus555minus
1275GgtA
51015840Flanking
(minus1275)
SNP
C=04263931
1000
Genom
es
rs191547130
g178131017CgtT
cminus555minus
1158GgtA
51015840Flanking
(minus1158)
SNP
T=000
051
1000
Genom
esrs188422217
g178131003AgtG
cminus555minus
1144TgtC
51015840Flanking
(minus114
4)SN
PG=000143
1000
Genom
esrs143047764
g178130865AgtG
cminus555minus
1006Tgt
C51015840Flanking
(minus1006)
SNP
G=000
6915
1000
Genom
esrs74432849
g178130766CgtA
cminus555minus
907GgtT
51015840Flanking
(minus907)
SNP
nars116
79252
g178130691CgtG
cminus555minus
832GgtC
51015840Flanking
(minus832)
SNP
nars12993217
g178130516AgtG
cminus555minus
657TgtC
51015840Flanking
(minus657)
SNP
nars115644
826
g17813044
2TgtA
cminus555minus
583AgtT
51015840Flanking
(minus583)
SNP
A=0015133
1000
Genom
esrs140803524
g178130431GgtA
cminus555minus
572CgtT
51015840Flanking
(minus572)
SNP
A=000
4610
1000
Genom
es
rs776844
20g178130427TgtC
cminus555minus
568AgtG
51015840Flanking
(minus568)
SNP
C=0033974
C=0190(84)
1000
Genom
es[24]
rs183651094
g178130336AgtT
cminus555minus
477TgtA
51015840Flanking
(minus477)
SNP
T=000184
1000
Genom
es
rs35652124
sect
(rs57695243)
g178130073TgtC
cminus555minus
214AgtG
51015840Flanking
(minus214)
SNP
C=03512767
T=0429(84)
T=0413181Dagger
C=033827Dagger
C=03517
69
1000
Genom
es[24]
[22]
[23]
[25]
rs6706649
sectg178130071CgtT
cminus555minus
212GgtA
51015840Flanking
(minus212)
SNP
T=0078170
T=004
8(84)
T=004
821Dagger
T=00753Dagger
1000
Genom
es[24]
[22]
[23]
rs1506
48896
g178130047CgtG
cminus555minus
188GgtC
51015840Flanking
(minus188)
SNP
G=000235
G=000
6(84)
1000
Genom
es[24]
rs6721961
sect
(rs117801448)
g178130037TgtC
TgtG
cminus555minus
178AgtC
AgtG
51015840Flanking
(minus178)
SNP
T=0150328
T=0283124Dagger
T=031325Dagger
T=0321(84)
1000
Genom
es[22]
[23]
[24]
8 Oxidative Medicine and Cellular Longevity
Table3Con
tinued
IDMap
onchromosom
e2lowast
(HGVS
name)
Positionin
Nfe2
l2
(HGVS
name)
Region
s(position
from
mRN
A)
Varia
tioncla
ssand
consequences
(HGVS
name)dagger
Minor
allelefrequ
ency
(MAF)
MAcoun
tsDagger(coh
ort
size)
MAFsources
rs20134560
4g178129924178129925insG
cminus555-66minus555-65insC
51015840Flanking
(minus66minus65)
Insertion
G=0017939
1000
Genom
es
rs200432479
g178129741178
129742delAA
cminus438minus437delT
Texon
1UTR
-51015840(118minus119
)Dele
tion
-=000378
-=000
6(84)
1000
Genom
es[24]
rs75485459
g178129608CgtA
cminus304GgtT
Exon
1UTR
-51015840(252)
SNP
nars192086766
g17812946
6CgtT
cminus162GgtA
Exon
1UTR
-51015840(394)
SNP
T=002248
1000
Genom
esmdash
g17812944
2GgtA
cminus138CgtT
Exon
1UTR
-51015840(418)
SNP
A=0012(84)
[24]
rs7166
8246
g17812940
0delG
cminus96delC
Exon
1UTR
-51015840(460)
Dele
tion
nars187291840
g178129399CgtT
cminus95GgtA
Exon
1UTR
-51015840(461)
SNP
T=0054912
01000
Genom
es
rs143406266
g178129391178129393delGGC
cminus89minus87delGCC
Exon
1UTR
-51015840(467minus469)
Dele
tion
-=064
4282Dagger
-=0589(84)
[22]
[24]
rs182428269
g178098918GgtA
c127Cgt
TEx
on2(682)
Cns(pArg43Trp)
A=000
051
1000
Genom
esrs35248500
g178098917CgtT
c128GgtA
Exon
2(683)
Cns(pArg43Gln)
T=000
613
1000
Genom
esrs1135118
g178098831CgtT
c214GgtA
Exon
2(769)
Cns(pAla72Th
r)TDagger
[23]
rs19969166
0g178098829AgtT
c216Tgt
AEx
on2(771)
Cs(pAla72=)
namdash
g178098769GgtA
c276Cgt
TEx
on2(831)
Cs(pIle92=)
TDagger[23]
rs5031039
g178098750AgtG
c295Tgt
CEx
on2(850)
Cns(pSer99P
ro)
G=0Dagger
[23]
rs200239262
g178098017CgtG
c363GgtC
Exon
3(918)
Cns(pGln121H
is)na
rs183034165
g178098008TgtC
c372GgtA
Exon
3(927)
Cs(pAla124=
)T=000
092
T=000
6(84)
1000
Genom
es[24]
rs199970826
g178097996CgtT
c384GgtA
Exon
3(939)
Cs(pPro128=
)T=000
051
1000
Genom
esrs201992337
g178097260CgtT
c454GgtA
Exon
4(100
9)Cn
s(pGlu152Lys)
nars201589693
g178097251CgtT
c463GgtA
Exon
4(1018)
Cns(pV
al155Ile)
T=000
051
1000
Genom
es
rs35577826
g178097185AgtC
c529Tgt
GEx
on4(1084)
Cns(pLeu177Va
l)C=000143
Alt0005Dagger
1000
Genom
es[23]
rs181513314
g178096710CgtT
c621GgtA
Exon
5(1176)
Cn(pLeu207=
)T=000
051
1000
Genom
esrs60132461
g178096675TgtC
c656AgtG
Exon
5(1211)
Cns(pLys219Arg)
C=000184
1000
Genom
es
rs139187151
g178096634GgtA
c697Cgt
TEx
on5(1252)
Cns(pPro233Ser)
A=000
051
A=0012Dagger
(84)
1000
Genom
es[24]
rs35557421
g178096620d
elT
c711delA
Exon
5(1266)
Fram
eshiftdeletio
n(pLys237=fs)
na
rs34154613
g178096529CgtT
c802GgtA
Exon
5(1357)
Cns(pV
al268M
et)
T=000184
1000
Genom
esrs141363120
g178096
406GgtA
c925Cgt
TEx
on5(14
80)
Cns(pLeu309Ph
e)A=000378
1000
Genom
esrs201661476
g178096380AgtC
c951Tgt
GEx
on5(1506)
Cns(pIle
317M
et)
na
Oxidative Medicine and Cellular Longevity 9
Table3Con
tinued
IDMap
onchromosom
e2lowast
(HGVS
name)
Positionin
Nfe2
l2
(HGVS
name)
Region
s(position
from
mRN
A)
Varia
tioncla
ssand
consequences
(HGVS
name)dagger
Minor
allelefrequ
ency
(MAF)
MAcoun
tsDagger(coh
ort
size)
MAFsources
rs199673454
g178096309TgtA
c1022AgtT
Exon
5(1577)
Cns(pAs
p341Va
l)A=000
051
1000
Genom
esrs35007548
g178096299GgtA
c1032CgtT
Exon
5(1587)
Cs(pSer344=
)A=000
092
1000
Genom
esrs200209692
g178096287TgtC
c104
4AgtG
Exon
5(1599)
Cs(pLeu348=
)C=000
051
1000
Genom
es
mdashg178096237CgtA
c1094GgtT
Exon
5(1649)
Cns(pSer365Ile
)A=0125273
A=000
6Dagger(84)
PubM
ed[24]
rs201214197
g178096171CgtT
c1160GgtA
Exon
5(1715)
Cns(pSer387Asn)
T=000
051
1000
Genom
esrs200494292
g178096165TgtC
c1166AgtG
Exon
5(1721)
Cns(pLys389Arg)
nars186171287
g178096115GgtA
c1216CgtT
Exon
5(1771)
Cns(pPro4
06Ser)
A=000
051
1000
Genom
esrs182276775
g178096062CgtA
c1269GgtT
Exon
5(1824)
Cns(pGlu423A
sp)
A=000
051
1000
Genom
esrs201560221
g17809604
8TgtC
c1283AgtG
Exon
5(1838)
Cns(pLys428Arg)
nars189238236
g178096043AgtG
c1288TgtC
Exon
5(1843)
Cs(pLeu430=
)G=000
051
1000
Genom
esrs184287392
g178096022GgtA
c1309CgtT
Exon
5(1864)
Cns(pArg437Trp)
A=000
051
1000
Genom
esrs201871588
g178095986GgtA
c1345CgtT
Exon
5(19
00)
Cns(pAg449Cy
s)na
rs181294188
g178095985TgtC
c1346GgtA
Exon
5(19
01)
Cns(pArg44
9His)
T=000
092
1000
Genom
esrs20169046
6g178095973TgtA
c1358AgtT
Exon
5(19
13)
Cns(pHis4
53Leu)
A=000
051
1000
Genom
esrs105704
4(rs52789869)
g178095781CgtT
c1550GgtA
Exon
5(2105)
Cns(pArg517Lys)
TDagger[23]
rs200750800
g178095603AgtG
c1728TgtC
Exon
5(2283)
Cs(pTyr576=
)na
rs200175942
g178095567AgtG
c1764TgtC
Exon
5(2319)
Cs(pAsp588=
)G=000
051
1000
Genom
esrs77547666
g178095495GgtC
clowast18Cgt
GEx
on5UTR
-31015840(2391)
SNP
C=000
6915
1000
Genom
esrs73031353
g178095425TgtC
clowast88AgtG
Exon
5UTR
-31015840(2461)
SNP
C=000184
1000
Genom
esrs675944
3g178095345TgtC
clowast168AgtG
Exon
5UTR
-31015840(2541)
SNP
C=000
419
1000
Genom
esrs188674558
g178095279CgtA
clowast234GgtT
Exon
5UTR
-31015840(2607)
SNP
A=000
051
1000
Genom
esrs77685897
g178095247AgtG
clowast266TgtC
Exon
5UTR
-31015840(2639)
SNP
G=000
092
1000
Genom
esrs1057092
g178095162TgtG
clowast351AgtC
Exon
5UTR
-31015840(2724)
SNP
nars3197704
g178095162TgtG
clowast351AgtC
Exon
5UTR
-31015840(2724)
SNP
nars184701151
g178095159TgtC
clowast354AgtG
Exon
5UTR
-31015840(2727)
SNP
C=000276
1000
Genom
esrs11543307
g178095153AgtG
clowast360TgtC
Exon
5UTR
-31015840(2733)
SNP
nars111874043
g178095146AgtG
clowast367TgtC
Exon
5UTR
-31015840(2740)
SNP
nars34012004
g178095102AgtC
clowast411TgtG
Exon
5UTR
-31015840(2784)
SNP
C=0071
[24]
10 Oxidative Medicine and Cellular Longevity
Table3Con
tinued
IDMap
onchromosom
e2lowast
(HGVS
name)
Positionin
Nfe2
l2
(HGVS
name)
Region
s(position
from
mRN
A)
Varia
tioncla
ssand
consequences
(HGVS
name)dagger
Minor
allelefrequ
ency
(MAF)
MAcoun
tsDagger(coh
ort
size)
MAFsources
rs201481890
g178095090178095091in
sTclowast
422lowast423insA
Exon
5UTR
-31015840
(2795minus
2796)
Insertion
na
rs3082500
g178095089178095090d
elTT
delTTinsT
clowast423lowast424d
elAAinsA
Exon
5UTR
-31015840
(2796minus
2797)
Dele
tion
insertion
na
rs71792546
(rs71796710)
g178095079d
elT
clowast425delA
Exon
5UTR
-31015840(2798)
Dele
tion
na
rs1057106
g178095078AgtC
AgtT
clowast435TgtA
TgtG
Exon
5UTR
-31015840(2808)
SNP
na
rs34176791
g178095076AgtC
clowast437TgtG
Exon
5UTR
-31015840(2810)
SNP
C=000235
1000
Genom
esrs35911553
g178095045CgtT
clowast46
8GgtA
Exon
5UTR
-31015840(2841)
SNP
T=000
6915
1000
Genom
esSequ
ence
varia
tions
inup
stream
andexon
sofh
uman
NRF
2fro
m655varia
tions
availablein
publicdatabase
asof
Decem
ber2012
(583
activ
esomeSN
Psmergedge14
SNPs
citedin
PubM
ed)lowastNCB
Ireference
sequ
ence
NC
0000
0211
(Hom
osapiens
chromosom
e2G
RCh37p
10prim
arya
ssem
bly)spanning
178095033ndash1781298
59bp
(com
plem
ent34827
bpforexons
andintro
ns)51015840-Flank
ingregions
startat17812
9860
bp(minus1)in
transcrip
tvariant
1HGVS
Hum
anGenom
eVa
riatio
nSo
ciety
Position
sinvaria
nt1(NM
006164
4)2859
bpsectSN
Pscitedin
PubM
edE
xon11781298
59ndash17812
9260
(600
bpT
TS=1781293
04)exon
2178098999ndash
178098733(267
bp)exon
3178098067ndash1780979
78(90b
p)exon41780973
11ndash178097120
(192b
p)and
exon
5178096736ndash
178095033(1704b
p)daggerProteinam
inoacid
(aa)
resid
uesiniso
form
1(N
P00
61552605
aa)Cn
scoding
-non
syno
nymou
sCs
cod
ing-syno
nymou
sDaggerheterozygositydetectednano
tavailable
Oxidative Medicine and Cellular Longevity 11
Table4Hum
anNRF
2SN
Psassociated
with
diseaser
isk
IDMap
onchromosom
e2 (H
GVS
name)
Region
class
Dise
asea
ssociatio
nandreferences
Risk
allelesa
ndstatistics
Ethn
icgrou
p(num
bero
fcase)
rs7557529
g178135097CgtT
minus5238
Ggt
AParkinsonrsquos
disease(2010
[26])
G=0718252Dagger
inhaplotype
OR=090
or040
CI=
060ndash140or
03ndash06
Swedish
Polish
Caucasian
(357)
rs2886162
g178133165AgtG
minus3306
TgtC
Breastcancer
survival(2012[27])
TT=0324
OR=16
87C
I=1105ndash275
FinlandKB
CP(452)
Chronicg
astritisgastric
ulcer(2007
[28])
GDagger
GDaggerin
haplotypefor
ulcer(OR=
252C
I=119ndash
545)
Japanese
(159)
P14methylationin
gastric
cancer
Hpyloriinfectio
n(2008[29])
Gin
haplotype
OR=290C
I=114ndash
736
Japanese
(209)
Gastriccancer
inHpylori-n
egative
cases(2008
[30])
Ain
haplotype
119875=0022
Japanese
(209)
rs35652124
(rs57695243)
g178130073TgtC
minus214Agt
G(previou
slyminus653[23]
orminus686[22])
Ulcerativec
olitis(2008
[31])
AG
(OR=045C
I=022ndash0
93)
G(O
R=257C
I=10
1ndash660)
Japanese
(89)
Lupu
swith
neph
ritisin
female(2010
[25])
GA
OR=18
1CI
=10
4ndash312
Mexican
mestizo(362)
Parkinsonrsquos
disease(2010
[26])
A=0884312Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
COPD
(2010[32])
G=052Dagger
hazard
ratio
=095C
I=091ndash0
99
(Haplotype)
German
(69)
Gastriculcer(2007
[28])
GDaggerin
haplotype
OR=252C
I=119ndash
545
Japanese
(159)
P14methylationin
gastric
cancer
H
pyloriinfection(2008[29])
Gin
haplotype
OR=290C
I=114ndash
736
Japanese
(209)
Gastriccancer
inHpylori-negative
cases(2008
[30])
Gin
haplotype119875=0022
Japanese
(209)
rs6706
649
g178130071CgtT
minus212Ggt
A(previou
slyminus651[23]
orminus684[22])
Ulcerativec
olitis(2008
[31])
AG
(OR=045C
I=022ndash0
93)
G(O
R=257C
I=10
1ndash660)
Japanese
(89)
Maternalacetaminop
henandasthma
(2010[33])
A=02321137Dagger
OR=17
3CI
=12
2245
UKALSPA
C(gt40
00mothersgt
5000
child
ren)
12 Oxidative Medicine and Cellular Longevity
Table4Con
tinued
IDMap
onchromosom
e2 (H
GVS
name)
Region
class
Dise
asea
ssociatio
nandreferences
Risk
allelesa
ndstatistics
Ethn
icgrou
p(num
bero
fcase)
COPD
(2010[32])
G=098Daggerin
haplotype
hazard
ratio
=095C
I=091ndash0
99
German
(69)
Parkinsonrsquos
disease(2010
[26])
G=0972343Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
03ndash06
Swedish
Polish
Caucasian
(357)
Acutelun
ginjury
follo
wingtrauma
(2007[23])
CA=0119Dagger
OR=644
CI=
134ndash
3080
Caucasia
nAfrican-
American
(164
)
Ann
ualF
EV1decline
(2011[34])
A=0082in
haplotype
Japanese
(915)
Vitiligo(2008[35])
CAA
AOR=17
24C
I=13
5ndash221
Chinese(300)
COPD
(2010[32])
G=073Daggerin
haplotype
hazard
ratio
=095C
I=091ndash0
99
German
(69)
rs6721961
(rs117801448)
g178130037TgtC
TgtG
minus178Agt
GAgt
C(previou
slyminus617[23]
orminus650[22])
Parkinsonrsquos
disease(2010
[26])
C=0980346Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
Postm
enop
ausalvenou
sthrombo
embo
lism
(2011[36])
A=033311
OR=25CI
=370ndash8570
Caucasia
n(161)
Breastcancer
survivalandNRF
2proteinexpressio
n(2012[27])
AA
OR=4656CI
=13
5ndash1606
FinlandKB
CP(452)
Acutelun
ginjury-related
mortality
followingsyste
micinflammatory
respon
sesynd
rome(2012
[37])
GG
OR=97
3CI
=12
7ndash7480
Caucasia
n(750)
Infection-indu
cedasthma(
2012
[38])
AC
OR=0437CI
=028ndash0
80
Hun
garia
nCaucasia
nGypsy
(307)
rs143406266
g178129391178
129393delGGC
Exon
1(467ndash4
69G
CC)
COPD
(2010[32])
GCC
4=053Dagger
hazard
ratio
=095C
I=091ndash0
99
Taiwanese(69)
rs2886161
g178127839Tgt
ClowastIntro
n1
Parkinsonrsquos
disease(2010
[26])
T=0878309Dagger
inhaplotype
OR=09or
04CI
=06ndash
14or
03ndash06
Swedish
Polish
Caucasian
(357)
rs2364723
g178126546Ggt
CIntro
n1
Basaland
smoker
FEV
1(200
9[39])
CI=minus6360simminus1780C
=0525Dagger
Also
ashaplotype
Netherla
nd(2542)
rs2364722
g178124787Agt
GIntro
n1
Ann
ualF
EV1decline
(2011[34])
A=0082in
haplotype
Japanese
(915)
rs13001694
g178118990Agt
GIntro
n1
Basaland
smoker
FEV
1(200
9[39])
G=040
11578Dagger
inhaplotype
Netherla
nd(2542)
Oxidative Medicine and Cellular Longevity 13
Table4Con
tinued
IDMap
onchromosom
e2 (H
GVS
name)
Region
class
Dise
asea
ssociatio
nandreferences
Risk
allelesa
ndstatistics
Ethn
icgrou
p(num
bero
fcase)
Breastcancer
[40]
Twith
NQO1N
OS3H
O1risk
allelesOR=15
6CI
=097ndash251
Caucasia
nandothers(505)
rs1806
649
(rs58745895)
g178118152Cgt
TIntro
n1
Basaland
smoker
FEV
1(200
9[39])
T=02631119Daggerin
haplotype
CI=minus8730ndash
(minus17
0)Netherla
nd(2542)
Parkinsonrsquos
disease(2010
[26])
T=0422148Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
Particulatem
attera
ndasthmaCO
PDadmiss
ion(2012[41])
Cwith
lowvitamin
Clevel(OR=
31CI
=15
0ndash630)
UK(209)
rs4243387
(rs6003846
4)g178117765Cgt
TIntro
n1
Basaland
smoker
FEV
1(200
9[39])
T=00914
25Daggerin
haplotype
Netherla
nd(2542)
rs1962142
(rs58448508)
g178113484Agt
GIntro
n1
Ann
ualF
EV1decline
(2011[34])
A=0082in
haplotype
Japanese
(915)
Breastcancer
NRF
2andARE
expressio
n(2012[27])
A(119875=0036)
FinlandKB
CP(452)
rs6726395
(rs57309289)
g178103229Agt
GIntro
n1
Smok
ing-relatedFE
V1decline
and
annu
alFE
V1decline
(2011[34])
G=0884Dagger
A=0082in
haplotype
Japanese
(915)
Basaland
smoker
FEV
1(200
9[39])
G=046
41764Daggerin
haplotype
Netherla
nd(2542)
rs2001350
(rs17515179
rs60883775)
g178100
425Cgt
TIntro
n1
Ann
ualF
EV1decline
(2011[34])
T=0082in
haplotype
Japanese
(915)
Parkinsonrsquos
disease(2010
[26])
T=0986350Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
rs10183914
(rs58731187
rs61374844
)g17809766
6Cgt
TIntro
n3
Parkinsonrsquos
disease(2010
[26])
T=0536188Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
rs2706110
g178092162Tgt
C31015840Flanking
Breastcancer
(2012[27])
TT
OR=2079CI
=118ndash368
FinlandKB
CP(452)
rs2588882
g178087165Ggt
T31015840Flanking
Infection-indu
cedasthma(
2012
[38])
TG
OR=0290CI
=013ndash0
62
Hun
garia
nCaucasia
nGypsy
(307)
minus686in
reference[22]
=minus653in
reference[23]
=currentlyminus214minus684in
reference[22]
=minus651inreference[23]
=currentlyminus212minus651inreference[22]
=minus617in
reference[23]
=currentlyminus178Ch
romosom
econtig(in
tron
31015840flank
ing)
orreversed
(51015840flank
ingprom
oter)a
llelesinbo
ldhave
been
used
inthetextand
TableOR
odds
ratio
CI95confi
denceinterval
14 Oxidative Medicine and Cellular Longevity
minus212GG (98 rs67006649 previously minus684minus651) minus178Callele (73) and GCC
4(53) was predicted to increase
respiratory failure development (hazard ratio = 095 CI091ndash099) in German COPD patients [32] Significant inter-action was also identified between an intronic SNP Gallele (rs6726395 g178103229AgtG 884 frequency) andsmoking status on FEV
1decline relative to the reference
AA allele in the above Japanese cohort [34] Siedlinskiet al [39] reported that the CC genotype of anotherintronic SNP (rs2364723 g178126546GgtC) was associatedwith a lower FEV
1level compared to the wild-type geno-
type (GG) in two Netherland cohorts (CI minus636minus178frequency = 0525 and pooled cohort size = 2542) ThisSNP alone or as a haplotype with 4 more intronic SNPs(rs13001694Grs1806649Trs4243387Trs6726395G)was alsoassociated with high FEV
1levels in individuals that ever
smoked [39] In a Hungarian population of childhoodasthma SNPs at minus178 (CA) and 31015840 flanking (rs2588882TG)loci were inversely associated with infection-induced asthma(OR 0437 CI 028ndash080 OR 0290 CI 013ndash062 resp) andthese SNPs significantly influenced an asthma-environmentalpollution interaction [38]The intronic SNP rs1806649 (CgtT)was associated but not significantly with an increased risk ofhospitalization during high-level particulate matter (PM
10)
periods in asthma or COPD patients (119899 = 209) of the UnitedKingdom (UK) [41] Asthma andCOPDadmission rateswererelated to the increase in environmental PM
10concentration
Importantly effects of interaction between prenatal stressand NRF2 SNPs on descendant pulmonary health wereinvestigated by the Avalon Longitudinal Study in the UKmaternal smoking during pregnancy was not associated withlung function change determined by maximum mild expi-ratory flow (FEF
25ndash75) or with asthma incidence in school-aged children and this relation was not modified by NRF2SNP genotypes [42]However early gestation acetaminophenexposure significantly influenced the risk of asthma andwheezing at the age of 7 years in gt4000 mothers and gt5000children [33] When maternal copies of the minus212A allele werepresent association with asthma (11374891 OR 173 CI122ndash245) and wheezing (11494949 OR 153 95 CI 106ndash220) was significantly increased [33]
Gastrointestinal Disorders While there was no evidence inlung cancer cases studies in Japanese populations suggesteda potential association of NRF2 variations with gastro-intestinal tumorigenesisHelicobacter pylori (H pylori) causesgastritis which can lead to gastric atrophy and cancer Ingastric epithelium from the Japanese cancer cohorts (39gastric cancers 46 controls) H pylori infection was posi-tively correlated with aberrant CpG island methylation oftumor suppressor genes (eg p14) and minus214Gminus212G orminus214Aminus212G NRF2 haplotype was significantly associatedwith increased (OR 290 95 CI 114ndash736) or decreased(OR 033 95 CI 013ndash088) risk of the CpG methyla-tion respectively in the H pylori-infected patients [29]Further study from the same investigators determined thatminus214Aminus212G allele carriers had significantly (119875 = 0022)reduced risk of gastric cancer inH pylori-negative cases [30]The minus214AGminus212AG genotypes were negatively associated
(OR 045 CI 022ndash093) and the minus214Gminus212G genotypeswere positively associated (chronic continuous phenotypeOR 257 CI 101ndash660) with ulcerative colitis (89 patients 141controls) in a Japanese population [31]
Autoimmune Disorders Systemic lupus erythematosus (SLE)is a long-term autoimmune disease more frequently found infemales than in males It affects organs including skin jointskidneys and brain and nephritis is an aggressive character-istic in some patients Genome-wide association studies inhumans identified a suggestive quantitative trait locus nearNRF2 [43] A study of a Mexican Mestizo population (362patients with childhood-onset SLE 379 controls and 212nephritis diagnosed) determined that lupus with nephritiswas significantly (OR 181 CI 104ndash312) associated with theminus214GA SNP in females [25] The same SNPs were notclosely associated with SLE risk in a Japanese cohort [22]Vitiligo is a skin condition in which there is a loss of browncolor (pigment) from areas of skin resulting in irregularwhite patches It is thought to be an autoimmune diseasecaused by loss of cells (melanocytes) that produce brownpigment A study indicated that theminus178A allele increased therisk of vitiligo dose-dependently (OR 1724 95 CI 135ndash221for CA OR 2902 CI 162ndash519 for AA) [35]
Female Disorders It is well known that estrogen metabolites(eg catechols) cause ROS formation suggesting correla-tion of NRF2 and downstream effectors in postmenopausalmammary cancer In a study of a Finish population (KuopioBreast Cancer Project 119899 = 452 patients 370 controls)the minus178AA homozygous genotype (OR 4656 CI = 135ndash1606) and 31015840 flanking rs2706110 (TT OR 2079 CI 118ndash368) genotype were associated with increased risk of breastcancer while the 51015840 flanking minus3306TT homozygous allelewas significantly associated with lower survival (frequency =71219 OR 1687 CI 1105ndash275) [27] suggesting that NRF2genetic polymorphisms affect susceptibility and outcome ofthe patients The minus178A allele carriers together with intronicrs1962142A allele carriers were associated with lowered tissuelevels of NRF2 proteins [27] In postmenopausal women theminus178A allele (OR 179 95CI 370ndash8570) appeared tomodifythe risk of venous thromboembolism caused by oral estrogentherapy (AA or AC frequency = 333) as demonstratedby the French ESTHER study (161 cases 474 controls) [36]An intronic rs1806649CgtT SNP did not associate with breastcancer risk in postmenopausal women [40] However whenthis SNP and other at-risk alleles of ARE-responsive genes(NQO1 HO-1 NOS3) were combined there was a significantgene-dose effect on the breast cancer risk [40] Althoughcoding region SNPs in NRF2 and KEAP1 were identifiedin the Japanese endometrial adenocarcinoma patients noassociation of NRF2 SNPs with the disease was found [44]
Neurodegenerative Diseases Oxidative stress is known to beinvolved in Parkinsonrsquos disease (PD) presumably due toproduction of ROS fromhigh-dopaminemetabolism and lowlevels of antioxidants in the substantia nigra of the brainInvestigators found a protective NRF2 haplotype consistingof four 51015840 flanking SNPs (minus5238Gminus214Aminus212Gminus178C)
Oxidative Medicine and Cellular Longevity 15
and 4 intronic SNPs (rs2886161Ars1806649Ars2001350Ars10183914A) from Swedish (OR 09 CI 060ndash140) and Polish(OR 04 CI 030ndash060) populations (total 165+192 PD cases190+192 controls) [26]The investigators also suggested thatNRF2 haplotype alleles were associated with 2 years earlierage of Alzheimerrsquos disease (AD) onset 4 years earlier age ofposterior subcapsular cataract surgery and 4 years later ageof cortical cataract surgery while they were not significantlyrelated to AD or age-related cataract risk [45]
34 Genetic Mutations in Mouse Nrf2 Tsang et al [46]compiled 673 SNPs in 55 mouse strains and constructedtheir phylogenetic tree to correlate and clarify the originsof strains based on the assembled mouse genome sequenceand SNP data [20 47 48] Recently using the completegenome sequence of C57BL6J (B6) mouse as a referencehigh-density SNP screening in other laboratory strains orin panels of strains has been published (see [17]) Althoughmillions of mouse SNPs (gt10089892 as of December 2012)and haplotype mappings from more than 120 strains havebeen published as valuable references for dissecting thegenetic basis of complex traits [49ndash51] little attention hasbeen paid to polymorphisms of Nrf2 and their correlationwith disease phenotypes
Figure 3 demonstrates the proximal promoter region(minus1 to minus950)51015840-UTR (exon 1 up to TSS) and proteinsequence of mouse Nrf2 based on GRCm38p1 PrimaryAssembly (75704641ndash75675513 bp) mRNA variant1 and protein (NP 035032 encoded by NM 010902234ndash2027 bp) sequences Genetic variations in the Nrf2genome of inbred strains collected from public databasesare listed in Tables S2 and 5 (See Supplementary TableS2) Overall 968 genetic mutations are compiled forNrf2 gene and 5 kb upstream2 kb downstream regions785 SNPs between B6 and another 16 strains wereacquired from the Mouse Phenome Database (MPDhttpphenomejaxorg dbqrtn=snpret1) and additionalSNPs and other mutations were acquired from NCBI dbSNP(httpwwwncbinlmnihgovsnpterm=mus+musculus20nfe2l2) In total 132 mutations are in the promoter (37in proximal 1 kb) 49 in exons (38 in coding region 19 Cns)727 in introns and 60 in the 31015840 flanking region Excludingmutations in the 51015840 and 31015840 flanking sequences murine Nrf2sequences appear to be more highly variable (1 variationper 375 bp) than much of the mouse genome which has anapproximate frequency of one SNP per every 245 bp (httpwwwinformaticsjaxorgmgihomehomepagesstatsallstatsshtmlallstats snp)
Nrf2 was found to be a susceptibility gene from genome-wide linkage analysis in a murine model of hyperoxia-induced ALI [52] A promoter SNP minus103TgtC (previouslypublished as minus336TgtC) in Nrf2 was found and predicted toadd an additional Sp1 binding site in hyperoxia-susceptibleB6 mice but not in resistant C3HHeJ mice [52] Genotypesfrom the SNP and from simple-sequence length polymor-phism markers of the Nrf2 locus (D2Mit248 and D2Mit94)cosegregated in the B6C3F
2mouse cohort [52] and Nrf2
deficient mice were significantly more susceptible to ALI
sub-phenotypes caused by hyperoxia than similarly exposedwild-type mice supporting Nrf2 as a contributor to thephenotypic traits [53] Although no other functional analyseson Nrf2 SNPs or haplotype association studies have beenconducted in inbred mice strains bearing haplotypes suchas multiple Cns in functional domains (eg F71L L451VH543Q and L575M) may be useful to elucidate the role ofNrf2 in differential susceptibility to oxidative diseases
4 Oncogenic Somatic Mutations in HumanNF-E2-Related Factor 2
Somatic mutation is a change in the DNA of somatic cellsthat affects derived cells but is not inherited by offspringEfforts to discover somatic mutations have provided insightinto mutagenesis and cancer development Lung cancerparticularly non-small cell lung cancer (NSCLC) is theleading cause of cancer death worldwide Somatic mutationsof NRF2 and KEAP1 discovered in lung cancer patients havedetermined the oncogenic potential ofNRF2 [54 55] KEAP1somatic mutations were associated with its reduced proteinlevels in lung cancer tissues and cells [56 57] Investigationsof NSCLC in various ethnic populations as well as cancersin gastrointestine breast and prostate have coordinatelydemonstrated that multiple Cns somatic mutations in KEAP1cause dysfunction of the translated protein and in turnconstitutive activation of NRF2 increasing risk of neoplasiaand chemoresistance [12 55 58 59] Somatic mutations ofNRF2 have been detected in various cancer tissues (largelysquamous cell carcinomas) in Asian populations (Table 6)NRF2 mutations were significantly associated with NSCLCcases (squamous cell lung carcinoma adenocarcinoma) ofthe Japanese (107 [54]) the Chinese (23 [60]) andthe Koreans (8 [61]) as well as with lung cancer celllines Smoking history was also correlated with mutationoccurrence in all of the studies [54 60 61] In addition tolung cancers laryngeal squamous carcinoma (13 in [61])esophageal squamous cancer (ESC 22 in [60] 114 in[61]) head and neck cancers (25 in [54]) skin (117 casein [61]) and oral cancer cell lines had somatic changesin NRF2 In contrast to wide-spread KEAP1 mutationsmutations in NRF2 were clustered in DLGETGE motifs ofthe Neh2 domain which are critical in the ldquohinge and latchrdquomodel of KEAP1 binding [12] Similar to KEAP1 somaticmutations it has been postulated that NRF2 mutations incancer cells lead to NRF2 accumulation by suppressing itsubiquitination or KEAP1 binding which eventually confersmalignant potential and resistance to chemotherapy
Most variable sites in NRF2 included aa residues 29 (AspD) 31 (Gly G) 77 (Asp D) and 79 (Glu E) (Table 6)Residue 33 (Ser S) in the Neh2 domain is mutated by eithergenetic or somatic processes (Figure 4) Cns in the EDGFmotif of NRF2 was experimentally determined to impairrecognition of KEAP1 [54] NRF2 mutations were signif-icantly correlated with increased (25-fold) copy number(31 of mutants versus 3 wild types) in Japanese NSCLCcases [63] Aberrant mutation of NRF2 also led to increasedexpression of downstream effectors includingRagD known to
16 Oxidative Medicine and Cellular Longevity
pL575M
pF71L
pH543QpL451V
pA151P pA151V pN159D pQ198delpV105D
pA204T pS224I225S226del pT253S pS278ApD305G
pM409V pR410HpP388L pA389G pT395I
Genomic DNA (reversed contig GRCm38 p1 C57BL6JNM 0109023)
Protein (NP 035032)1
51101151201251301351401451501551
minus400
minus500
minus50
minus100
minus200
minus300
minus450
minus350
minus250
minus150
minus950
minus900
minus850
minus800
minus750
minus700
151
101151201
2001
g minus470gg minusg minus
c minus41c minus
c
c
c c
gg minus 6C
g minus125insTTC g minus118insC
g minus281delC
g minus63delCg minus1 (75704642 c minus234)
Translation start site (c +1)Termination codon (c +2025 +2027)
c minus136 minus134in-frame stop codonc minus8585
g minus41
246
440
minus381
minus11
minus91 minus90
minus332
minus153
minus98
c minus197c minus209 minus202
gminus942 minus899 (cminus1175 minus1132) CpG islands [71]
g 460CgtT
g minus103CCgtT [51]
CgtTCgtG
g minus320CgtT
CgtT
CgtT
CgtTg minus426GgtA
GgtACgtA
CgtA
GgtA
g minus238GgtTTgtC
TgtC
TgtC
TgtCTgtG
g minus229AgtG
CgtG
CgtG
GgtC
gminus826 minus654 (cminus1059 ) CpG islands [71]minus887
Figure 3 DNA (partial promoter and exons 1 and 5) and protein sequence ofmouseNrf2 SNPs and amino acid residues for non-synonymousSNPs are marked Promoter regions bearing 5CpG islands are underlined
151
201251301351401451501551601
Genetic mutation (Table 3 and Figure 2)Somatic mutation (Table 6)
Neh2
Neh3
Neh4Neh5
Neh6
Neh1
Protein isoform 1 (NP 006155)
101151
Figure 4 Genetic and somatic mutation loci in human NRF2 protein
Oxidative Medicine and Cellular Longevity 17
Table5Mou
seup
stream
andexon
varia
tions
ofNfe2
l2locusin17
inbred
strainsR
eference
sequ
ence
isC5
7BL6J
(B6strain
1)geno
me(GI14933824975547698ndash75513576)SNPalleleand
geno
typesareshow
nas
chromosom
econ
tigsequ
enceStrains
2129S1S
vlmJ3AJ
4AKR
J5BA
LBcJ6C3
HH
eJ7C
57BL
6NJ8CA
STEiJ9CB
AJ
10D
BA2J11FVBNJ12LPJ
13N
ODShiLtJ14N
ZOH
ILtJ
15P
WK
PhJ16SPR
ETEiJ
17W
SBEiJ
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs2566
08517
75705565
GA
(CT)
51015840Flankingminus924
NC
GG
GG
GG
GG
GG
GG
GG
AA
Grs47274959
75705562
AT
(TA
)51015840Flankingminus921
NC
AT
TA
TT
AA
TT
AT
AT
TT
Ars216940398
75705554ndash75705555
Tadd(A
add)
51015840Flankingminus913minus914
NC
CGrs239114134
75705540
CT(G
A)
51015840Flankingminus899
NC
CT
TC
TT
CC
TT
CT
CT
CT
Crs46
461765
75705528
TG(A
C)oA
C
51015840Flankingminus887
NC
TG
GT
GG
TT
GG
TG
TG
GG
Trs5144
9853
75705525
CA(G
T)
51015840Flankingminus884
NC
CA
AC
AA
CC
AA
CA
CA
CC
Crs249093111
75705512
CT(G
A)
51015840Flankingminus871
NC
CT
TC
TT
CC
TT
CT
CT
TT
Crs263745200
75705510
CG(G
C)
51015840Flankingminus869
NC
GG
GG
GG
GG
GG
GG
GG
CG
Grs45651867
75705498
TG(A
C)
51015840Flankingminus857
NC
TG
GT
GG
TT
GG
TG
TG
GG
Trs219997531
75705495
GA
(CT)
51015840Flankingminus854
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs251918379
75705451
TA(A
T)
51015840Flankingminus810
NC
TT
TT
TT
TT
TT
TT
TT
TA
Trs27978306
75705449
TA(A
T)
51015840Flankingminus808
NC
TA
AT
AA
TT
AA
TA
TA
TT
Trs216087412
75705429
TG(A
C)lowast
51015840Flankingminus788
NC
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
GTlowast
Tlowast
rs261229914
75705423ndash75705424
AGdel(CT
del)
51015840Flankingminus783minus784
NC
AGrs27978307
75705400
CT(G
A)
51015840Flankingminus759
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs243167395
75705359
GA
(CT)
51015840Flankingminus718
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs27978308
75705307
GA
(CT)
51015840Flankingminus66
6NC
GG
GG
GG
GG
GG
GG
GG
AG
Grs254744
09875705294
AT
(TA
)51015840Flankingminus653
NC
AA
AA
AA
AA
AA
AA
AA
AT
Ars228133419
75705179
GT
(CA
)51015840Flankingminus538
NC
GG
GG
GG
GG
GG
GG
GG
GT
Grs214719520
75705111
GA
(CT)
51015840Flankingminus470
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs2440
87730
75705101
GA
(CT)
51015840Flankingminus46
0NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs227781699
75705081
CG(G
C)
51015840Flankingminus44
0NC
CC
CC
CC
CC
CC
CC
CC
GC
Crs257870353
75705067
CT(G
A)
51015840Flankingminus426
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs24487744
075705022
AG
(TC)
51015840Flankingminus381
NC
AA
AA
AA
AA
AA
AA
AA
AG
Ars234628138
75704973
AC
(TG
)51015840Flankingminus332
NC
AA
AA
AA
AA
AA
AA
AA
AC
A
18 Oxidative Medicine and Cellular Longevity
Table5Con
tinued
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs247275247
75704961
GA
(CT)
51015840Flankingminus320
NC
GG
GG
GG
GG
GG
GG
AG
GG
Grs247519047
75704922
Gdel(Cdel)
51015840Flankingminus281
NC
Grs27978309
(rs51915758)
75704887
GA
C(C
TG
)51015840Flankingminus246
NC
GA
AG
AA
GG
AA
GA
GA
CC
G
rs218139102
75704879
CA(G
T)
51015840Flankingminus238
NC
CC
CC
CC
CC
CC
CC
CC
AC
Crs251990355
75704870
TC(A
G)
51015840Flankingminus229
NC
TT
TT
TT
TT
TT
TT
TT
TC
Trs238746955
75704794
AG
(TC)
51015840Flankingminus153
NC
AA
AA
AA
AA
AA
AA
AA
GA
Ars221664
405
75704786
AG
(TC)
51015840Flankingminus145
NC
AA
AA
AA
AA
AA
AA
AA
AG
Ars217054035
75704766
ndash75704767
GAAadd(TTC
add)
51015840Flankingminus125minus126
NC
GA
rs248182931
75704759ndash75704760
Gadd(C
add)
51015840Flankingminus118
minus119
NC
AGrs217197904
75704744
GA
(CT)
51015840Flankingminus103
(+)S
p1[23]
GA
AG
AA
GG
AA
GA
AA
GG
Ars2644
9364975704704
Gdel(Cdel)
51015840Flankingminus63
NC
Grs2404
81112
757046
82CT(G
A)
51015840Flankingminus41
NC
CC
CC
CC
CC
CC
CC
CC
CT
Crs27978310
75704617
AG
(TC)
UTR
-525
NC
AA
AA
AA
AA
AA
AA
AA
GG
Ars27978311
75704610
CG(G
C)
UTR
-532
NC
GG
GG
GG
GG
GG
GG
GG
GG
Crs27978312
757046
05GA
(CT)
UTR
-537
NC
GA
AG
AA
GG
AA
GA
GA
GG
Grs214784220
757044
99AG
(TC)
UTR
-5143
NC
AA
AA
AA
AA
AA
AA
AA
GG
Ars244146318
757044
98GT
(CA
)UTR
-5144
NC
GG
GG
GG
GG
GG
GG
GG
GT
Grs27978313
757044
97GC
(CG
)UTR
-5145
NC
GC
CG
CC
GC
CC
GC
CC
CC
Crs215431944
757044
93CT(G
A)
UTR
-5149
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs257015697
757044
49GA
(CT)
UTR
-5193
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs252234782
757044
19GT
(CA
)UTR
-5223
NC
Grs1346
0861
75679262
AG
(TC)
Exon
244
6Cn
sF71L
AG
GA
GG
AG
GG
AG
AG
GG
Grs1346
0859
75679202
GA
(CT)lowast
Exon
2506
CsH91=
GA
AG
AA
GG
AA
GA
GA
GG
Grs27978436
75679187
GA
(CT)lowast
Exon
2521
CsT9
6=G
GG
GG
GG
AG
GG
GG
GA
GG
rs226131070
75679178
GA
(CT)
Exon
2530
CsS99=
GG
GG
GG
GG
GG
GG
GG
GA
Grs227683834
75678576
AT
(TA
)Ex
on3
547
CnsV
105D
AA
AA
AA
AA
AA
AA
AA
AT
Ars257321187
75678506
TC(A
G)
Exon
3617
CsP128=
TT
TT
TT
TC
TT
TT
TT
TT
Trs27978444
75678500
TA(A
T)
Exon
3623
CsV130=
TT
TT
TT
TA
TT
TT
TT
AT
Trs227743136
75677686
GA
(CT)
Exon
4662
CsH143=
GG
GG
GG
GG
GG
GG
GG
GA
Grs215327202
75677664
CG(G
C)
Exon
4684
CnsA
151P
CC
CC
CC
CG
CC
CC
CC
CC
Crs247539755
75677663
GA
(CT)
Exon
4685
CnsA
151V
GG
GG
GG
GA
GG
GG
GG
GG
Grs233164
668
75677640
TC(A
G)
Exon
4708
CnsN
159D
TT
TT
TT
TT
TT
TT
TT
TC
T
rs2340
95816
75677162ndash75677160
GCT
del
(AGCdel)
Exon
5826minus
828
Cds-in
delQ
198
GCT
Oxidative Medicine and Cellular Longevity 19
Table5Con
tinued
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs27978452
75677145
CT(G
A)
Exon
5843
CnsA
204T
CC
CC
CC
CC
CC
CC
CC
CC
T
rs225047937
75677086ndash75677078
GAG
ATCG
ATdel
(ATG
CATC
TCdel)
Exon
5902minus
910
Cds-in
del
(S224I225S226)
GAG
ATC
GAT
rs241469868
75676998
TA(G
T)
Exon
5990
CnT2
53S
TT
TT
TT
TT
TT
TT
TT
TA
Trs221602571
75676923
AC
(TG
)Ex
on5
1065
CnsS
278A
AA
AA
AA
AA
AA
AA
AA
AC
Ars252576472
75676876
GA
(CT)
Exon
51112
CsS293=
GG
GG
GG
GG
GG
GG
GG
GA
Grs250802933
75676841
TC(A
G)
Exon
5114
7Cn
sD305G
TT
TT
TT
TT
TT
TT
TT
TC
Trs225425698
75676792
CT(G
A)
Exon
5119
6Cs
P321=
CC
CC
CC
CC
CC
CC
CC
CT
Crs13459064
75676732
CT(G
A)
Exon
51256
CsT3
41=
CT
TC
TT
CT
TT
CT
CT
TC
Crs27978453
75676717
CT(G
A)
Exon
51271
CsA346=
CC
CC
CC
CC
CC
CC
CC
TC
Crs214335034
75676636
AG
(TC)
Exon
51352
CsD373=
AA
AA
AA
AA
AA
AA
AA
AG
Ars24958364
4756766
03AC
(TG
)Ex
on5
1385
CsP3
84=
AA
AA
AA
AA
AA
AA
AA
AC
Ars27978454
75676592
GA
(CT)
Exon
51396
CnsP
388L
GG
GG
GG
GG
GG
GG
GG
AG
Grs215384328
75676589
GC
(CG
)Ex
on5
1399
CnsA
389G
GG
GG
GG
GG
GG
GG
GG
GC
Grs251728286
75676571
GA
(CT)
Exon
51417
CnsT
395I
Grs247602334
75676567
TC(A
G)
Exon
51421
CsV396=
TT
TT
TT
TT
TT
TT
TT
TC
Trs234216231
75676530
TC(A
G)
Exon
51458
CnsM
409V
TT
TT
TT
TT
TT
TT
TT
TC
Trs212904337
75676526
CT(G
A)
Exon
51462
CnsR
410H
CC
CC
CC
CC
CC
CC
CC
CT
Crs252650779
75676522
TC(A
G)
Exon
51466
CsE4
11=
TT
TT
TT
TT
TT
TT
TT
TC
Trs231273560
75676516
TC(A
G)
Exon
51472
CsQ413=
TT
TT
TT
TT
TT
TT
TT
TC
Trs257886949
756764
13GT
(CA
)Ex
on5
1575
CsR4
48=
GG
GG
GG
GG
GG
GG
GG
GT
Grs241537608
756764
04GC
(CG
)Ex
on5
1584
CnsL
451V
GG
GG
GG
GG
GG
GG
GG
GC
Grs227619071
75676312
TC(A
G)
Exon
51676
CsQ481=
TT
TT
TT
TT
TT
TT
TT
TC
Trs258913831
75676290
AG
(TC)
Exon
51698
CsL4
89=
AA
AA
AA
AA
AA
AA
AA
AG
Ars225593319
75676126
AT
(TA
)Ex
on5
1862
CnsH
543Q
Ars4223233
75676105
GA
(CT)
Exon
51883
CsS550=
GG
GG
GG
GA
GG
GG
GG
GG
Grs4223232
75676032
GT
(CA
)Ex
on5
1956
CnsL
575M
Grs4223231
75675816
CG
UTR
-32172
NC
GG
GG
GG
GG
GG
GG
GG
GC
Grs1346
860
75675682
AT
UTR
-32306
NC
ASequ
ence
varia
tions
inmou
seNr
f2wereob
tained
from
Mou
sePh
enom
eDatabase(http
ph
enom
ejaxorgSNP)
andNCB
ISNPdatabase
(http
wwwncbinlm
nihgovSNP)N
CBIreference
sequ
ence
isMus
Musculusstra
inC5
7BL6J
chromosom
e2GRC
m38p1(NC
0000
687
GI37209910875704641ndash756755192
9123
bp)To
tal968
genetic
mutations
arerepo
rted
inNr
f2gene
and5k
bup
stream
(ge75704
642minus
1)and2k
bdo
wnstre
am(le75675512)sequences
asof
Janu
ary2003A
llSN
Psandallelesa
representedas
geno
miccontig
(reversedsequ
ence
indicated)E
xon175704
641ndash75704364(278
bpT
TS=75704
408)
exon
275679431ndash756791
65(267
bp)exon
375678579ndash75678490(90b
p)exon475677713ndash756775
46(168
bp)andexon
57567718
4ndash75678849
(1666
bp)Proteinam
inoacid
(aa)
resid
uesinNP00
61552
(597
aa)Cs
cod
ing-syno
nymou
sCn
scoding
-non
syno
nymou
sAminoacid
A-alanineD-aspartic
acidE
-glutamicacidF-phenylalanineG
-glycineH
-histidineI-iso
leucineL-leu
cineand
M-m
ethion
ineN-
asparagineP-prolin
eQ-glutamineR-arginineS-serineT-threon
ineandV-valin
elowastErrorsin
databasesfi
xedNC
notcon
firmed
20 Oxidative Medicine and Cellular Longevity
Table 6 NRF2 somatic mutations revealed in various human cancers
Domain Amino acid residues DNA mutation Cancer types (cases) ReferencesLocus Wild type Mutant
DLG motif
24 W (Trp) C (Cys) c72GgtCGgtT NSCLC neck ESC [54 62]K (Lys) c72TgtC ESC [62]
26 Q (Gln) E (Glu) c76CgtG NSCLC ESC [54 62]
27 D (Asp) G (Gly)lowast c80GgtA NSCLC [60]Y (Tyr) c79GgtT ESC [61]
28 I (Ile) T (Thr) c83CgtT NSCLC [54]
29 D (Asp) G (Gly) c86AgtG Head and neck ESC [54 62]H (His) c85GgtC NSCLC laynx [60 61]
30 L (Leu) F (Phe) c88CgtT NSCLC ESC [54 62]
31 G (Gly) A (Ala) c92GgtC NSCLC ESC skin [54 60ndash62]
32 V (Val) T (Thr) c95TgtG NSCLC [54]del c93 95delAGT ESC [61]
33ndash36 S-R-E-V S-R-E-V-S-R-E-Vlowast c97 108dupAGTCGAGCCGTA ESC [61]
34 R (Arg) Q (Gln) c101GgtA NCSCL [54 60 61]P (Pro) c101GgtC NSCLC [63]
ETGF motif
75 Q (Gln) H (His) c225AgtC Head and neck ESC [54 62]
77 D (Asp)
V (Val) c230AgtT NSCLC ESC [54 60 62]G (Gly) c230AgtG ESC [62]A (Ala) c230AgtC NSCLC [61]N (Asn) c229GgtA Larynx [61]
78 E (Glu) K (Lys) c232GgtA NSCLC ESC [54 62]
79 E (Glu)
K (Lys) c235GgtA NSCLC ESC [54 61 62]Q (Gln) c235GgtC NSCLC ESC [54 60ndash62]G (Gly) c236AgtG Larynx [61]E-E c234 236dupAGA ESC [61]
80 T (Thr) K (Lys) c239CgtACgtG NSCLC ESC [54 61 62]
80 T (Thr)P (Phe) c238AgtC ESC [62]I (Ile) c239CgtT Head and neck [54]A (Ala) c238AgtG NSCLC [63]
81 G (Gly) V (Val) c242GgtT ESC [61]D (Asp) c242GgtA NSCLC ESC [60ndash62]
82 E (Glu)
D (Asp) c246AgtT ESC oral cancer cell line [54 62]G (Gly) c245AgtG NSCLC [54]Q (Gln)lowast c244GgtC NSCLC ESC [60 61]V (Val)lowast c245AgtT ESC [61]
83 F (Phe) L (Leu)lowast c247TgtC NSCLC [60]NSCLC non-small cell lung cancer ESC esophageal squamous cancer and lowasterrors in reference fixed Number of cases 82 NSCLC and 10 ESC in [62] 125NSCLC 70 ESC 23 larynx and 17 skin in [61] 103 NSCLC and 12 head and neck in [54] 90 NSCLC in [63] 103 NSCLC in [60]
be involved in squamous lung cancer cell proliferation [64]suggesting that the mutation is functional and overcomesKEAP1 inhibition Singh et al [65] determined in vitrothat RNAi-mediated depletion of NRF2 in lung cancer cellsenhanced ROS production and susceptibility to cell deathby ionizing radiation These studies support the concept thatelevated NRF2 and ARE responsiveness provides cancer cellswith proliferative advantage for malignant transformation
and undue protection from anti-cancer therapy Onco-genic epidermal growth factor receptor (EGFR) signaling isrecently found to be critical in NRF2-mediated proliferationof NSCLC cells [66]
Collectively ldquogain of functionrdquo mutations in NRF2 thatreduce KEAP1 recognition are suggested to be predictivemarkers for poor responsiveness to chemotherapy and radi-ation therapy Although NRF2-mediated cellular defense
Oxidative Medicine and Cellular Longevity 21
processes are essential in the initiation stage enhancedNRF2-ARE activity in advanced stages of cancer developmentmay create a favorable intracellular environment for tumorcell growth and survival [67 68] In this context NRF2may be a potential molecular target for the treatment ofradio-resistance cancers especially those that have ldquoloss offunctionrdquo mutations in EGFR KRAS or KEAP1 as well asldquogain of functionrdquo mutations in NRF2
5 Epigenetic Alterations of NF-E2-RelatedFactor 2
Epigenetic modifications are alterations of molecules inter-acting with genes without changes to the primary DNAsequence They include post-translational modification ofhistones DNA methylation events chromatin conforma-tional changes and alterations to noncoding regulatoryRNAs Epigenetic alterations are stable and often inheritablebut are reversible and may affect expression of the geneDysregulation or defects in epigenetic processes particularlyhypermethylation of tumor suppressor gene promoters (egCpG islands) or histone modifications are thought to beassociated with carcinogenesis Investigators have reportedhypermethylation in CpG islands of KEAP1 which wereassociated with reduced KEAP1 expression in human cancersfrom lung prostate colon and so forth [69ndash72] Similar tosomatic mutations epigenetic changes on KEAP1 impairedthe function of its encoded protein leading to constitutiveNRF2 activation
Supporting the role of ldquopathogenic mutationsrdquo in NRF2expression of Nrf2 and downstream Nqo1 was suppressedin prostate tumors of mice (transgenic adenocarcinomaof mouse prostate TRAMP) Among 15 promoter CpGislands located between minus942 and minus654 (cminus1175 cminus1132and cminus1059 cminus887 gap in cminus1131 cminus1060 see Figure 3)hypermethylation of the first 5 CpG islands (minus942 minus899 andcminus1175 cminus1132) was significantly associated with tumorige-nesis [73] Moreover treatment with inhibitors for DNAmethyltransferase and histone deacetylase restored Nrf2expression in these tumor cells [73] A dietary phytochemicalcurcumin known as a DNA hypomethylation agent restoredepigenetically silent Nrf2 expression through CpG demethy-lation in carcinogen-induced mouse tumor cells [74]
The whole genome epigenetic datasets for 5 species arepublicly accessible at NCBI Epigenomics [75 76]The humanNRF2 epigenome of primary cells (breast penis) and H1stem cell line as well as mouse Nrf2 CpG island methylationdata for sperm blood and cerebellum are currently avail-able (httpwwwncbinlmnihgovepigenomics) Althoughno direct evidence of disease-associated epigenetic modu-lation has been identified in human NRF2 various phyto-chemical NRF2 agonists such as sulforaphane and curcuminhave shown their roles in DNA methylation and histonemodification (see reviews by Lee and colleagues eg [77])Taken together epigenetic modifications of theNRF2KEAP1axle are predicted to cause dysregulation of ARE-mediatedcellular defense leading to deleterious health effects and
phytochemical antioxidants as epigenetic modulators forNRF2 are suggested to be useful in cancer prevention
6 Conclusions
NRF2 is evolutionally conserved with high-sequence homol-ogy in many species However it is a highly mutable geneand numerous genetic variants have been discovered inhuman ethnic groups Importantly certain SNPs or haplo-types have been identified in various diseases as ldquoat-riskrdquoalleles and are related to functional alterations In additionto genetic variations multiple somatic mutations identifiedin the KEAP1 recognition domain of NRF2 in cancer cellshave been found to be oncogenic due to dysregulation ofNRF2 homeostasis by its excess ldquogain of functionrdquo Epigeneticalteration of theNRF2 is under investigation and is predictedto have pathogenic influences as learned from mouse andphytochemical agonist studies Continuous updates of Nrf2allelic variants in inbred mouse strains will provide a usefultool for effective experimental designs formodels of oxidativedisorders to provide insight into the disease mechanisms andintervention strategies
Abbreviations
AD Alzheimerrsquos diseaseALI acute lung injuryARE antioxidant response elementbZIP basic leucine zippercds coding DNA sequencesCI confidence intervalCOPD chronic obstructive pulmonary diseaseESC esophageal squamous cancerFEV1 forced expiratory volume in one second
Gbp giga base pairsGST glutathione S-transferaseGWAS genome-wide association studyHGVS Human Genome Variation SocietyHO-1 heme oxygenase-1119867 119901119910119897119900119903119894 119867119890119897119894119888119900119887119886119888119905119890119903 119901119910119897119900119903119894KEAP1 Kelch-like ECH activating protein 1MPD Mouse Phenome DatabaseMRP multidrug resistance proteinNCBI National Center for Biotechnology InformationNeh NRF2-ECH homologyNfe2l2 nuclear factor (erythroid-derived 2)-like 2Nrf2 NF-E2-related factor 2NSCLC nonsmall cell lung cancerNQO1 NAD(P)Hquinone oxidoreductase 1OR odds ratioPD Parkinsonrsquos diseasePM particulate matterROS reactive oxygen speciesSLE systemic lupus erythematosusSNP single nucleotide polymorphismSOD superoxide dismutaseURT untranslated region
22 Oxidative Medicine and Cellular Longevity
Disclosure
Authorrsquos contribution to the Work was done as part of theAuthorrsquos official duties as a NIH employee and is a Work ofthe United States Government Therefore copyright may notbe established in the United States
Conflict of Interests
The author declares that there is no conflict of interests
Acknowledgments
The research related to this paper was supported by theIntramural Research Program of the National Institutes ofHealth of the National Institute of Environmental HealthSciences (NIEHS) Drs Steven Kleeberger and StephanieLondon at the NIEHS provided excellent critical review ofthis paper The author thanks Mrs Jacqui Marzec for herhelpful comments and English editing
References
[1] J Y Chan M C Cheung P Moi K Chan and Y W KanldquoChromosomal localization of the humanNF-E2 family of bZIPtranscription factors by fluorescence in situ hybridizationrdquoHuman Genetics vol 95 no 3 pp 265ndash269 1995
[2] K Itoh K Igarashi N Hayashi M Nishizawa and MYamamoto ldquoCloning and characterization of a novel erythroidcell-derived CNC family transcription factor heterodimerizingwith the small Maf family proteinsrdquo Molecular and CellularBiology vol 15 no 8 pp 4184ndash4193 1995
[3] W O Osburn and T W Kensler ldquoNRF2 signaling an adaptiveresponse pathway for protection against environmental toxicinsultsrdquoMutation Research vol 659 no 1-2 pp 31ndash39 2008
[4] D Papp K Lenti D Modos D Fazekas Z Dul D Tureiet al ldquoThe NRF2-related interactome and regulome containmultifunctional proteins and fine-tuned autoregulatory loopsrdquoFEBS Letters vol 586 no 13 pp 1795ndash1802 2012
[5] X Wang D J Tomso B N Chorley et al ldquoIdentificationof polymorphic antioxidant response elements in the humangenomerdquo Human Molecular Genetics vol 16 no 10 pp 1188ndash1200 2007
[6] D Malhotra E Portales-Casamar A Singh et al ldquoGlobalmapping of binding sites for NRF2 identifies novel targets incell survival response through ChIP-Seq profiling and networkanalysisrdquo Nucleic Acids Research vol 38 no 17 pp 5718ndash57342010
[7] K Chan R Lu J C Chang and Y W Kan ldquoNRF2 a memberof the NFE2 family of transcription factors is not essential formurine erythropoiesis growth and developmentrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 93 no 24 pp 13943ndash13948 1996
[8] K Itoh T Chiba S Takahashi et al ldquoAn NRF2small Mafheterodimer mediates the induction of phase II detoxifyingenzyme genes through antioxidant response elementsrdquo Bio-chemical and Biophysical Research Communications vol 236no 2 pp 313ndash322 1997
[9] FMartin J M vanDeursen R A Shivdasani CW Jackson AG Troutman and P A Ney ldquoErythroid maturation and globin
gene expression inmicewith combined deficiency ofNF-E2 andNrf-2rdquo Blood vol 91 no 9 pp 3459ndash3466 1998
[10] K Itoh N Wakabayashi Y Katoh et al ldquoKEAP1 repressesnuclear activation of antioxidant responsive elements by NRF2through binding to the amino-terminal Neh2 domainrdquo Genesand Development vol 13 no 1 pp 76ndash86 1999
[11] A Kobayashi M I Kang H Okawa et al ldquoOxidative stresssensor KEAP1 functions as an adaptor for Cul3-based E3 ligaseto regulate proteasomal degradation of NRF2rdquo Molecular andCellular Biology vol 24 no 16 pp 7130ndash7139 2004
[12] K Taguchi H Motohashi and M Yamamoto ldquoMolecularmechanisms of the KEAP1-NRF2 pathway in stress responseand cancer evolutionrdquo Genes to Cells vol 16 no 2 pp 123ndash1402011
[13] K Mukaigasa L T Nguyen L Li H Nakajima M Yamamotoand M Kobayashi ldquoGenetic evidence of an evolutionarilyconserved role for NRF2 in the protection against oxidativestressrdquoMolecular and Cellular Biology vol 32 no 21 pp 4455ndash4461 2012
[14] JMaher andMYamamoto ldquoThe rise of antioxidant signalingmdashthe evolution and hormetic actions of NRF2rdquo Toxicology andApplied Pharmacology vol 244 no 1 pp 4ndash15 2010
[15] S C Lo X Li M T Henzl L J Beamer and M HanninkldquoStructure of the KEAP1NRF2 interface provides mechanisticinsight intoNRF2 signalingrdquoThe EMBO Journal vol 25 no 15pp 3605ndash3617 2006
[16] V A McKusick ldquoMendelian inheritance in man and its onlineversion OMIMrdquo American Journal of Human Genetics vol 80no 4 pp 588ndash604 2007
[17] B Yalcin D J Adams J Flint and T M Keane ldquoNext-generation sequencing of experimental mouse strainsrdquo Mam-malian Genome vol 23 no 9-10 pp 490ndash498 2012
[18] D R Bentley S Balasubramanian H P Swerdlow et alldquoAccurate whole human genome sequencing using reversibleterminator chemistryrdquo Nature vol 456 no 7218 pp 53ndash592008
[19] P S G Chain D V Grafham R S Fulton et al ldquoGenomeproject standards in a new era of sequencingrdquo Science vol 326no 5950 pp 236ndash237 2009
[20] RHWaterstonK Lindblad-Toh E Birney J Rogers J F AbrilP Agarwal et al ldquoInitial sequencing and comparative analysis ofthe mouse genomerdquo Nature vol 420 pp 520ndash562 2002
[21] T M Keane L Goodstadt P Danecek M A White K WongB Yalcin et al ldquoMouse genomic variation and its effect onphenotypes and gene regulationrdquoNature vol 477 pp 289ndash2942011
[22] T Yamamoto K Yoh A Kobayashi et al ldquoIdentification ofpolymorphisms in the promoter region of the human NRF2generdquo Biochemical and Biophysical Research Communicationsvol 321 no 1 pp 72ndash79 2004
[23] J M Marzec J D Christie S P Reddy et al ldquoFunctionalpolymorphisms in the transcription factor NRF2 in humansincrease the risk of acute lung injuryrdquo The FASEB Journal vol21 no 9 pp 2237ndash2246 2007
[24] H Fukushima-Uesaka Y Saito K Maekawa et al ldquoGeneticvariations and haplotype structures of transcriptional factorNRF2 and its cytosolic reservoir protein KEAP1 in JapaneserdquoDrug Metabolism and Pharmacokinetics vol 22 no 3 pp 212ndash219 2007
[25] E J Cordova R Velazquez-Cruz F Centeno V Baca and LOrozco ldquoThe NRF2 gene variant -653GA is associated with
Oxidative Medicine and Cellular Longevity 23
nephritis in childhood-onset systemic lupus erythematosusrdquoLupus vol 19 no 10 pp 1237ndash1242 2010
[26] M von Otter S Landgren S Nilsson et al ldquoAssociation ofNRF2-encoding NFE2L2 haplotypes with Parkinsonrsquos diseaserdquoBMCMedical Genetics vol 11 no 1 article 36 2010
[27] JMHartikainenM TengstromVMKosmaV L Kinnula AMannermaa andY Soini ldquoGenetic polymorphisms and proteinexpression of NRF2 and sulfiredoxin predict survival outcomesin breast cancerrdquo Cancer Research vol 72 pp 5537ndash5546 2012
[28] T Arisawa T Tahara T Shibata et al ldquoThe relationshipbetween Helicobacter pylori infection and promoter polymor-phism of the NRF2 gene in chronic gastritisrdquo InternationalJournal of Molecular Medicine vol 19 no 1 pp 143ndash148 2007
[29] T Arisawa T Tahara T Shibata et al ldquoThe influence ofpromoter polymorphism of nuclear factor-erythroid 2-relatedfactor 2 gene on the aberrant DNA methylation in gastricepitheliumrdquo Oncology Reports vol 19 no 1 pp 211ndash216 2008
[30] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene pro-moter polymorphism and gastric carcinogenesisrdquo Hepato-Gastroenterology vol 55 no 82-83 pp 750ndash754 2008
[31] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene promoterpolymorphism is associated with ulcerative colitis in a Japanesepopulationrdquo Hepato-Gastroenterology vol 55 no 82-83 pp394ndash397 2008
[32] C C Hua L C Chang J C Tseng C M Chu Y C Liu andW B Shieh ldquoFunctional haplotypes in the promoter region oftranscription factor NRF2 in chronic obstructive pulmonarydiseaserdquo Disease Markers vol 28 no 3 pp 185ndash193 2010
[33] S O Shaheen R B Newson S M Ring M J Rose-ZerilliJ W Holloway and A J Henderson ldquoPrenatal and infantacetaminophen exposure antioxidant gene polymorphismsand childhood asthmardquo The Journal of Allergy and ClinicalImmunology vol 126 no 6 pp 1141e7ndash1148e7 2010
[34] H Masuko T Sakamoto Y Kaneko et al ldquoLower FEV1 innon-COPD nonasthmatic subjects association with smokingannual decline in FEV1 total IgE levels and TSLP genotypesrdquoInternational Journal of Chronic Obstructive Pulmonary Diseasevol 6 pp 181ndash189 2011
[35] C P Guan M N Zhou A E Xu et al ldquoThe susceptibilityto vitiligo is associated with NF-E2-related factor 2 (NRF2)gene polymorphisms a study on Chinese Han populationrdquoExperimental Dermatology vol 17 no 12 pp 1059ndash1062 2008
[36] J Bouligand O Cabaret M Canonico et al ldquoEffect of NFE2L2genetic polymorphism on the association between oral estrogentherapy and the risk of venous thromboembolism in post-menopausal womenrdquo Clinical Pharmacology amp Therapeuticsvol 89 no 1 pp 60ndash64 2011
[37] D S OrsquoMahony B J Glavan T D Holden C Fong R A BlackG Rona et al ldquoInflammation and immune-related candidategene associations with acute lung injury susceptibility andseverity a validation studyrdquo PLoS ONE vol 7 no 12 Article IDe51104 2012
[38] I Ungvari E Hadadi V Virag et al ldquoRelationship between airpollutionNFE2L2 gene polymorphisms and childhood asthmain aHungarian populationrdquo Journal of CommunityGenetics vol3 no 1 pp 25ndash33 2012
[39] M SiedlinskiD S Postma JMA Boer et al ldquoLevel and courseof FEV1 in relation to polymorphisms inNFE2L2 andKEAP1 inthe general populationrdquo Respiratory Research vol 10 article 732009
[40] C C Hong C B Ambrosone J Ahn et al ldquoGenetic variabilityin iron-related oxidative stress pathways (NRF2 NQ01 NOS3
and HO-1) iron intake and risk of postmenopausal breastcancerrdquo Cancer Epidemiology Biomarkers and Prevention vol16 no 9 pp 1784ndash1794 2007
[41] C Canova C Dunster F J Kelly C Minelli P L Shah CCaneja et al ldquoPM10-induced hospital admissions for asthmaand chronic obstructive pulmonary disease the modifyingeffect of individual characteristicsrdquo Epidemiology vol 23 no 4pp 607ndash615 2012
[42] A J Henderson R B Newson M Rose-Zerilli S M RingJ W Holloway and S O Shaheen ldquoMaternal NRF2 andgluthathione-S-transferase polymorphisms donotmodify asso-ciations of prenatal tobacco smoke exposure with asthma andlung function in school-aged childrenrdquo Thorax vol 65 no 10pp 897ndash902 2010
[43] C Xing A L Sestak J A Kelly et al ldquoLocalization andreplication of the systemic lupus erythematosus linkage signalat 4p16 interaction with 2p11 12q24 and 19q13 in EuropeanAmericansrdquo Human Genetics vol 120 no 5 pp 623ndash631 2007
[44] T F Wong K Yoshinaga Y Monma K Ito H NiikuraS Nagase et al ldquoAssociation of KEAP1 and NRF2 geneticmutations and polymorphisms with endometrioid endometrialadenocarcinoma survivalrdquo International Journal of Gynecologi-cal Cancer vol 21 no 8 pp 1428ndash1435 2011
[45] M von Otter S Landgren S Nilsson et al ldquoNRF2-encodingNFE2L2 haplotypes influence disease progression but not riskin Alzheimerrsquos disease and age-related cataractrdquoMechanisms ofAgeing and Development vol 131 no 2 pp 105ndash110 2010
[46] S Tsang Z Sun B Luke et al ldquoA comprehensive SNP-basedgenetic analysis of inbred mouse strainsrdquoMammalian Genomevol 16 no 7 pp 476ndash480 2005
[47] C M Wade E J Kulbokas III A W Kirby et al ldquoThe mosaicstructure of variation in the laboratory mouse genomerdquoNaturevol 420 no 6915 pp 574ndash578 2002
[48] T Wiltshire M T Pletcher S Batalov et al ldquoGenome-widesingle-nucleotide polymorphism analysis defines haplotypepatterns in mouserdquo Proceedings of the National Academy ofSciences of the United States of America vol 100 no 6 pp 3380ndash3385 2003
[49] K A Frazer E Eskin H M Kang et al ldquoA sequence-basedvariation map of 827 million SNPs in inbred mouse strainsrdquoNature vol 448 no 7157 pp 1050ndash1053 2007
[50] T P Maddatu S C Grubb C J Bult andM A Bogue ldquoMousephenome database (MPD)rdquo Nucleic Acids Research vol 40 ppD887ndashD894 2007
[51] A Kirby H M Kang C M Wade et al ldquoFine mapping in 94inbred mouse strains using a high-density haplotype resourcerdquoGenetics vol 185 no 3 pp 1081ndash1095 2010
[52] H Y Cho A E Jedlicka S P M Reddy L Y Zhang T WKensler and S R Kleeberger ldquoLinkage analysis of susceptibilityto hyperoxia NRF2 is a candidate generdquo American Journal ofRespiratory Cell and Molecular Biology vol 26 no 1 pp 42ndash512002
[53] H Y Cho A E Jedlicka S P M Reddy et al ldquoRole of NRF2in protection against hyperoxic lung injury in micerdquo AmericanJournal of Respiratory Cell and Molecular Biology vol 26 no 2pp 175ndash182 2002
[54] T Shibata T Ohta K I Tong et al ldquoCancer related mutationsin NRF2 impair its recognition by KEAP1-Cul3 E3 ligase andpromote malignancyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 36 pp13568ndash13573 2008
24 Oxidative Medicine and Cellular Longevity
[55] A Singh V Misra R K Thimmulappa et al ldquoDysfunctionalKEAP1-NRF2 interaction in non-small-cell lung cancerrdquo PLoSMedicine vol 3 no 10 article e420 2006
[56] T Ohta K Iijima M Miyamoto et al ldquoLoss of KEAP1 functionactivates NRF2 and provides advantages for lung cancer cellgrowthrdquo Cancer Research vol 68 no 5 pp 1303ndash1309 2008
[57] B Padmanabhan K I Tong T Ohta et al ldquoStructural basisfor defects of KEAP1 activity provoked by its point mutationsin lung cancerrdquoMolecular Cell vol 21 no 5 pp 689ndash700 2006
[58] T Shibata A Kokubu M Gotoh et al ldquoGenetic alteration ofKEAP1 confers constitutive NRF2 activation and resistance tochemotherapy in gallbladder cancerrdquoGastroenterology vol 135no 4 pp 1358e4ndash1368e4 2008
[59] N J Yoo H R Kim Y R Kim C H An and S H LeeldquoSomatic mutations of the KEAP1 gene in common solidcancersrdquo Histopathology vol 60 no 6 pp 943ndash952 2012
[60] Y Hu Y Ju D Lin Z Wang Y Huang S Zhang et alldquoMutation of the NRF2 gene in non-small cell lung cancerrdquoMolecular Biology Reports vol 39 no 4 pp 4743ndash4747 2012
[61] Y R Kim J EOhM S Kim et al ldquoOncogenicNRF2mutationsin squamous cell carcinomas of oesophagus and skinrdquo TheJournal of Pathology vol 220 no 4 pp 446ndash451 2010
[62] T Shibata A Kokubu S Saito et al ldquoNRF2 mutation confersmalignant potential and resistance to chemoradiation therapyin advanced esophageal squamous cancerrdquo Neoplasia vol 13pp 864ndash873 2011
[63] H Sasaki M Shitara K Yokota Y Hikosaka S MoriyamaM Yano et al ldquoIncreased NRF2 gene (NFE2L2) copy numbercorrelates with mutations in lung squamous cell carcinomasrdquoMolecular Medicine Reports vol 6 no 2 pp 391ndash394 2012
[64] H Sasaki M Shitara K Yokota Y Hikosaka S Moriyama MYano et al ldquoRagD gene expression andNRF2mutations in lungsquamous cell carcinomasrdquo Oncology Letters vol 4 pp 1167ndash1170 2012
[65] A Singh M Bodas N Wakabayashi F Bunz and S BiswalldquoGain of NRF2 function in non-small-cell lung cancer cellsconfers radioresistancerdquo Antioxidants and Redox Signaling vol13 no 11 pp 1627ndash1637 2010
[66] T Yamadori Y Ishii SHommaYMorishimaKKurishimaKItoh et al ldquoMolecular mechanisms for the regulation of NRF2-mediated cell proliferation in non-small-cell lung cancersrdquoOncogene vol 31 pp 4768ndash4777 2012
[67] A K Bauer H Y Cho L Miller-Degraff C Walker K HelmsJ Fostel et al ldquoTargeted deletion of NRF2 reduces urethane-induced lung tumor development in micerdquo PLoS ONE vol 6no 10 Article ID e26590 2011
[68] H Y Cho and S R Kleeberger ldquoNRF2 protects against airwaydisordersrdquo Toxicology and Applied Pharmacology vol 244 no1 pp 43ndash56 2010
[69] R Wang J An F Ji H Jiao H Sun and D Zhou ldquoHyperme-thylation of the KEAP1 gene in human lung cancer cell linesand lung cancer tissuesrdquo Biochemical and Biophysical ResearchCommunications vol 373 no 1 pp 151ndash154 2008
[70] P Zhang A Singh S Yegnasubramanian et al ldquoLoss ofkelch-like ECH-associated protein 1 function in prostate cancercells causes chemoresistance and radioresistance and promotestumor growthrdquoMolecular Cancer Therapeutics vol 9 no 2 pp336ndash346 2010
[71] N Hanada T Takahata Q Zhou et al ldquoMethylation of theKEAP1 gene promoter region in human colorectal cancerrdquoBMCCancer vol 12 article 66 2012
[72] L A Muscarella R Barbano V DrsquoAngelo et al ldquoRegulationof KEAP1 expression by promoter methylation in malignantgliomas and association with patientrsquos outcomerdquo Epigeneticsvol 6 no 3 pp 317ndash325 2011
[73] S Yu T O Khor K L Cheung et al ldquoNRF2 expressionis regulated by epigenetic mechanisms in prostate cancer ofTRAMP micerdquo PLoS ONE vol 5 no 1 Article ID e8579 2010
[74] T O Khor Y Huang T Y Wu L Shu J Lee and A N KongldquoPharmacodynamics of curcumin as DNA hypomethylationagent in restoring the expression of NRF2 via promoter CpGsdemethylationrdquo Biochemical Pharmacology vol 82 no 9 pp1073ndash1078 2011
[75] I M Fingerman L McDaniel X Zhang et al ldquoNCBI epige-nomics a new public resource for exploring epigenomic datasetsrdquo Nucleic Acids Research vol 39 supplemen 1 pp D908ndashD912 2011
[76] I M Fingerman X Zhang W Ratzat N Husain R F Cohenand G D Schuler ldquoNCBI epigenomics whatrsquos new for 2013rdquoNucleic Acids Research vol 41 no 1 pp D221ndashD225 2013
[77] J H Lee T O Khor L Shu Z Y Su F Fuentes andA N Kong ldquoDietary phytochemicals and cancer preventionNRF2 signaling epigenetics and cell death mechanisms inblocking cancer initiation and progressionrdquo Pharmacology ampTherapeutics vol 137 no 2 pp 153ndash171 2012
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Oxidative Medicine and Cellular Longevity 7Ta
ble3Geneticmutations
inprom
oter
andexon
sofh
uman
NRF
2
IDMap
onchromosom
e2lowast
(HGVS
name)
Positionin
Nfe2
l2
(HGVS
name)
Region
s(position
from
mRN
A)
Varia
tioncla
ssand
consequences
(HGVS
name)dagger
Minor
allelefrequ
ency
(MAF)
MAcoun
tsDagger(coh
ort
size)
MAFsources
rs16865105
g178136629AgtC
cminus555minus
6770Tgt
G51015840Flanking
(minus6770)
SNP
C=01928421
1000
Genom
esrs7557529
g178135097CgtT
cminus555minus
5238GgtA
51015840Flanking
(minus5238)
SNP
nars6750320
g178131796CgtT
cminus555minus
1937GgtA
51015840Flanking
(minus1937)
SNP
T=0030266
1000
Genom
esrs18116
2518
g178131774TgtC
cminus555minus
1915AgtG
51015840Flanking
(minus1915)
SNP
C=000
051
1000
Genom
esrs1900
44775
g178131746CgtT
cminus555minus
1887GgtA
51015840Flanking
(minus1887)
SNP
T=000
051
1000
Genom
esrs185117338
g178131704AgtG
cminus555minus
1845Tgt
C51015840Flanking
(minus1845)
SNP
G=000
051
1000
Genom
esrs149947189
g178131697CgtT
cminus555minus
1838GgtA
51015840Flanking
(minus1838)
SNP
T=000
092
1000
Genom
esrs139771244
g178131625AgtG
cminus555minus
1766Tgt
C51015840Flanking
(minus1766)
SNP
G=000
092
1000
Genom
esrs6747203
g17813160
4CgtG
cminus555minus
1745GgtC
51015840Flanking
(minus1745)
SNP
G=000
613
1000
Genom
esrs193101749
g178131504TgtC
cminus555minus
1645AgtG
51015840Flanking
(minus1645)
SNP
C=000
4610
1000
Genom
esrs190630762
g178131495GgtC
cminus555minus
1636Cgt
G51015840Flanking
(minus1636)
SNP
C=000
092
1000
Genom
esrs183764
402
g178131366CgtA
cminus555minus
1507GgtT
51015840Flanking
(minus1507)
SNP
A=000
051
1000
Genom
esrs19122296
4g178131211GgtA
cminus555minus
1352Cgt
T51015840Flanking
(minus1352)
SNP
A=000
092
1000
Genom
esrs187137522
g178131165TgtG
cminus555minus
1306AgtC
51015840Flanking
(minus1306)
SNP
G=000
092
1000
Genom
esrs182620359
g178131158AgtG
cminus555minus
1299Tgt
C51015840Flanking
(minus1299)
SNP
G=000
051
1000
Genom
esrs4893819
(rs61433302)
g178131134CgtT
cminus555minus
1275GgtA
51015840Flanking
(minus1275)
SNP
C=04263931
1000
Genom
es
rs191547130
g178131017CgtT
cminus555minus
1158GgtA
51015840Flanking
(minus1158)
SNP
T=000
051
1000
Genom
esrs188422217
g178131003AgtG
cminus555minus
1144TgtC
51015840Flanking
(minus114
4)SN
PG=000143
1000
Genom
esrs143047764
g178130865AgtG
cminus555minus
1006Tgt
C51015840Flanking
(minus1006)
SNP
G=000
6915
1000
Genom
esrs74432849
g178130766CgtA
cminus555minus
907GgtT
51015840Flanking
(minus907)
SNP
nars116
79252
g178130691CgtG
cminus555minus
832GgtC
51015840Flanking
(minus832)
SNP
nars12993217
g178130516AgtG
cminus555minus
657TgtC
51015840Flanking
(minus657)
SNP
nars115644
826
g17813044
2TgtA
cminus555minus
583AgtT
51015840Flanking
(minus583)
SNP
A=0015133
1000
Genom
esrs140803524
g178130431GgtA
cminus555minus
572CgtT
51015840Flanking
(minus572)
SNP
A=000
4610
1000
Genom
es
rs776844
20g178130427TgtC
cminus555minus
568AgtG
51015840Flanking
(minus568)
SNP
C=0033974
C=0190(84)
1000
Genom
es[24]
rs183651094
g178130336AgtT
cminus555minus
477TgtA
51015840Flanking
(minus477)
SNP
T=000184
1000
Genom
es
rs35652124
sect
(rs57695243)
g178130073TgtC
cminus555minus
214AgtG
51015840Flanking
(minus214)
SNP
C=03512767
T=0429(84)
T=0413181Dagger
C=033827Dagger
C=03517
69
1000
Genom
es[24]
[22]
[23]
[25]
rs6706649
sectg178130071CgtT
cminus555minus
212GgtA
51015840Flanking
(minus212)
SNP
T=0078170
T=004
8(84)
T=004
821Dagger
T=00753Dagger
1000
Genom
es[24]
[22]
[23]
rs1506
48896
g178130047CgtG
cminus555minus
188GgtC
51015840Flanking
(minus188)
SNP
G=000235
G=000
6(84)
1000
Genom
es[24]
rs6721961
sect
(rs117801448)
g178130037TgtC
TgtG
cminus555minus
178AgtC
AgtG
51015840Flanking
(minus178)
SNP
T=0150328
T=0283124Dagger
T=031325Dagger
T=0321(84)
1000
Genom
es[22]
[23]
[24]
8 Oxidative Medicine and Cellular Longevity
Table3Con
tinued
IDMap
onchromosom
e2lowast
(HGVS
name)
Positionin
Nfe2
l2
(HGVS
name)
Region
s(position
from
mRN
A)
Varia
tioncla
ssand
consequences
(HGVS
name)dagger
Minor
allelefrequ
ency
(MAF)
MAcoun
tsDagger(coh
ort
size)
MAFsources
rs20134560
4g178129924178129925insG
cminus555-66minus555-65insC
51015840Flanking
(minus66minus65)
Insertion
G=0017939
1000
Genom
es
rs200432479
g178129741178
129742delAA
cminus438minus437delT
Texon
1UTR
-51015840(118minus119
)Dele
tion
-=000378
-=000
6(84)
1000
Genom
es[24]
rs75485459
g178129608CgtA
cminus304GgtT
Exon
1UTR
-51015840(252)
SNP
nars192086766
g17812946
6CgtT
cminus162GgtA
Exon
1UTR
-51015840(394)
SNP
T=002248
1000
Genom
esmdash
g17812944
2GgtA
cminus138CgtT
Exon
1UTR
-51015840(418)
SNP
A=0012(84)
[24]
rs7166
8246
g17812940
0delG
cminus96delC
Exon
1UTR
-51015840(460)
Dele
tion
nars187291840
g178129399CgtT
cminus95GgtA
Exon
1UTR
-51015840(461)
SNP
T=0054912
01000
Genom
es
rs143406266
g178129391178129393delGGC
cminus89minus87delGCC
Exon
1UTR
-51015840(467minus469)
Dele
tion
-=064
4282Dagger
-=0589(84)
[22]
[24]
rs182428269
g178098918GgtA
c127Cgt
TEx
on2(682)
Cns(pArg43Trp)
A=000
051
1000
Genom
esrs35248500
g178098917CgtT
c128GgtA
Exon
2(683)
Cns(pArg43Gln)
T=000
613
1000
Genom
esrs1135118
g178098831CgtT
c214GgtA
Exon
2(769)
Cns(pAla72Th
r)TDagger
[23]
rs19969166
0g178098829AgtT
c216Tgt
AEx
on2(771)
Cs(pAla72=)
namdash
g178098769GgtA
c276Cgt
TEx
on2(831)
Cs(pIle92=)
TDagger[23]
rs5031039
g178098750AgtG
c295Tgt
CEx
on2(850)
Cns(pSer99P
ro)
G=0Dagger
[23]
rs200239262
g178098017CgtG
c363GgtC
Exon
3(918)
Cns(pGln121H
is)na
rs183034165
g178098008TgtC
c372GgtA
Exon
3(927)
Cs(pAla124=
)T=000
092
T=000
6(84)
1000
Genom
es[24]
rs199970826
g178097996CgtT
c384GgtA
Exon
3(939)
Cs(pPro128=
)T=000
051
1000
Genom
esrs201992337
g178097260CgtT
c454GgtA
Exon
4(100
9)Cn
s(pGlu152Lys)
nars201589693
g178097251CgtT
c463GgtA
Exon
4(1018)
Cns(pV
al155Ile)
T=000
051
1000
Genom
es
rs35577826
g178097185AgtC
c529Tgt
GEx
on4(1084)
Cns(pLeu177Va
l)C=000143
Alt0005Dagger
1000
Genom
es[23]
rs181513314
g178096710CgtT
c621GgtA
Exon
5(1176)
Cn(pLeu207=
)T=000
051
1000
Genom
esrs60132461
g178096675TgtC
c656AgtG
Exon
5(1211)
Cns(pLys219Arg)
C=000184
1000
Genom
es
rs139187151
g178096634GgtA
c697Cgt
TEx
on5(1252)
Cns(pPro233Ser)
A=000
051
A=0012Dagger
(84)
1000
Genom
es[24]
rs35557421
g178096620d
elT
c711delA
Exon
5(1266)
Fram
eshiftdeletio
n(pLys237=fs)
na
rs34154613
g178096529CgtT
c802GgtA
Exon
5(1357)
Cns(pV
al268M
et)
T=000184
1000
Genom
esrs141363120
g178096
406GgtA
c925Cgt
TEx
on5(14
80)
Cns(pLeu309Ph
e)A=000378
1000
Genom
esrs201661476
g178096380AgtC
c951Tgt
GEx
on5(1506)
Cns(pIle
317M
et)
na
Oxidative Medicine and Cellular Longevity 9
Table3Con
tinued
IDMap
onchromosom
e2lowast
(HGVS
name)
Positionin
Nfe2
l2
(HGVS
name)
Region
s(position
from
mRN
A)
Varia
tioncla
ssand
consequences
(HGVS
name)dagger
Minor
allelefrequ
ency
(MAF)
MAcoun
tsDagger(coh
ort
size)
MAFsources
rs199673454
g178096309TgtA
c1022AgtT
Exon
5(1577)
Cns(pAs
p341Va
l)A=000
051
1000
Genom
esrs35007548
g178096299GgtA
c1032CgtT
Exon
5(1587)
Cs(pSer344=
)A=000
092
1000
Genom
esrs200209692
g178096287TgtC
c104
4AgtG
Exon
5(1599)
Cs(pLeu348=
)C=000
051
1000
Genom
es
mdashg178096237CgtA
c1094GgtT
Exon
5(1649)
Cns(pSer365Ile
)A=0125273
A=000
6Dagger(84)
PubM
ed[24]
rs201214197
g178096171CgtT
c1160GgtA
Exon
5(1715)
Cns(pSer387Asn)
T=000
051
1000
Genom
esrs200494292
g178096165TgtC
c1166AgtG
Exon
5(1721)
Cns(pLys389Arg)
nars186171287
g178096115GgtA
c1216CgtT
Exon
5(1771)
Cns(pPro4
06Ser)
A=000
051
1000
Genom
esrs182276775
g178096062CgtA
c1269GgtT
Exon
5(1824)
Cns(pGlu423A
sp)
A=000
051
1000
Genom
esrs201560221
g17809604
8TgtC
c1283AgtG
Exon
5(1838)
Cns(pLys428Arg)
nars189238236
g178096043AgtG
c1288TgtC
Exon
5(1843)
Cs(pLeu430=
)G=000
051
1000
Genom
esrs184287392
g178096022GgtA
c1309CgtT
Exon
5(1864)
Cns(pArg437Trp)
A=000
051
1000
Genom
esrs201871588
g178095986GgtA
c1345CgtT
Exon
5(19
00)
Cns(pAg449Cy
s)na
rs181294188
g178095985TgtC
c1346GgtA
Exon
5(19
01)
Cns(pArg44
9His)
T=000
092
1000
Genom
esrs20169046
6g178095973TgtA
c1358AgtT
Exon
5(19
13)
Cns(pHis4
53Leu)
A=000
051
1000
Genom
esrs105704
4(rs52789869)
g178095781CgtT
c1550GgtA
Exon
5(2105)
Cns(pArg517Lys)
TDagger[23]
rs200750800
g178095603AgtG
c1728TgtC
Exon
5(2283)
Cs(pTyr576=
)na
rs200175942
g178095567AgtG
c1764TgtC
Exon
5(2319)
Cs(pAsp588=
)G=000
051
1000
Genom
esrs77547666
g178095495GgtC
clowast18Cgt
GEx
on5UTR
-31015840(2391)
SNP
C=000
6915
1000
Genom
esrs73031353
g178095425TgtC
clowast88AgtG
Exon
5UTR
-31015840(2461)
SNP
C=000184
1000
Genom
esrs675944
3g178095345TgtC
clowast168AgtG
Exon
5UTR
-31015840(2541)
SNP
C=000
419
1000
Genom
esrs188674558
g178095279CgtA
clowast234GgtT
Exon
5UTR
-31015840(2607)
SNP
A=000
051
1000
Genom
esrs77685897
g178095247AgtG
clowast266TgtC
Exon
5UTR
-31015840(2639)
SNP
G=000
092
1000
Genom
esrs1057092
g178095162TgtG
clowast351AgtC
Exon
5UTR
-31015840(2724)
SNP
nars3197704
g178095162TgtG
clowast351AgtC
Exon
5UTR
-31015840(2724)
SNP
nars184701151
g178095159TgtC
clowast354AgtG
Exon
5UTR
-31015840(2727)
SNP
C=000276
1000
Genom
esrs11543307
g178095153AgtG
clowast360TgtC
Exon
5UTR
-31015840(2733)
SNP
nars111874043
g178095146AgtG
clowast367TgtC
Exon
5UTR
-31015840(2740)
SNP
nars34012004
g178095102AgtC
clowast411TgtG
Exon
5UTR
-31015840(2784)
SNP
C=0071
[24]
10 Oxidative Medicine and Cellular Longevity
Table3Con
tinued
IDMap
onchromosom
e2lowast
(HGVS
name)
Positionin
Nfe2
l2
(HGVS
name)
Region
s(position
from
mRN
A)
Varia
tioncla
ssand
consequences
(HGVS
name)dagger
Minor
allelefrequ
ency
(MAF)
MAcoun
tsDagger(coh
ort
size)
MAFsources
rs201481890
g178095090178095091in
sTclowast
422lowast423insA
Exon
5UTR
-31015840
(2795minus
2796)
Insertion
na
rs3082500
g178095089178095090d
elTT
delTTinsT
clowast423lowast424d
elAAinsA
Exon
5UTR
-31015840
(2796minus
2797)
Dele
tion
insertion
na
rs71792546
(rs71796710)
g178095079d
elT
clowast425delA
Exon
5UTR
-31015840(2798)
Dele
tion
na
rs1057106
g178095078AgtC
AgtT
clowast435TgtA
TgtG
Exon
5UTR
-31015840(2808)
SNP
na
rs34176791
g178095076AgtC
clowast437TgtG
Exon
5UTR
-31015840(2810)
SNP
C=000235
1000
Genom
esrs35911553
g178095045CgtT
clowast46
8GgtA
Exon
5UTR
-31015840(2841)
SNP
T=000
6915
1000
Genom
esSequ
ence
varia
tions
inup
stream
andexon
sofh
uman
NRF
2fro
m655varia
tions
availablein
publicdatabase
asof
Decem
ber2012
(583
activ
esomeSN
Psmergedge14
SNPs
citedin
PubM
ed)lowastNCB
Ireference
sequ
ence
NC
0000
0211
(Hom
osapiens
chromosom
e2G
RCh37p
10prim
arya
ssem
bly)spanning
178095033ndash1781298
59bp
(com
plem
ent34827
bpforexons
andintro
ns)51015840-Flank
ingregions
startat17812
9860
bp(minus1)in
transcrip
tvariant
1HGVS
Hum
anGenom
eVa
riatio
nSo
ciety
Position
sinvaria
nt1(NM
006164
4)2859
bpsectSN
Pscitedin
PubM
edE
xon11781298
59ndash17812
9260
(600
bpT
TS=1781293
04)exon
2178098999ndash
178098733(267
bp)exon
3178098067ndash1780979
78(90b
p)exon41780973
11ndash178097120
(192b
p)and
exon
5178096736ndash
178095033(1704b
p)daggerProteinam
inoacid
(aa)
resid
uesiniso
form
1(N
P00
61552605
aa)Cn
scoding
-non
syno
nymou
sCs
cod
ing-syno
nymou
sDaggerheterozygositydetectednano
tavailable
Oxidative Medicine and Cellular Longevity 11
Table4Hum
anNRF
2SN
Psassociated
with
diseaser
isk
IDMap
onchromosom
e2 (H
GVS
name)
Region
class
Dise
asea
ssociatio
nandreferences
Risk
allelesa
ndstatistics
Ethn
icgrou
p(num
bero
fcase)
rs7557529
g178135097CgtT
minus5238
Ggt
AParkinsonrsquos
disease(2010
[26])
G=0718252Dagger
inhaplotype
OR=090
or040
CI=
060ndash140or
03ndash06
Swedish
Polish
Caucasian
(357)
rs2886162
g178133165AgtG
minus3306
TgtC
Breastcancer
survival(2012[27])
TT=0324
OR=16
87C
I=1105ndash275
FinlandKB
CP(452)
Chronicg
astritisgastric
ulcer(2007
[28])
GDagger
GDaggerin
haplotypefor
ulcer(OR=
252C
I=119ndash
545)
Japanese
(159)
P14methylationin
gastric
cancer
Hpyloriinfectio
n(2008[29])
Gin
haplotype
OR=290C
I=114ndash
736
Japanese
(209)
Gastriccancer
inHpylori-n
egative
cases(2008
[30])
Ain
haplotype
119875=0022
Japanese
(209)
rs35652124
(rs57695243)
g178130073TgtC
minus214Agt
G(previou
slyminus653[23]
orminus686[22])
Ulcerativec
olitis(2008
[31])
AG
(OR=045C
I=022ndash0
93)
G(O
R=257C
I=10
1ndash660)
Japanese
(89)
Lupu
swith
neph
ritisin
female(2010
[25])
GA
OR=18
1CI
=10
4ndash312
Mexican
mestizo(362)
Parkinsonrsquos
disease(2010
[26])
A=0884312Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
COPD
(2010[32])
G=052Dagger
hazard
ratio
=095C
I=091ndash0
99
(Haplotype)
German
(69)
Gastriculcer(2007
[28])
GDaggerin
haplotype
OR=252C
I=119ndash
545
Japanese
(159)
P14methylationin
gastric
cancer
H
pyloriinfection(2008[29])
Gin
haplotype
OR=290C
I=114ndash
736
Japanese
(209)
Gastriccancer
inHpylori-negative
cases(2008
[30])
Gin
haplotype119875=0022
Japanese
(209)
rs6706
649
g178130071CgtT
minus212Ggt
A(previou
slyminus651[23]
orminus684[22])
Ulcerativec
olitis(2008
[31])
AG
(OR=045C
I=022ndash0
93)
G(O
R=257C
I=10
1ndash660)
Japanese
(89)
Maternalacetaminop
henandasthma
(2010[33])
A=02321137Dagger
OR=17
3CI
=12
2245
UKALSPA
C(gt40
00mothersgt
5000
child
ren)
12 Oxidative Medicine and Cellular Longevity
Table4Con
tinued
IDMap
onchromosom
e2 (H
GVS
name)
Region
class
Dise
asea
ssociatio
nandreferences
Risk
allelesa
ndstatistics
Ethn
icgrou
p(num
bero
fcase)
COPD
(2010[32])
G=098Daggerin
haplotype
hazard
ratio
=095C
I=091ndash0
99
German
(69)
Parkinsonrsquos
disease(2010
[26])
G=0972343Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
03ndash06
Swedish
Polish
Caucasian
(357)
Acutelun
ginjury
follo
wingtrauma
(2007[23])
CA=0119Dagger
OR=644
CI=
134ndash
3080
Caucasia
nAfrican-
American
(164
)
Ann
ualF
EV1decline
(2011[34])
A=0082in
haplotype
Japanese
(915)
Vitiligo(2008[35])
CAA
AOR=17
24C
I=13
5ndash221
Chinese(300)
COPD
(2010[32])
G=073Daggerin
haplotype
hazard
ratio
=095C
I=091ndash0
99
German
(69)
rs6721961
(rs117801448)
g178130037TgtC
TgtG
minus178Agt
GAgt
C(previou
slyminus617[23]
orminus650[22])
Parkinsonrsquos
disease(2010
[26])
C=0980346Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
Postm
enop
ausalvenou
sthrombo
embo
lism
(2011[36])
A=033311
OR=25CI
=370ndash8570
Caucasia
n(161)
Breastcancer
survivalandNRF
2proteinexpressio
n(2012[27])
AA
OR=4656CI
=13
5ndash1606
FinlandKB
CP(452)
Acutelun
ginjury-related
mortality
followingsyste
micinflammatory
respon
sesynd
rome(2012
[37])
GG
OR=97
3CI
=12
7ndash7480
Caucasia
n(750)
Infection-indu
cedasthma(
2012
[38])
AC
OR=0437CI
=028ndash0
80
Hun
garia
nCaucasia
nGypsy
(307)
rs143406266
g178129391178
129393delGGC
Exon
1(467ndash4
69G
CC)
COPD
(2010[32])
GCC
4=053Dagger
hazard
ratio
=095C
I=091ndash0
99
Taiwanese(69)
rs2886161
g178127839Tgt
ClowastIntro
n1
Parkinsonrsquos
disease(2010
[26])
T=0878309Dagger
inhaplotype
OR=09or
04CI
=06ndash
14or
03ndash06
Swedish
Polish
Caucasian
(357)
rs2364723
g178126546Ggt
CIntro
n1
Basaland
smoker
FEV
1(200
9[39])
CI=minus6360simminus1780C
=0525Dagger
Also
ashaplotype
Netherla
nd(2542)
rs2364722
g178124787Agt
GIntro
n1
Ann
ualF
EV1decline
(2011[34])
A=0082in
haplotype
Japanese
(915)
rs13001694
g178118990Agt
GIntro
n1
Basaland
smoker
FEV
1(200
9[39])
G=040
11578Dagger
inhaplotype
Netherla
nd(2542)
Oxidative Medicine and Cellular Longevity 13
Table4Con
tinued
IDMap
onchromosom
e2 (H
GVS
name)
Region
class
Dise
asea
ssociatio
nandreferences
Risk
allelesa
ndstatistics
Ethn
icgrou
p(num
bero
fcase)
Breastcancer
[40]
Twith
NQO1N
OS3H
O1risk
allelesOR=15
6CI
=097ndash251
Caucasia
nandothers(505)
rs1806
649
(rs58745895)
g178118152Cgt
TIntro
n1
Basaland
smoker
FEV
1(200
9[39])
T=02631119Daggerin
haplotype
CI=minus8730ndash
(minus17
0)Netherla
nd(2542)
Parkinsonrsquos
disease(2010
[26])
T=0422148Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
Particulatem
attera
ndasthmaCO
PDadmiss
ion(2012[41])
Cwith
lowvitamin
Clevel(OR=
31CI
=15
0ndash630)
UK(209)
rs4243387
(rs6003846
4)g178117765Cgt
TIntro
n1
Basaland
smoker
FEV
1(200
9[39])
T=00914
25Daggerin
haplotype
Netherla
nd(2542)
rs1962142
(rs58448508)
g178113484Agt
GIntro
n1
Ann
ualF
EV1decline
(2011[34])
A=0082in
haplotype
Japanese
(915)
Breastcancer
NRF
2andARE
expressio
n(2012[27])
A(119875=0036)
FinlandKB
CP(452)
rs6726395
(rs57309289)
g178103229Agt
GIntro
n1
Smok
ing-relatedFE
V1decline
and
annu
alFE
V1decline
(2011[34])
G=0884Dagger
A=0082in
haplotype
Japanese
(915)
Basaland
smoker
FEV
1(200
9[39])
G=046
41764Daggerin
haplotype
Netherla
nd(2542)
rs2001350
(rs17515179
rs60883775)
g178100
425Cgt
TIntro
n1
Ann
ualF
EV1decline
(2011[34])
T=0082in
haplotype
Japanese
(915)
Parkinsonrsquos
disease(2010
[26])
T=0986350Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
rs10183914
(rs58731187
rs61374844
)g17809766
6Cgt
TIntro
n3
Parkinsonrsquos
disease(2010
[26])
T=0536188Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
rs2706110
g178092162Tgt
C31015840Flanking
Breastcancer
(2012[27])
TT
OR=2079CI
=118ndash368
FinlandKB
CP(452)
rs2588882
g178087165Ggt
T31015840Flanking
Infection-indu
cedasthma(
2012
[38])
TG
OR=0290CI
=013ndash0
62
Hun
garia
nCaucasia
nGypsy
(307)
minus686in
reference[22]
=minus653in
reference[23]
=currentlyminus214minus684in
reference[22]
=minus651inreference[23]
=currentlyminus212minus651inreference[22]
=minus617in
reference[23]
=currentlyminus178Ch
romosom
econtig(in
tron
31015840flank
ing)
orreversed
(51015840flank
ingprom
oter)a
llelesinbo
ldhave
been
used
inthetextand
TableOR
odds
ratio
CI95confi
denceinterval
14 Oxidative Medicine and Cellular Longevity
minus212GG (98 rs67006649 previously minus684minus651) minus178Callele (73) and GCC
4(53) was predicted to increase
respiratory failure development (hazard ratio = 095 CI091ndash099) in German COPD patients [32] Significant inter-action was also identified between an intronic SNP Gallele (rs6726395 g178103229AgtG 884 frequency) andsmoking status on FEV
1decline relative to the reference
AA allele in the above Japanese cohort [34] Siedlinskiet al [39] reported that the CC genotype of anotherintronic SNP (rs2364723 g178126546GgtC) was associatedwith a lower FEV
1level compared to the wild-type geno-
type (GG) in two Netherland cohorts (CI minus636minus178frequency = 0525 and pooled cohort size = 2542) ThisSNP alone or as a haplotype with 4 more intronic SNPs(rs13001694Grs1806649Trs4243387Trs6726395G)was alsoassociated with high FEV
1levels in individuals that ever
smoked [39] In a Hungarian population of childhoodasthma SNPs at minus178 (CA) and 31015840 flanking (rs2588882TG)loci were inversely associated with infection-induced asthma(OR 0437 CI 028ndash080 OR 0290 CI 013ndash062 resp) andthese SNPs significantly influenced an asthma-environmentalpollution interaction [38]The intronic SNP rs1806649 (CgtT)was associated but not significantly with an increased risk ofhospitalization during high-level particulate matter (PM
10)
periods in asthma or COPD patients (119899 = 209) of the UnitedKingdom (UK) [41] Asthma andCOPDadmission rateswererelated to the increase in environmental PM
10concentration
Importantly effects of interaction between prenatal stressand NRF2 SNPs on descendant pulmonary health wereinvestigated by the Avalon Longitudinal Study in the UKmaternal smoking during pregnancy was not associated withlung function change determined by maximum mild expi-ratory flow (FEF
25ndash75) or with asthma incidence in school-aged children and this relation was not modified by NRF2SNP genotypes [42]However early gestation acetaminophenexposure significantly influenced the risk of asthma andwheezing at the age of 7 years in gt4000 mothers and gt5000children [33] When maternal copies of the minus212A allele werepresent association with asthma (11374891 OR 173 CI122ndash245) and wheezing (11494949 OR 153 95 CI 106ndash220) was significantly increased [33]
Gastrointestinal Disorders While there was no evidence inlung cancer cases studies in Japanese populations suggesteda potential association of NRF2 variations with gastro-intestinal tumorigenesisHelicobacter pylori (H pylori) causesgastritis which can lead to gastric atrophy and cancer Ingastric epithelium from the Japanese cancer cohorts (39gastric cancers 46 controls) H pylori infection was posi-tively correlated with aberrant CpG island methylation oftumor suppressor genes (eg p14) and minus214Gminus212G orminus214Aminus212G NRF2 haplotype was significantly associatedwith increased (OR 290 95 CI 114ndash736) or decreased(OR 033 95 CI 013ndash088) risk of the CpG methyla-tion respectively in the H pylori-infected patients [29]Further study from the same investigators determined thatminus214Aminus212G allele carriers had significantly (119875 = 0022)reduced risk of gastric cancer inH pylori-negative cases [30]The minus214AGminus212AG genotypes were negatively associated
(OR 045 CI 022ndash093) and the minus214Gminus212G genotypeswere positively associated (chronic continuous phenotypeOR 257 CI 101ndash660) with ulcerative colitis (89 patients 141controls) in a Japanese population [31]
Autoimmune Disorders Systemic lupus erythematosus (SLE)is a long-term autoimmune disease more frequently found infemales than in males It affects organs including skin jointskidneys and brain and nephritis is an aggressive character-istic in some patients Genome-wide association studies inhumans identified a suggestive quantitative trait locus nearNRF2 [43] A study of a Mexican Mestizo population (362patients with childhood-onset SLE 379 controls and 212nephritis diagnosed) determined that lupus with nephritiswas significantly (OR 181 CI 104ndash312) associated with theminus214GA SNP in females [25] The same SNPs were notclosely associated with SLE risk in a Japanese cohort [22]Vitiligo is a skin condition in which there is a loss of browncolor (pigment) from areas of skin resulting in irregularwhite patches It is thought to be an autoimmune diseasecaused by loss of cells (melanocytes) that produce brownpigment A study indicated that theminus178A allele increased therisk of vitiligo dose-dependently (OR 1724 95 CI 135ndash221for CA OR 2902 CI 162ndash519 for AA) [35]
Female Disorders It is well known that estrogen metabolites(eg catechols) cause ROS formation suggesting correla-tion of NRF2 and downstream effectors in postmenopausalmammary cancer In a study of a Finish population (KuopioBreast Cancer Project 119899 = 452 patients 370 controls)the minus178AA homozygous genotype (OR 4656 CI = 135ndash1606) and 31015840 flanking rs2706110 (TT OR 2079 CI 118ndash368) genotype were associated with increased risk of breastcancer while the 51015840 flanking minus3306TT homozygous allelewas significantly associated with lower survival (frequency =71219 OR 1687 CI 1105ndash275) [27] suggesting that NRF2genetic polymorphisms affect susceptibility and outcome ofthe patients The minus178A allele carriers together with intronicrs1962142A allele carriers were associated with lowered tissuelevels of NRF2 proteins [27] In postmenopausal women theminus178A allele (OR 179 95CI 370ndash8570) appeared tomodifythe risk of venous thromboembolism caused by oral estrogentherapy (AA or AC frequency = 333) as demonstratedby the French ESTHER study (161 cases 474 controls) [36]An intronic rs1806649CgtT SNP did not associate with breastcancer risk in postmenopausal women [40] However whenthis SNP and other at-risk alleles of ARE-responsive genes(NQO1 HO-1 NOS3) were combined there was a significantgene-dose effect on the breast cancer risk [40] Althoughcoding region SNPs in NRF2 and KEAP1 were identifiedin the Japanese endometrial adenocarcinoma patients noassociation of NRF2 SNPs with the disease was found [44]
Neurodegenerative Diseases Oxidative stress is known to beinvolved in Parkinsonrsquos disease (PD) presumably due toproduction of ROS fromhigh-dopaminemetabolism and lowlevels of antioxidants in the substantia nigra of the brainInvestigators found a protective NRF2 haplotype consistingof four 51015840 flanking SNPs (minus5238Gminus214Aminus212Gminus178C)
Oxidative Medicine and Cellular Longevity 15
and 4 intronic SNPs (rs2886161Ars1806649Ars2001350Ars10183914A) from Swedish (OR 09 CI 060ndash140) and Polish(OR 04 CI 030ndash060) populations (total 165+192 PD cases190+192 controls) [26]The investigators also suggested thatNRF2 haplotype alleles were associated with 2 years earlierage of Alzheimerrsquos disease (AD) onset 4 years earlier age ofposterior subcapsular cataract surgery and 4 years later ageof cortical cataract surgery while they were not significantlyrelated to AD or age-related cataract risk [45]
34 Genetic Mutations in Mouse Nrf2 Tsang et al [46]compiled 673 SNPs in 55 mouse strains and constructedtheir phylogenetic tree to correlate and clarify the originsof strains based on the assembled mouse genome sequenceand SNP data [20 47 48] Recently using the completegenome sequence of C57BL6J (B6) mouse as a referencehigh-density SNP screening in other laboratory strains orin panels of strains has been published (see [17]) Althoughmillions of mouse SNPs (gt10089892 as of December 2012)and haplotype mappings from more than 120 strains havebeen published as valuable references for dissecting thegenetic basis of complex traits [49ndash51] little attention hasbeen paid to polymorphisms of Nrf2 and their correlationwith disease phenotypes
Figure 3 demonstrates the proximal promoter region(minus1 to minus950)51015840-UTR (exon 1 up to TSS) and proteinsequence of mouse Nrf2 based on GRCm38p1 PrimaryAssembly (75704641ndash75675513 bp) mRNA variant1 and protein (NP 035032 encoded by NM 010902234ndash2027 bp) sequences Genetic variations in the Nrf2genome of inbred strains collected from public databasesare listed in Tables S2 and 5 (See Supplementary TableS2) Overall 968 genetic mutations are compiled forNrf2 gene and 5 kb upstream2 kb downstream regions785 SNPs between B6 and another 16 strains wereacquired from the Mouse Phenome Database (MPDhttpphenomejaxorg dbqrtn=snpret1) and additionalSNPs and other mutations were acquired from NCBI dbSNP(httpwwwncbinlmnihgovsnpterm=mus+musculus20nfe2l2) In total 132 mutations are in the promoter (37in proximal 1 kb) 49 in exons (38 in coding region 19 Cns)727 in introns and 60 in the 31015840 flanking region Excludingmutations in the 51015840 and 31015840 flanking sequences murine Nrf2sequences appear to be more highly variable (1 variationper 375 bp) than much of the mouse genome which has anapproximate frequency of one SNP per every 245 bp (httpwwwinformaticsjaxorgmgihomehomepagesstatsallstatsshtmlallstats snp)
Nrf2 was found to be a susceptibility gene from genome-wide linkage analysis in a murine model of hyperoxia-induced ALI [52] A promoter SNP minus103TgtC (previouslypublished as minus336TgtC) in Nrf2 was found and predicted toadd an additional Sp1 binding site in hyperoxia-susceptibleB6 mice but not in resistant C3HHeJ mice [52] Genotypesfrom the SNP and from simple-sequence length polymor-phism markers of the Nrf2 locus (D2Mit248 and D2Mit94)cosegregated in the B6C3F
2mouse cohort [52] and Nrf2
deficient mice were significantly more susceptible to ALI
sub-phenotypes caused by hyperoxia than similarly exposedwild-type mice supporting Nrf2 as a contributor to thephenotypic traits [53] Although no other functional analyseson Nrf2 SNPs or haplotype association studies have beenconducted in inbred mice strains bearing haplotypes suchas multiple Cns in functional domains (eg F71L L451VH543Q and L575M) may be useful to elucidate the role ofNrf2 in differential susceptibility to oxidative diseases
4 Oncogenic Somatic Mutations in HumanNF-E2-Related Factor 2
Somatic mutation is a change in the DNA of somatic cellsthat affects derived cells but is not inherited by offspringEfforts to discover somatic mutations have provided insightinto mutagenesis and cancer development Lung cancerparticularly non-small cell lung cancer (NSCLC) is theleading cause of cancer death worldwide Somatic mutationsof NRF2 and KEAP1 discovered in lung cancer patients havedetermined the oncogenic potential ofNRF2 [54 55] KEAP1somatic mutations were associated with its reduced proteinlevels in lung cancer tissues and cells [56 57] Investigationsof NSCLC in various ethnic populations as well as cancersin gastrointestine breast and prostate have coordinatelydemonstrated that multiple Cns somatic mutations in KEAP1cause dysfunction of the translated protein and in turnconstitutive activation of NRF2 increasing risk of neoplasiaand chemoresistance [12 55 58 59] Somatic mutations ofNRF2 have been detected in various cancer tissues (largelysquamous cell carcinomas) in Asian populations (Table 6)NRF2 mutations were significantly associated with NSCLCcases (squamous cell lung carcinoma adenocarcinoma) ofthe Japanese (107 [54]) the Chinese (23 [60]) andthe Koreans (8 [61]) as well as with lung cancer celllines Smoking history was also correlated with mutationoccurrence in all of the studies [54 60 61] In addition tolung cancers laryngeal squamous carcinoma (13 in [61])esophageal squamous cancer (ESC 22 in [60] 114 in[61]) head and neck cancers (25 in [54]) skin (117 casein [61]) and oral cancer cell lines had somatic changesin NRF2 In contrast to wide-spread KEAP1 mutationsmutations in NRF2 were clustered in DLGETGE motifs ofthe Neh2 domain which are critical in the ldquohinge and latchrdquomodel of KEAP1 binding [12] Similar to KEAP1 somaticmutations it has been postulated that NRF2 mutations incancer cells lead to NRF2 accumulation by suppressing itsubiquitination or KEAP1 binding which eventually confersmalignant potential and resistance to chemotherapy
Most variable sites in NRF2 included aa residues 29 (AspD) 31 (Gly G) 77 (Asp D) and 79 (Glu E) (Table 6)Residue 33 (Ser S) in the Neh2 domain is mutated by eithergenetic or somatic processes (Figure 4) Cns in the EDGFmotif of NRF2 was experimentally determined to impairrecognition of KEAP1 [54] NRF2 mutations were signif-icantly correlated with increased (25-fold) copy number(31 of mutants versus 3 wild types) in Japanese NSCLCcases [63] Aberrant mutation of NRF2 also led to increasedexpression of downstream effectors includingRagD known to
16 Oxidative Medicine and Cellular Longevity
pL575M
pF71L
pH543QpL451V
pA151P pA151V pN159D pQ198delpV105D
pA204T pS224I225S226del pT253S pS278ApD305G
pM409V pR410HpP388L pA389G pT395I
Genomic DNA (reversed contig GRCm38 p1 C57BL6JNM 0109023)
Protein (NP 035032)1
51101151201251301351401451501551
minus400
minus500
minus50
minus100
minus200
minus300
minus450
minus350
minus250
minus150
minus950
minus900
minus850
minus800
minus750
minus700
151
101151201
2001
g minus470gg minusg minus
c minus41c minus
c
c
c c
gg minus 6C
g minus125insTTC g minus118insC
g minus281delC
g minus63delCg minus1 (75704642 c minus234)
Translation start site (c +1)Termination codon (c +2025 +2027)
c minus136 minus134in-frame stop codonc minus8585
g minus41
246
440
minus381
minus11
minus91 minus90
minus332
minus153
minus98
c minus197c minus209 minus202
gminus942 minus899 (cminus1175 minus1132) CpG islands [71]
g 460CgtT
g minus103CCgtT [51]
CgtTCgtG
g minus320CgtT
CgtT
CgtT
CgtTg minus426GgtA
GgtACgtA
CgtA
GgtA
g minus238GgtTTgtC
TgtC
TgtC
TgtCTgtG
g minus229AgtG
CgtG
CgtG
GgtC
gminus826 minus654 (cminus1059 ) CpG islands [71]minus887
Figure 3 DNA (partial promoter and exons 1 and 5) and protein sequence ofmouseNrf2 SNPs and amino acid residues for non-synonymousSNPs are marked Promoter regions bearing 5CpG islands are underlined
151
201251301351401451501551601
Genetic mutation (Table 3 and Figure 2)Somatic mutation (Table 6)
Neh2
Neh3
Neh4Neh5
Neh6
Neh1
Protein isoform 1 (NP 006155)
101151
Figure 4 Genetic and somatic mutation loci in human NRF2 protein
Oxidative Medicine and Cellular Longevity 17
Table5Mou
seup
stream
andexon
varia
tions
ofNfe2
l2locusin17
inbred
strainsR
eference
sequ
ence
isC5
7BL6J
(B6strain
1)geno
me(GI14933824975547698ndash75513576)SNPalleleand
geno
typesareshow
nas
chromosom
econ
tigsequ
enceStrains
2129S1S
vlmJ3AJ
4AKR
J5BA
LBcJ6C3
HH
eJ7C
57BL
6NJ8CA
STEiJ9CB
AJ
10D
BA2J11FVBNJ12LPJ
13N
ODShiLtJ14N
ZOH
ILtJ
15P
WK
PhJ16SPR
ETEiJ
17W
SBEiJ
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs2566
08517
75705565
GA
(CT)
51015840Flankingminus924
NC
GG
GG
GG
GG
GG
GG
GG
AA
Grs47274959
75705562
AT
(TA
)51015840Flankingminus921
NC
AT
TA
TT
AA
TT
AT
AT
TT
Ars216940398
75705554ndash75705555
Tadd(A
add)
51015840Flankingminus913minus914
NC
CGrs239114134
75705540
CT(G
A)
51015840Flankingminus899
NC
CT
TC
TT
CC
TT
CT
CT
CT
Crs46
461765
75705528
TG(A
C)oA
C
51015840Flankingminus887
NC
TG
GT
GG
TT
GG
TG
TG
GG
Trs5144
9853
75705525
CA(G
T)
51015840Flankingminus884
NC
CA
AC
AA
CC
AA
CA
CA
CC
Crs249093111
75705512
CT(G
A)
51015840Flankingminus871
NC
CT
TC
TT
CC
TT
CT
CT
TT
Crs263745200
75705510
CG(G
C)
51015840Flankingminus869
NC
GG
GG
GG
GG
GG
GG
GG
CG
Grs45651867
75705498
TG(A
C)
51015840Flankingminus857
NC
TG
GT
GG
TT
GG
TG
TG
GG
Trs219997531
75705495
GA
(CT)
51015840Flankingminus854
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs251918379
75705451
TA(A
T)
51015840Flankingminus810
NC
TT
TT
TT
TT
TT
TT
TT
TA
Trs27978306
75705449
TA(A
T)
51015840Flankingminus808
NC
TA
AT
AA
TT
AA
TA
TA
TT
Trs216087412
75705429
TG(A
C)lowast
51015840Flankingminus788
NC
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
GTlowast
Tlowast
rs261229914
75705423ndash75705424
AGdel(CT
del)
51015840Flankingminus783minus784
NC
AGrs27978307
75705400
CT(G
A)
51015840Flankingminus759
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs243167395
75705359
GA
(CT)
51015840Flankingminus718
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs27978308
75705307
GA
(CT)
51015840Flankingminus66
6NC
GG
GG
GG
GG
GG
GG
GG
AG
Grs254744
09875705294
AT
(TA
)51015840Flankingminus653
NC
AA
AA
AA
AA
AA
AA
AA
AT
Ars228133419
75705179
GT
(CA
)51015840Flankingminus538
NC
GG
GG
GG
GG
GG
GG
GG
GT
Grs214719520
75705111
GA
(CT)
51015840Flankingminus470
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs2440
87730
75705101
GA
(CT)
51015840Flankingminus46
0NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs227781699
75705081
CG(G
C)
51015840Flankingminus44
0NC
CC
CC
CC
CC
CC
CC
CC
GC
Crs257870353
75705067
CT(G
A)
51015840Flankingminus426
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs24487744
075705022
AG
(TC)
51015840Flankingminus381
NC
AA
AA
AA
AA
AA
AA
AA
AG
Ars234628138
75704973
AC
(TG
)51015840Flankingminus332
NC
AA
AA
AA
AA
AA
AA
AA
AC
A
18 Oxidative Medicine and Cellular Longevity
Table5Con
tinued
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs247275247
75704961
GA
(CT)
51015840Flankingminus320
NC
GG
GG
GG
GG
GG
GG
AG
GG
Grs247519047
75704922
Gdel(Cdel)
51015840Flankingminus281
NC
Grs27978309
(rs51915758)
75704887
GA
C(C
TG
)51015840Flankingminus246
NC
GA
AG
AA
GG
AA
GA
GA
CC
G
rs218139102
75704879
CA(G
T)
51015840Flankingminus238
NC
CC
CC
CC
CC
CC
CC
CC
AC
Crs251990355
75704870
TC(A
G)
51015840Flankingminus229
NC
TT
TT
TT
TT
TT
TT
TT
TC
Trs238746955
75704794
AG
(TC)
51015840Flankingminus153
NC
AA
AA
AA
AA
AA
AA
AA
GA
Ars221664
405
75704786
AG
(TC)
51015840Flankingminus145
NC
AA
AA
AA
AA
AA
AA
AA
AG
Ars217054035
75704766
ndash75704767
GAAadd(TTC
add)
51015840Flankingminus125minus126
NC
GA
rs248182931
75704759ndash75704760
Gadd(C
add)
51015840Flankingminus118
minus119
NC
AGrs217197904
75704744
GA
(CT)
51015840Flankingminus103
(+)S
p1[23]
GA
AG
AA
GG
AA
GA
AA
GG
Ars2644
9364975704704
Gdel(Cdel)
51015840Flankingminus63
NC
Grs2404
81112
757046
82CT(G
A)
51015840Flankingminus41
NC
CC
CC
CC
CC
CC
CC
CC
CT
Crs27978310
75704617
AG
(TC)
UTR
-525
NC
AA
AA
AA
AA
AA
AA
AA
GG
Ars27978311
75704610
CG(G
C)
UTR
-532
NC
GG
GG
GG
GG
GG
GG
GG
GG
Crs27978312
757046
05GA
(CT)
UTR
-537
NC
GA
AG
AA
GG
AA
GA
GA
GG
Grs214784220
757044
99AG
(TC)
UTR
-5143
NC
AA
AA
AA
AA
AA
AA
AA
GG
Ars244146318
757044
98GT
(CA
)UTR
-5144
NC
GG
GG
GG
GG
GG
GG
GG
GT
Grs27978313
757044
97GC
(CG
)UTR
-5145
NC
GC
CG
CC
GC
CC
GC
CC
CC
Crs215431944
757044
93CT(G
A)
UTR
-5149
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs257015697
757044
49GA
(CT)
UTR
-5193
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs252234782
757044
19GT
(CA
)UTR
-5223
NC
Grs1346
0861
75679262
AG
(TC)
Exon
244
6Cn
sF71L
AG
GA
GG
AG
GG
AG
AG
GG
Grs1346
0859
75679202
GA
(CT)lowast
Exon
2506
CsH91=
GA
AG
AA
GG
AA
GA
GA
GG
Grs27978436
75679187
GA
(CT)lowast
Exon
2521
CsT9
6=G
GG
GG
GG
AG
GG
GG
GA
GG
rs226131070
75679178
GA
(CT)
Exon
2530
CsS99=
GG
GG
GG
GG
GG
GG
GG
GA
Grs227683834
75678576
AT
(TA
)Ex
on3
547
CnsV
105D
AA
AA
AA
AA
AA
AA
AA
AT
Ars257321187
75678506
TC(A
G)
Exon
3617
CsP128=
TT
TT
TT
TC
TT
TT
TT
TT
Trs27978444
75678500
TA(A
T)
Exon
3623
CsV130=
TT
TT
TT
TA
TT
TT
TT
AT
Trs227743136
75677686
GA
(CT)
Exon
4662
CsH143=
GG
GG
GG
GG
GG
GG
GG
GA
Grs215327202
75677664
CG(G
C)
Exon
4684
CnsA
151P
CC
CC
CC
CG
CC
CC
CC
CC
Crs247539755
75677663
GA
(CT)
Exon
4685
CnsA
151V
GG
GG
GG
GA
GG
GG
GG
GG
Grs233164
668
75677640
TC(A
G)
Exon
4708
CnsN
159D
TT
TT
TT
TT
TT
TT
TT
TC
T
rs2340
95816
75677162ndash75677160
GCT
del
(AGCdel)
Exon
5826minus
828
Cds-in
delQ
198
GCT
Oxidative Medicine and Cellular Longevity 19
Table5Con
tinued
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs27978452
75677145
CT(G
A)
Exon
5843
CnsA
204T
CC
CC
CC
CC
CC
CC
CC
CC
T
rs225047937
75677086ndash75677078
GAG
ATCG
ATdel
(ATG
CATC
TCdel)
Exon
5902minus
910
Cds-in
del
(S224I225S226)
GAG
ATC
GAT
rs241469868
75676998
TA(G
T)
Exon
5990
CnT2
53S
TT
TT
TT
TT
TT
TT
TT
TA
Trs221602571
75676923
AC
(TG
)Ex
on5
1065
CnsS
278A
AA
AA
AA
AA
AA
AA
AA
AC
Ars252576472
75676876
GA
(CT)
Exon
51112
CsS293=
GG
GG
GG
GG
GG
GG
GG
GA
Grs250802933
75676841
TC(A
G)
Exon
5114
7Cn
sD305G
TT
TT
TT
TT
TT
TT
TT
TC
Trs225425698
75676792
CT(G
A)
Exon
5119
6Cs
P321=
CC
CC
CC
CC
CC
CC
CC
CT
Crs13459064
75676732
CT(G
A)
Exon
51256
CsT3
41=
CT
TC
TT
CT
TT
CT
CT
TC
Crs27978453
75676717
CT(G
A)
Exon
51271
CsA346=
CC
CC
CC
CC
CC
CC
CC
TC
Crs214335034
75676636
AG
(TC)
Exon
51352
CsD373=
AA
AA
AA
AA
AA
AA
AA
AG
Ars24958364
4756766
03AC
(TG
)Ex
on5
1385
CsP3
84=
AA
AA
AA
AA
AA
AA
AA
AC
Ars27978454
75676592
GA
(CT)
Exon
51396
CnsP
388L
GG
GG
GG
GG
GG
GG
GG
AG
Grs215384328
75676589
GC
(CG
)Ex
on5
1399
CnsA
389G
GG
GG
GG
GG
GG
GG
GG
GC
Grs251728286
75676571
GA
(CT)
Exon
51417
CnsT
395I
Grs247602334
75676567
TC(A
G)
Exon
51421
CsV396=
TT
TT
TT
TT
TT
TT
TT
TC
Trs234216231
75676530
TC(A
G)
Exon
51458
CnsM
409V
TT
TT
TT
TT
TT
TT
TT
TC
Trs212904337
75676526
CT(G
A)
Exon
51462
CnsR
410H
CC
CC
CC
CC
CC
CC
CC
CT
Crs252650779
75676522
TC(A
G)
Exon
51466
CsE4
11=
TT
TT
TT
TT
TT
TT
TT
TC
Trs231273560
75676516
TC(A
G)
Exon
51472
CsQ413=
TT
TT
TT
TT
TT
TT
TT
TC
Trs257886949
756764
13GT
(CA
)Ex
on5
1575
CsR4
48=
GG
GG
GG
GG
GG
GG
GG
GT
Grs241537608
756764
04GC
(CG
)Ex
on5
1584
CnsL
451V
GG
GG
GG
GG
GG
GG
GG
GC
Grs227619071
75676312
TC(A
G)
Exon
51676
CsQ481=
TT
TT
TT
TT
TT
TT
TT
TC
Trs258913831
75676290
AG
(TC)
Exon
51698
CsL4
89=
AA
AA
AA
AA
AA
AA
AA
AG
Ars225593319
75676126
AT
(TA
)Ex
on5
1862
CnsH
543Q
Ars4223233
75676105
GA
(CT)
Exon
51883
CsS550=
GG
GG
GG
GA
GG
GG
GG
GG
Grs4223232
75676032
GT
(CA
)Ex
on5
1956
CnsL
575M
Grs4223231
75675816
CG
UTR
-32172
NC
GG
GG
GG
GG
GG
GG
GG
GC
Grs1346
860
75675682
AT
UTR
-32306
NC
ASequ
ence
varia
tions
inmou
seNr
f2wereob
tained
from
Mou
sePh
enom
eDatabase(http
ph
enom
ejaxorgSNP)
andNCB
ISNPdatabase
(http
wwwncbinlm
nihgovSNP)N
CBIreference
sequ
ence
isMus
Musculusstra
inC5
7BL6J
chromosom
e2GRC
m38p1(NC
0000
687
GI37209910875704641ndash756755192
9123
bp)To
tal968
genetic
mutations
arerepo
rted
inNr
f2gene
and5k
bup
stream
(ge75704
642minus
1)and2k
bdo
wnstre
am(le75675512)sequences
asof
Janu
ary2003A
llSN
Psandallelesa
representedas
geno
miccontig
(reversedsequ
ence
indicated)E
xon175704
641ndash75704364(278
bpT
TS=75704
408)
exon
275679431ndash756791
65(267
bp)exon
375678579ndash75678490(90b
p)exon475677713ndash756775
46(168
bp)andexon
57567718
4ndash75678849
(1666
bp)Proteinam
inoacid
(aa)
resid
uesinNP00
61552
(597
aa)Cs
cod
ing-syno
nymou
sCn
scoding
-non
syno
nymou
sAminoacid
A-alanineD-aspartic
acidE
-glutamicacidF-phenylalanineG
-glycineH
-histidineI-iso
leucineL-leu
cineand
M-m
ethion
ineN-
asparagineP-prolin
eQ-glutamineR-arginineS-serineT-threon
ineandV-valin
elowastErrorsin
databasesfi
xedNC
notcon
firmed
20 Oxidative Medicine and Cellular Longevity
Table 6 NRF2 somatic mutations revealed in various human cancers
Domain Amino acid residues DNA mutation Cancer types (cases) ReferencesLocus Wild type Mutant
DLG motif
24 W (Trp) C (Cys) c72GgtCGgtT NSCLC neck ESC [54 62]K (Lys) c72TgtC ESC [62]
26 Q (Gln) E (Glu) c76CgtG NSCLC ESC [54 62]
27 D (Asp) G (Gly)lowast c80GgtA NSCLC [60]Y (Tyr) c79GgtT ESC [61]
28 I (Ile) T (Thr) c83CgtT NSCLC [54]
29 D (Asp) G (Gly) c86AgtG Head and neck ESC [54 62]H (His) c85GgtC NSCLC laynx [60 61]
30 L (Leu) F (Phe) c88CgtT NSCLC ESC [54 62]
31 G (Gly) A (Ala) c92GgtC NSCLC ESC skin [54 60ndash62]
32 V (Val) T (Thr) c95TgtG NSCLC [54]del c93 95delAGT ESC [61]
33ndash36 S-R-E-V S-R-E-V-S-R-E-Vlowast c97 108dupAGTCGAGCCGTA ESC [61]
34 R (Arg) Q (Gln) c101GgtA NCSCL [54 60 61]P (Pro) c101GgtC NSCLC [63]
ETGF motif
75 Q (Gln) H (His) c225AgtC Head and neck ESC [54 62]
77 D (Asp)
V (Val) c230AgtT NSCLC ESC [54 60 62]G (Gly) c230AgtG ESC [62]A (Ala) c230AgtC NSCLC [61]N (Asn) c229GgtA Larynx [61]
78 E (Glu) K (Lys) c232GgtA NSCLC ESC [54 62]
79 E (Glu)
K (Lys) c235GgtA NSCLC ESC [54 61 62]Q (Gln) c235GgtC NSCLC ESC [54 60ndash62]G (Gly) c236AgtG Larynx [61]E-E c234 236dupAGA ESC [61]
80 T (Thr) K (Lys) c239CgtACgtG NSCLC ESC [54 61 62]
80 T (Thr)P (Phe) c238AgtC ESC [62]I (Ile) c239CgtT Head and neck [54]A (Ala) c238AgtG NSCLC [63]
81 G (Gly) V (Val) c242GgtT ESC [61]D (Asp) c242GgtA NSCLC ESC [60ndash62]
82 E (Glu)
D (Asp) c246AgtT ESC oral cancer cell line [54 62]G (Gly) c245AgtG NSCLC [54]Q (Gln)lowast c244GgtC NSCLC ESC [60 61]V (Val)lowast c245AgtT ESC [61]
83 F (Phe) L (Leu)lowast c247TgtC NSCLC [60]NSCLC non-small cell lung cancer ESC esophageal squamous cancer and lowasterrors in reference fixed Number of cases 82 NSCLC and 10 ESC in [62] 125NSCLC 70 ESC 23 larynx and 17 skin in [61] 103 NSCLC and 12 head and neck in [54] 90 NSCLC in [63] 103 NSCLC in [60]
be involved in squamous lung cancer cell proliferation [64]suggesting that the mutation is functional and overcomesKEAP1 inhibition Singh et al [65] determined in vitrothat RNAi-mediated depletion of NRF2 in lung cancer cellsenhanced ROS production and susceptibility to cell deathby ionizing radiation These studies support the concept thatelevated NRF2 and ARE responsiveness provides cancer cellswith proliferative advantage for malignant transformation
and undue protection from anti-cancer therapy Onco-genic epidermal growth factor receptor (EGFR) signaling isrecently found to be critical in NRF2-mediated proliferationof NSCLC cells [66]
Collectively ldquogain of functionrdquo mutations in NRF2 thatreduce KEAP1 recognition are suggested to be predictivemarkers for poor responsiveness to chemotherapy and radi-ation therapy Although NRF2-mediated cellular defense
Oxidative Medicine and Cellular Longevity 21
processes are essential in the initiation stage enhancedNRF2-ARE activity in advanced stages of cancer developmentmay create a favorable intracellular environment for tumorcell growth and survival [67 68] In this context NRF2may be a potential molecular target for the treatment ofradio-resistance cancers especially those that have ldquoloss offunctionrdquo mutations in EGFR KRAS or KEAP1 as well asldquogain of functionrdquo mutations in NRF2
5 Epigenetic Alterations of NF-E2-RelatedFactor 2
Epigenetic modifications are alterations of molecules inter-acting with genes without changes to the primary DNAsequence They include post-translational modification ofhistones DNA methylation events chromatin conforma-tional changes and alterations to noncoding regulatoryRNAs Epigenetic alterations are stable and often inheritablebut are reversible and may affect expression of the geneDysregulation or defects in epigenetic processes particularlyhypermethylation of tumor suppressor gene promoters (egCpG islands) or histone modifications are thought to beassociated with carcinogenesis Investigators have reportedhypermethylation in CpG islands of KEAP1 which wereassociated with reduced KEAP1 expression in human cancersfrom lung prostate colon and so forth [69ndash72] Similar tosomatic mutations epigenetic changes on KEAP1 impairedthe function of its encoded protein leading to constitutiveNRF2 activation
Supporting the role of ldquopathogenic mutationsrdquo in NRF2expression of Nrf2 and downstream Nqo1 was suppressedin prostate tumors of mice (transgenic adenocarcinomaof mouse prostate TRAMP) Among 15 promoter CpGislands located between minus942 and minus654 (cminus1175 cminus1132and cminus1059 cminus887 gap in cminus1131 cminus1060 see Figure 3)hypermethylation of the first 5 CpG islands (minus942 minus899 andcminus1175 cminus1132) was significantly associated with tumorige-nesis [73] Moreover treatment with inhibitors for DNAmethyltransferase and histone deacetylase restored Nrf2expression in these tumor cells [73] A dietary phytochemicalcurcumin known as a DNA hypomethylation agent restoredepigenetically silent Nrf2 expression through CpG demethy-lation in carcinogen-induced mouse tumor cells [74]
The whole genome epigenetic datasets for 5 species arepublicly accessible at NCBI Epigenomics [75 76]The humanNRF2 epigenome of primary cells (breast penis) and H1stem cell line as well as mouse Nrf2 CpG island methylationdata for sperm blood and cerebellum are currently avail-able (httpwwwncbinlmnihgovepigenomics) Althoughno direct evidence of disease-associated epigenetic modu-lation has been identified in human NRF2 various phyto-chemical NRF2 agonists such as sulforaphane and curcuminhave shown their roles in DNA methylation and histonemodification (see reviews by Lee and colleagues eg [77])Taken together epigenetic modifications of theNRF2KEAP1axle are predicted to cause dysregulation of ARE-mediatedcellular defense leading to deleterious health effects and
phytochemical antioxidants as epigenetic modulators forNRF2 are suggested to be useful in cancer prevention
6 Conclusions
NRF2 is evolutionally conserved with high-sequence homol-ogy in many species However it is a highly mutable geneand numerous genetic variants have been discovered inhuman ethnic groups Importantly certain SNPs or haplo-types have been identified in various diseases as ldquoat-riskrdquoalleles and are related to functional alterations In additionto genetic variations multiple somatic mutations identifiedin the KEAP1 recognition domain of NRF2 in cancer cellshave been found to be oncogenic due to dysregulation ofNRF2 homeostasis by its excess ldquogain of functionrdquo Epigeneticalteration of theNRF2 is under investigation and is predictedto have pathogenic influences as learned from mouse andphytochemical agonist studies Continuous updates of Nrf2allelic variants in inbred mouse strains will provide a usefultool for effective experimental designs formodels of oxidativedisorders to provide insight into the disease mechanisms andintervention strategies
Abbreviations
AD Alzheimerrsquos diseaseALI acute lung injuryARE antioxidant response elementbZIP basic leucine zippercds coding DNA sequencesCI confidence intervalCOPD chronic obstructive pulmonary diseaseESC esophageal squamous cancerFEV1 forced expiratory volume in one second
Gbp giga base pairsGST glutathione S-transferaseGWAS genome-wide association studyHGVS Human Genome Variation SocietyHO-1 heme oxygenase-1119867 119901119910119897119900119903119894 119867119890119897119894119888119900119887119886119888119905119890119903 119901119910119897119900119903119894KEAP1 Kelch-like ECH activating protein 1MPD Mouse Phenome DatabaseMRP multidrug resistance proteinNCBI National Center for Biotechnology InformationNeh NRF2-ECH homologyNfe2l2 nuclear factor (erythroid-derived 2)-like 2Nrf2 NF-E2-related factor 2NSCLC nonsmall cell lung cancerNQO1 NAD(P)Hquinone oxidoreductase 1OR odds ratioPD Parkinsonrsquos diseasePM particulate matterROS reactive oxygen speciesSLE systemic lupus erythematosusSNP single nucleotide polymorphismSOD superoxide dismutaseURT untranslated region
22 Oxidative Medicine and Cellular Longevity
Disclosure
Authorrsquos contribution to the Work was done as part of theAuthorrsquos official duties as a NIH employee and is a Work ofthe United States Government Therefore copyright may notbe established in the United States
Conflict of Interests
The author declares that there is no conflict of interests
Acknowledgments
The research related to this paper was supported by theIntramural Research Program of the National Institutes ofHealth of the National Institute of Environmental HealthSciences (NIEHS) Drs Steven Kleeberger and StephanieLondon at the NIEHS provided excellent critical review ofthis paper The author thanks Mrs Jacqui Marzec for herhelpful comments and English editing
References
[1] J Y Chan M C Cheung P Moi K Chan and Y W KanldquoChromosomal localization of the humanNF-E2 family of bZIPtranscription factors by fluorescence in situ hybridizationrdquoHuman Genetics vol 95 no 3 pp 265ndash269 1995
[2] K Itoh K Igarashi N Hayashi M Nishizawa and MYamamoto ldquoCloning and characterization of a novel erythroidcell-derived CNC family transcription factor heterodimerizingwith the small Maf family proteinsrdquo Molecular and CellularBiology vol 15 no 8 pp 4184ndash4193 1995
[3] W O Osburn and T W Kensler ldquoNRF2 signaling an adaptiveresponse pathway for protection against environmental toxicinsultsrdquoMutation Research vol 659 no 1-2 pp 31ndash39 2008
[4] D Papp K Lenti D Modos D Fazekas Z Dul D Tureiet al ldquoThe NRF2-related interactome and regulome containmultifunctional proteins and fine-tuned autoregulatory loopsrdquoFEBS Letters vol 586 no 13 pp 1795ndash1802 2012
[5] X Wang D J Tomso B N Chorley et al ldquoIdentificationof polymorphic antioxidant response elements in the humangenomerdquo Human Molecular Genetics vol 16 no 10 pp 1188ndash1200 2007
[6] D Malhotra E Portales-Casamar A Singh et al ldquoGlobalmapping of binding sites for NRF2 identifies novel targets incell survival response through ChIP-Seq profiling and networkanalysisrdquo Nucleic Acids Research vol 38 no 17 pp 5718ndash57342010
[7] K Chan R Lu J C Chang and Y W Kan ldquoNRF2 a memberof the NFE2 family of transcription factors is not essential formurine erythropoiesis growth and developmentrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 93 no 24 pp 13943ndash13948 1996
[8] K Itoh T Chiba S Takahashi et al ldquoAn NRF2small Mafheterodimer mediates the induction of phase II detoxifyingenzyme genes through antioxidant response elementsrdquo Bio-chemical and Biophysical Research Communications vol 236no 2 pp 313ndash322 1997
[9] FMartin J M vanDeursen R A Shivdasani CW Jackson AG Troutman and P A Ney ldquoErythroid maturation and globin
gene expression inmicewith combined deficiency ofNF-E2 andNrf-2rdquo Blood vol 91 no 9 pp 3459ndash3466 1998
[10] K Itoh N Wakabayashi Y Katoh et al ldquoKEAP1 repressesnuclear activation of antioxidant responsive elements by NRF2through binding to the amino-terminal Neh2 domainrdquo Genesand Development vol 13 no 1 pp 76ndash86 1999
[11] A Kobayashi M I Kang H Okawa et al ldquoOxidative stresssensor KEAP1 functions as an adaptor for Cul3-based E3 ligaseto regulate proteasomal degradation of NRF2rdquo Molecular andCellular Biology vol 24 no 16 pp 7130ndash7139 2004
[12] K Taguchi H Motohashi and M Yamamoto ldquoMolecularmechanisms of the KEAP1-NRF2 pathway in stress responseand cancer evolutionrdquo Genes to Cells vol 16 no 2 pp 123ndash1402011
[13] K Mukaigasa L T Nguyen L Li H Nakajima M Yamamotoand M Kobayashi ldquoGenetic evidence of an evolutionarilyconserved role for NRF2 in the protection against oxidativestressrdquoMolecular and Cellular Biology vol 32 no 21 pp 4455ndash4461 2012
[14] JMaher andMYamamoto ldquoThe rise of antioxidant signalingmdashthe evolution and hormetic actions of NRF2rdquo Toxicology andApplied Pharmacology vol 244 no 1 pp 4ndash15 2010
[15] S C Lo X Li M T Henzl L J Beamer and M HanninkldquoStructure of the KEAP1NRF2 interface provides mechanisticinsight intoNRF2 signalingrdquoThe EMBO Journal vol 25 no 15pp 3605ndash3617 2006
[16] V A McKusick ldquoMendelian inheritance in man and its onlineversion OMIMrdquo American Journal of Human Genetics vol 80no 4 pp 588ndash604 2007
[17] B Yalcin D J Adams J Flint and T M Keane ldquoNext-generation sequencing of experimental mouse strainsrdquo Mam-malian Genome vol 23 no 9-10 pp 490ndash498 2012
[18] D R Bentley S Balasubramanian H P Swerdlow et alldquoAccurate whole human genome sequencing using reversibleterminator chemistryrdquo Nature vol 456 no 7218 pp 53ndash592008
[19] P S G Chain D V Grafham R S Fulton et al ldquoGenomeproject standards in a new era of sequencingrdquo Science vol 326no 5950 pp 236ndash237 2009
[20] RHWaterstonK Lindblad-Toh E Birney J Rogers J F AbrilP Agarwal et al ldquoInitial sequencing and comparative analysis ofthe mouse genomerdquo Nature vol 420 pp 520ndash562 2002
[21] T M Keane L Goodstadt P Danecek M A White K WongB Yalcin et al ldquoMouse genomic variation and its effect onphenotypes and gene regulationrdquoNature vol 477 pp 289ndash2942011
[22] T Yamamoto K Yoh A Kobayashi et al ldquoIdentification ofpolymorphisms in the promoter region of the human NRF2generdquo Biochemical and Biophysical Research Communicationsvol 321 no 1 pp 72ndash79 2004
[23] J M Marzec J D Christie S P Reddy et al ldquoFunctionalpolymorphisms in the transcription factor NRF2 in humansincrease the risk of acute lung injuryrdquo The FASEB Journal vol21 no 9 pp 2237ndash2246 2007
[24] H Fukushima-Uesaka Y Saito K Maekawa et al ldquoGeneticvariations and haplotype structures of transcriptional factorNRF2 and its cytosolic reservoir protein KEAP1 in JapaneserdquoDrug Metabolism and Pharmacokinetics vol 22 no 3 pp 212ndash219 2007
[25] E J Cordova R Velazquez-Cruz F Centeno V Baca and LOrozco ldquoThe NRF2 gene variant -653GA is associated with
Oxidative Medicine and Cellular Longevity 23
nephritis in childhood-onset systemic lupus erythematosusrdquoLupus vol 19 no 10 pp 1237ndash1242 2010
[26] M von Otter S Landgren S Nilsson et al ldquoAssociation ofNRF2-encoding NFE2L2 haplotypes with Parkinsonrsquos diseaserdquoBMCMedical Genetics vol 11 no 1 article 36 2010
[27] JMHartikainenM TengstromVMKosmaV L Kinnula AMannermaa andY Soini ldquoGenetic polymorphisms and proteinexpression of NRF2 and sulfiredoxin predict survival outcomesin breast cancerrdquo Cancer Research vol 72 pp 5537ndash5546 2012
[28] T Arisawa T Tahara T Shibata et al ldquoThe relationshipbetween Helicobacter pylori infection and promoter polymor-phism of the NRF2 gene in chronic gastritisrdquo InternationalJournal of Molecular Medicine vol 19 no 1 pp 143ndash148 2007
[29] T Arisawa T Tahara T Shibata et al ldquoThe influence ofpromoter polymorphism of nuclear factor-erythroid 2-relatedfactor 2 gene on the aberrant DNA methylation in gastricepitheliumrdquo Oncology Reports vol 19 no 1 pp 211ndash216 2008
[30] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene pro-moter polymorphism and gastric carcinogenesisrdquo Hepato-Gastroenterology vol 55 no 82-83 pp 750ndash754 2008
[31] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene promoterpolymorphism is associated with ulcerative colitis in a Japanesepopulationrdquo Hepato-Gastroenterology vol 55 no 82-83 pp394ndash397 2008
[32] C C Hua L C Chang J C Tseng C M Chu Y C Liu andW B Shieh ldquoFunctional haplotypes in the promoter region oftranscription factor NRF2 in chronic obstructive pulmonarydiseaserdquo Disease Markers vol 28 no 3 pp 185ndash193 2010
[33] S O Shaheen R B Newson S M Ring M J Rose-ZerilliJ W Holloway and A J Henderson ldquoPrenatal and infantacetaminophen exposure antioxidant gene polymorphismsand childhood asthmardquo The Journal of Allergy and ClinicalImmunology vol 126 no 6 pp 1141e7ndash1148e7 2010
[34] H Masuko T Sakamoto Y Kaneko et al ldquoLower FEV1 innon-COPD nonasthmatic subjects association with smokingannual decline in FEV1 total IgE levels and TSLP genotypesrdquoInternational Journal of Chronic Obstructive Pulmonary Diseasevol 6 pp 181ndash189 2011
[35] C P Guan M N Zhou A E Xu et al ldquoThe susceptibilityto vitiligo is associated with NF-E2-related factor 2 (NRF2)gene polymorphisms a study on Chinese Han populationrdquoExperimental Dermatology vol 17 no 12 pp 1059ndash1062 2008
[36] J Bouligand O Cabaret M Canonico et al ldquoEffect of NFE2L2genetic polymorphism on the association between oral estrogentherapy and the risk of venous thromboembolism in post-menopausal womenrdquo Clinical Pharmacology amp Therapeuticsvol 89 no 1 pp 60ndash64 2011
[37] D S OrsquoMahony B J Glavan T D Holden C Fong R A BlackG Rona et al ldquoInflammation and immune-related candidategene associations with acute lung injury susceptibility andseverity a validation studyrdquo PLoS ONE vol 7 no 12 Article IDe51104 2012
[38] I Ungvari E Hadadi V Virag et al ldquoRelationship between airpollutionNFE2L2 gene polymorphisms and childhood asthmain aHungarian populationrdquo Journal of CommunityGenetics vol3 no 1 pp 25ndash33 2012
[39] M SiedlinskiD S Postma JMA Boer et al ldquoLevel and courseof FEV1 in relation to polymorphisms inNFE2L2 andKEAP1 inthe general populationrdquo Respiratory Research vol 10 article 732009
[40] C C Hong C B Ambrosone J Ahn et al ldquoGenetic variabilityin iron-related oxidative stress pathways (NRF2 NQ01 NOS3
and HO-1) iron intake and risk of postmenopausal breastcancerrdquo Cancer Epidemiology Biomarkers and Prevention vol16 no 9 pp 1784ndash1794 2007
[41] C Canova C Dunster F J Kelly C Minelli P L Shah CCaneja et al ldquoPM10-induced hospital admissions for asthmaand chronic obstructive pulmonary disease the modifyingeffect of individual characteristicsrdquo Epidemiology vol 23 no 4pp 607ndash615 2012
[42] A J Henderson R B Newson M Rose-Zerilli S M RingJ W Holloway and S O Shaheen ldquoMaternal NRF2 andgluthathione-S-transferase polymorphisms donotmodify asso-ciations of prenatal tobacco smoke exposure with asthma andlung function in school-aged childrenrdquo Thorax vol 65 no 10pp 897ndash902 2010
[43] C Xing A L Sestak J A Kelly et al ldquoLocalization andreplication of the systemic lupus erythematosus linkage signalat 4p16 interaction with 2p11 12q24 and 19q13 in EuropeanAmericansrdquo Human Genetics vol 120 no 5 pp 623ndash631 2007
[44] T F Wong K Yoshinaga Y Monma K Ito H NiikuraS Nagase et al ldquoAssociation of KEAP1 and NRF2 geneticmutations and polymorphisms with endometrioid endometrialadenocarcinoma survivalrdquo International Journal of Gynecologi-cal Cancer vol 21 no 8 pp 1428ndash1435 2011
[45] M von Otter S Landgren S Nilsson et al ldquoNRF2-encodingNFE2L2 haplotypes influence disease progression but not riskin Alzheimerrsquos disease and age-related cataractrdquoMechanisms ofAgeing and Development vol 131 no 2 pp 105ndash110 2010
[46] S Tsang Z Sun B Luke et al ldquoA comprehensive SNP-basedgenetic analysis of inbred mouse strainsrdquoMammalian Genomevol 16 no 7 pp 476ndash480 2005
[47] C M Wade E J Kulbokas III A W Kirby et al ldquoThe mosaicstructure of variation in the laboratory mouse genomerdquoNaturevol 420 no 6915 pp 574ndash578 2002
[48] T Wiltshire M T Pletcher S Batalov et al ldquoGenome-widesingle-nucleotide polymorphism analysis defines haplotypepatterns in mouserdquo Proceedings of the National Academy ofSciences of the United States of America vol 100 no 6 pp 3380ndash3385 2003
[49] K A Frazer E Eskin H M Kang et al ldquoA sequence-basedvariation map of 827 million SNPs in inbred mouse strainsrdquoNature vol 448 no 7157 pp 1050ndash1053 2007
[50] T P Maddatu S C Grubb C J Bult andM A Bogue ldquoMousephenome database (MPD)rdquo Nucleic Acids Research vol 40 ppD887ndashD894 2007
[51] A Kirby H M Kang C M Wade et al ldquoFine mapping in 94inbred mouse strains using a high-density haplotype resourcerdquoGenetics vol 185 no 3 pp 1081ndash1095 2010
[52] H Y Cho A E Jedlicka S P M Reddy L Y Zhang T WKensler and S R Kleeberger ldquoLinkage analysis of susceptibilityto hyperoxia NRF2 is a candidate generdquo American Journal ofRespiratory Cell and Molecular Biology vol 26 no 1 pp 42ndash512002
[53] H Y Cho A E Jedlicka S P M Reddy et al ldquoRole of NRF2in protection against hyperoxic lung injury in micerdquo AmericanJournal of Respiratory Cell and Molecular Biology vol 26 no 2pp 175ndash182 2002
[54] T Shibata T Ohta K I Tong et al ldquoCancer related mutationsin NRF2 impair its recognition by KEAP1-Cul3 E3 ligase andpromote malignancyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 36 pp13568ndash13573 2008
24 Oxidative Medicine and Cellular Longevity
[55] A Singh V Misra R K Thimmulappa et al ldquoDysfunctionalKEAP1-NRF2 interaction in non-small-cell lung cancerrdquo PLoSMedicine vol 3 no 10 article e420 2006
[56] T Ohta K Iijima M Miyamoto et al ldquoLoss of KEAP1 functionactivates NRF2 and provides advantages for lung cancer cellgrowthrdquo Cancer Research vol 68 no 5 pp 1303ndash1309 2008
[57] B Padmanabhan K I Tong T Ohta et al ldquoStructural basisfor defects of KEAP1 activity provoked by its point mutationsin lung cancerrdquoMolecular Cell vol 21 no 5 pp 689ndash700 2006
[58] T Shibata A Kokubu M Gotoh et al ldquoGenetic alteration ofKEAP1 confers constitutive NRF2 activation and resistance tochemotherapy in gallbladder cancerrdquoGastroenterology vol 135no 4 pp 1358e4ndash1368e4 2008
[59] N J Yoo H R Kim Y R Kim C H An and S H LeeldquoSomatic mutations of the KEAP1 gene in common solidcancersrdquo Histopathology vol 60 no 6 pp 943ndash952 2012
[60] Y Hu Y Ju D Lin Z Wang Y Huang S Zhang et alldquoMutation of the NRF2 gene in non-small cell lung cancerrdquoMolecular Biology Reports vol 39 no 4 pp 4743ndash4747 2012
[61] Y R Kim J EOhM S Kim et al ldquoOncogenicNRF2mutationsin squamous cell carcinomas of oesophagus and skinrdquo TheJournal of Pathology vol 220 no 4 pp 446ndash451 2010
[62] T Shibata A Kokubu S Saito et al ldquoNRF2 mutation confersmalignant potential and resistance to chemoradiation therapyin advanced esophageal squamous cancerrdquo Neoplasia vol 13pp 864ndash873 2011
[63] H Sasaki M Shitara K Yokota Y Hikosaka S MoriyamaM Yano et al ldquoIncreased NRF2 gene (NFE2L2) copy numbercorrelates with mutations in lung squamous cell carcinomasrdquoMolecular Medicine Reports vol 6 no 2 pp 391ndash394 2012
[64] H Sasaki M Shitara K Yokota Y Hikosaka S Moriyama MYano et al ldquoRagD gene expression andNRF2mutations in lungsquamous cell carcinomasrdquo Oncology Letters vol 4 pp 1167ndash1170 2012
[65] A Singh M Bodas N Wakabayashi F Bunz and S BiswalldquoGain of NRF2 function in non-small-cell lung cancer cellsconfers radioresistancerdquo Antioxidants and Redox Signaling vol13 no 11 pp 1627ndash1637 2010
[66] T Yamadori Y Ishii SHommaYMorishimaKKurishimaKItoh et al ldquoMolecular mechanisms for the regulation of NRF2-mediated cell proliferation in non-small-cell lung cancersrdquoOncogene vol 31 pp 4768ndash4777 2012
[67] A K Bauer H Y Cho L Miller-Degraff C Walker K HelmsJ Fostel et al ldquoTargeted deletion of NRF2 reduces urethane-induced lung tumor development in micerdquo PLoS ONE vol 6no 10 Article ID e26590 2011
[68] H Y Cho and S R Kleeberger ldquoNRF2 protects against airwaydisordersrdquo Toxicology and Applied Pharmacology vol 244 no1 pp 43ndash56 2010
[69] R Wang J An F Ji H Jiao H Sun and D Zhou ldquoHyperme-thylation of the KEAP1 gene in human lung cancer cell linesand lung cancer tissuesrdquo Biochemical and Biophysical ResearchCommunications vol 373 no 1 pp 151ndash154 2008
[70] P Zhang A Singh S Yegnasubramanian et al ldquoLoss ofkelch-like ECH-associated protein 1 function in prostate cancercells causes chemoresistance and radioresistance and promotestumor growthrdquoMolecular Cancer Therapeutics vol 9 no 2 pp336ndash346 2010
[71] N Hanada T Takahata Q Zhou et al ldquoMethylation of theKEAP1 gene promoter region in human colorectal cancerrdquoBMCCancer vol 12 article 66 2012
[72] L A Muscarella R Barbano V DrsquoAngelo et al ldquoRegulationof KEAP1 expression by promoter methylation in malignantgliomas and association with patientrsquos outcomerdquo Epigeneticsvol 6 no 3 pp 317ndash325 2011
[73] S Yu T O Khor K L Cheung et al ldquoNRF2 expressionis regulated by epigenetic mechanisms in prostate cancer ofTRAMP micerdquo PLoS ONE vol 5 no 1 Article ID e8579 2010
[74] T O Khor Y Huang T Y Wu L Shu J Lee and A N KongldquoPharmacodynamics of curcumin as DNA hypomethylationagent in restoring the expression of NRF2 via promoter CpGsdemethylationrdquo Biochemical Pharmacology vol 82 no 9 pp1073ndash1078 2011
[75] I M Fingerman L McDaniel X Zhang et al ldquoNCBI epige-nomics a new public resource for exploring epigenomic datasetsrdquo Nucleic Acids Research vol 39 supplemen 1 pp D908ndashD912 2011
[76] I M Fingerman X Zhang W Ratzat N Husain R F Cohenand G D Schuler ldquoNCBI epigenomics whatrsquos new for 2013rdquoNucleic Acids Research vol 41 no 1 pp D221ndashD225 2013
[77] J H Lee T O Khor L Shu Z Y Su F Fuentes andA N Kong ldquoDietary phytochemicals and cancer preventionNRF2 signaling epigenetics and cell death mechanisms inblocking cancer initiation and progressionrdquo Pharmacology ampTherapeutics vol 137 no 2 pp 153ndash171 2012
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
8 Oxidative Medicine and Cellular Longevity
Table3Con
tinued
IDMap
onchromosom
e2lowast
(HGVS
name)
Positionin
Nfe2
l2
(HGVS
name)
Region
s(position
from
mRN
A)
Varia
tioncla
ssand
consequences
(HGVS
name)dagger
Minor
allelefrequ
ency
(MAF)
MAcoun
tsDagger(coh
ort
size)
MAFsources
rs20134560
4g178129924178129925insG
cminus555-66minus555-65insC
51015840Flanking
(minus66minus65)
Insertion
G=0017939
1000
Genom
es
rs200432479
g178129741178
129742delAA
cminus438minus437delT
Texon
1UTR
-51015840(118minus119
)Dele
tion
-=000378
-=000
6(84)
1000
Genom
es[24]
rs75485459
g178129608CgtA
cminus304GgtT
Exon
1UTR
-51015840(252)
SNP
nars192086766
g17812946
6CgtT
cminus162GgtA
Exon
1UTR
-51015840(394)
SNP
T=002248
1000
Genom
esmdash
g17812944
2GgtA
cminus138CgtT
Exon
1UTR
-51015840(418)
SNP
A=0012(84)
[24]
rs7166
8246
g17812940
0delG
cminus96delC
Exon
1UTR
-51015840(460)
Dele
tion
nars187291840
g178129399CgtT
cminus95GgtA
Exon
1UTR
-51015840(461)
SNP
T=0054912
01000
Genom
es
rs143406266
g178129391178129393delGGC
cminus89minus87delGCC
Exon
1UTR
-51015840(467minus469)
Dele
tion
-=064
4282Dagger
-=0589(84)
[22]
[24]
rs182428269
g178098918GgtA
c127Cgt
TEx
on2(682)
Cns(pArg43Trp)
A=000
051
1000
Genom
esrs35248500
g178098917CgtT
c128GgtA
Exon
2(683)
Cns(pArg43Gln)
T=000
613
1000
Genom
esrs1135118
g178098831CgtT
c214GgtA
Exon
2(769)
Cns(pAla72Th
r)TDagger
[23]
rs19969166
0g178098829AgtT
c216Tgt
AEx
on2(771)
Cs(pAla72=)
namdash
g178098769GgtA
c276Cgt
TEx
on2(831)
Cs(pIle92=)
TDagger[23]
rs5031039
g178098750AgtG
c295Tgt
CEx
on2(850)
Cns(pSer99P
ro)
G=0Dagger
[23]
rs200239262
g178098017CgtG
c363GgtC
Exon
3(918)
Cns(pGln121H
is)na
rs183034165
g178098008TgtC
c372GgtA
Exon
3(927)
Cs(pAla124=
)T=000
092
T=000
6(84)
1000
Genom
es[24]
rs199970826
g178097996CgtT
c384GgtA
Exon
3(939)
Cs(pPro128=
)T=000
051
1000
Genom
esrs201992337
g178097260CgtT
c454GgtA
Exon
4(100
9)Cn
s(pGlu152Lys)
nars201589693
g178097251CgtT
c463GgtA
Exon
4(1018)
Cns(pV
al155Ile)
T=000
051
1000
Genom
es
rs35577826
g178097185AgtC
c529Tgt
GEx
on4(1084)
Cns(pLeu177Va
l)C=000143
Alt0005Dagger
1000
Genom
es[23]
rs181513314
g178096710CgtT
c621GgtA
Exon
5(1176)
Cn(pLeu207=
)T=000
051
1000
Genom
esrs60132461
g178096675TgtC
c656AgtG
Exon
5(1211)
Cns(pLys219Arg)
C=000184
1000
Genom
es
rs139187151
g178096634GgtA
c697Cgt
TEx
on5(1252)
Cns(pPro233Ser)
A=000
051
A=0012Dagger
(84)
1000
Genom
es[24]
rs35557421
g178096620d
elT
c711delA
Exon
5(1266)
Fram
eshiftdeletio
n(pLys237=fs)
na
rs34154613
g178096529CgtT
c802GgtA
Exon
5(1357)
Cns(pV
al268M
et)
T=000184
1000
Genom
esrs141363120
g178096
406GgtA
c925Cgt
TEx
on5(14
80)
Cns(pLeu309Ph
e)A=000378
1000
Genom
esrs201661476
g178096380AgtC
c951Tgt
GEx
on5(1506)
Cns(pIle
317M
et)
na
Oxidative Medicine and Cellular Longevity 9
Table3Con
tinued
IDMap
onchromosom
e2lowast
(HGVS
name)
Positionin
Nfe2
l2
(HGVS
name)
Region
s(position
from
mRN
A)
Varia
tioncla
ssand
consequences
(HGVS
name)dagger
Minor
allelefrequ
ency
(MAF)
MAcoun
tsDagger(coh
ort
size)
MAFsources
rs199673454
g178096309TgtA
c1022AgtT
Exon
5(1577)
Cns(pAs
p341Va
l)A=000
051
1000
Genom
esrs35007548
g178096299GgtA
c1032CgtT
Exon
5(1587)
Cs(pSer344=
)A=000
092
1000
Genom
esrs200209692
g178096287TgtC
c104
4AgtG
Exon
5(1599)
Cs(pLeu348=
)C=000
051
1000
Genom
es
mdashg178096237CgtA
c1094GgtT
Exon
5(1649)
Cns(pSer365Ile
)A=0125273
A=000
6Dagger(84)
PubM
ed[24]
rs201214197
g178096171CgtT
c1160GgtA
Exon
5(1715)
Cns(pSer387Asn)
T=000
051
1000
Genom
esrs200494292
g178096165TgtC
c1166AgtG
Exon
5(1721)
Cns(pLys389Arg)
nars186171287
g178096115GgtA
c1216CgtT
Exon
5(1771)
Cns(pPro4
06Ser)
A=000
051
1000
Genom
esrs182276775
g178096062CgtA
c1269GgtT
Exon
5(1824)
Cns(pGlu423A
sp)
A=000
051
1000
Genom
esrs201560221
g17809604
8TgtC
c1283AgtG
Exon
5(1838)
Cns(pLys428Arg)
nars189238236
g178096043AgtG
c1288TgtC
Exon
5(1843)
Cs(pLeu430=
)G=000
051
1000
Genom
esrs184287392
g178096022GgtA
c1309CgtT
Exon
5(1864)
Cns(pArg437Trp)
A=000
051
1000
Genom
esrs201871588
g178095986GgtA
c1345CgtT
Exon
5(19
00)
Cns(pAg449Cy
s)na
rs181294188
g178095985TgtC
c1346GgtA
Exon
5(19
01)
Cns(pArg44
9His)
T=000
092
1000
Genom
esrs20169046
6g178095973TgtA
c1358AgtT
Exon
5(19
13)
Cns(pHis4
53Leu)
A=000
051
1000
Genom
esrs105704
4(rs52789869)
g178095781CgtT
c1550GgtA
Exon
5(2105)
Cns(pArg517Lys)
TDagger[23]
rs200750800
g178095603AgtG
c1728TgtC
Exon
5(2283)
Cs(pTyr576=
)na
rs200175942
g178095567AgtG
c1764TgtC
Exon
5(2319)
Cs(pAsp588=
)G=000
051
1000
Genom
esrs77547666
g178095495GgtC
clowast18Cgt
GEx
on5UTR
-31015840(2391)
SNP
C=000
6915
1000
Genom
esrs73031353
g178095425TgtC
clowast88AgtG
Exon
5UTR
-31015840(2461)
SNP
C=000184
1000
Genom
esrs675944
3g178095345TgtC
clowast168AgtG
Exon
5UTR
-31015840(2541)
SNP
C=000
419
1000
Genom
esrs188674558
g178095279CgtA
clowast234GgtT
Exon
5UTR
-31015840(2607)
SNP
A=000
051
1000
Genom
esrs77685897
g178095247AgtG
clowast266TgtC
Exon
5UTR
-31015840(2639)
SNP
G=000
092
1000
Genom
esrs1057092
g178095162TgtG
clowast351AgtC
Exon
5UTR
-31015840(2724)
SNP
nars3197704
g178095162TgtG
clowast351AgtC
Exon
5UTR
-31015840(2724)
SNP
nars184701151
g178095159TgtC
clowast354AgtG
Exon
5UTR
-31015840(2727)
SNP
C=000276
1000
Genom
esrs11543307
g178095153AgtG
clowast360TgtC
Exon
5UTR
-31015840(2733)
SNP
nars111874043
g178095146AgtG
clowast367TgtC
Exon
5UTR
-31015840(2740)
SNP
nars34012004
g178095102AgtC
clowast411TgtG
Exon
5UTR
-31015840(2784)
SNP
C=0071
[24]
10 Oxidative Medicine and Cellular Longevity
Table3Con
tinued
IDMap
onchromosom
e2lowast
(HGVS
name)
Positionin
Nfe2
l2
(HGVS
name)
Region
s(position
from
mRN
A)
Varia
tioncla
ssand
consequences
(HGVS
name)dagger
Minor
allelefrequ
ency
(MAF)
MAcoun
tsDagger(coh
ort
size)
MAFsources
rs201481890
g178095090178095091in
sTclowast
422lowast423insA
Exon
5UTR
-31015840
(2795minus
2796)
Insertion
na
rs3082500
g178095089178095090d
elTT
delTTinsT
clowast423lowast424d
elAAinsA
Exon
5UTR
-31015840
(2796minus
2797)
Dele
tion
insertion
na
rs71792546
(rs71796710)
g178095079d
elT
clowast425delA
Exon
5UTR
-31015840(2798)
Dele
tion
na
rs1057106
g178095078AgtC
AgtT
clowast435TgtA
TgtG
Exon
5UTR
-31015840(2808)
SNP
na
rs34176791
g178095076AgtC
clowast437TgtG
Exon
5UTR
-31015840(2810)
SNP
C=000235
1000
Genom
esrs35911553
g178095045CgtT
clowast46
8GgtA
Exon
5UTR
-31015840(2841)
SNP
T=000
6915
1000
Genom
esSequ
ence
varia
tions
inup
stream
andexon
sofh
uman
NRF
2fro
m655varia
tions
availablein
publicdatabase
asof
Decem
ber2012
(583
activ
esomeSN
Psmergedge14
SNPs
citedin
PubM
ed)lowastNCB
Ireference
sequ
ence
NC
0000
0211
(Hom
osapiens
chromosom
e2G
RCh37p
10prim
arya
ssem
bly)spanning
178095033ndash1781298
59bp
(com
plem
ent34827
bpforexons
andintro
ns)51015840-Flank
ingregions
startat17812
9860
bp(minus1)in
transcrip
tvariant
1HGVS
Hum
anGenom
eVa
riatio
nSo
ciety
Position
sinvaria
nt1(NM
006164
4)2859
bpsectSN
Pscitedin
PubM
edE
xon11781298
59ndash17812
9260
(600
bpT
TS=1781293
04)exon
2178098999ndash
178098733(267
bp)exon
3178098067ndash1780979
78(90b
p)exon41780973
11ndash178097120
(192b
p)and
exon
5178096736ndash
178095033(1704b
p)daggerProteinam
inoacid
(aa)
resid
uesiniso
form
1(N
P00
61552605
aa)Cn
scoding
-non
syno
nymou
sCs
cod
ing-syno
nymou
sDaggerheterozygositydetectednano
tavailable
Oxidative Medicine and Cellular Longevity 11
Table4Hum
anNRF
2SN
Psassociated
with
diseaser
isk
IDMap
onchromosom
e2 (H
GVS
name)
Region
class
Dise
asea
ssociatio
nandreferences
Risk
allelesa
ndstatistics
Ethn
icgrou
p(num
bero
fcase)
rs7557529
g178135097CgtT
minus5238
Ggt
AParkinsonrsquos
disease(2010
[26])
G=0718252Dagger
inhaplotype
OR=090
or040
CI=
060ndash140or
03ndash06
Swedish
Polish
Caucasian
(357)
rs2886162
g178133165AgtG
minus3306
TgtC
Breastcancer
survival(2012[27])
TT=0324
OR=16
87C
I=1105ndash275
FinlandKB
CP(452)
Chronicg
astritisgastric
ulcer(2007
[28])
GDagger
GDaggerin
haplotypefor
ulcer(OR=
252C
I=119ndash
545)
Japanese
(159)
P14methylationin
gastric
cancer
Hpyloriinfectio
n(2008[29])
Gin
haplotype
OR=290C
I=114ndash
736
Japanese
(209)
Gastriccancer
inHpylori-n
egative
cases(2008
[30])
Ain
haplotype
119875=0022
Japanese
(209)
rs35652124
(rs57695243)
g178130073TgtC
minus214Agt
G(previou
slyminus653[23]
orminus686[22])
Ulcerativec
olitis(2008
[31])
AG
(OR=045C
I=022ndash0
93)
G(O
R=257C
I=10
1ndash660)
Japanese
(89)
Lupu
swith
neph
ritisin
female(2010
[25])
GA
OR=18
1CI
=10
4ndash312
Mexican
mestizo(362)
Parkinsonrsquos
disease(2010
[26])
A=0884312Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
COPD
(2010[32])
G=052Dagger
hazard
ratio
=095C
I=091ndash0
99
(Haplotype)
German
(69)
Gastriculcer(2007
[28])
GDaggerin
haplotype
OR=252C
I=119ndash
545
Japanese
(159)
P14methylationin
gastric
cancer
H
pyloriinfection(2008[29])
Gin
haplotype
OR=290C
I=114ndash
736
Japanese
(209)
Gastriccancer
inHpylori-negative
cases(2008
[30])
Gin
haplotype119875=0022
Japanese
(209)
rs6706
649
g178130071CgtT
minus212Ggt
A(previou
slyminus651[23]
orminus684[22])
Ulcerativec
olitis(2008
[31])
AG
(OR=045C
I=022ndash0
93)
G(O
R=257C
I=10
1ndash660)
Japanese
(89)
Maternalacetaminop
henandasthma
(2010[33])
A=02321137Dagger
OR=17
3CI
=12
2245
UKALSPA
C(gt40
00mothersgt
5000
child
ren)
12 Oxidative Medicine and Cellular Longevity
Table4Con
tinued
IDMap
onchromosom
e2 (H
GVS
name)
Region
class
Dise
asea
ssociatio
nandreferences
Risk
allelesa
ndstatistics
Ethn
icgrou
p(num
bero
fcase)
COPD
(2010[32])
G=098Daggerin
haplotype
hazard
ratio
=095C
I=091ndash0
99
German
(69)
Parkinsonrsquos
disease(2010
[26])
G=0972343Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
03ndash06
Swedish
Polish
Caucasian
(357)
Acutelun
ginjury
follo
wingtrauma
(2007[23])
CA=0119Dagger
OR=644
CI=
134ndash
3080
Caucasia
nAfrican-
American
(164
)
Ann
ualF
EV1decline
(2011[34])
A=0082in
haplotype
Japanese
(915)
Vitiligo(2008[35])
CAA
AOR=17
24C
I=13
5ndash221
Chinese(300)
COPD
(2010[32])
G=073Daggerin
haplotype
hazard
ratio
=095C
I=091ndash0
99
German
(69)
rs6721961
(rs117801448)
g178130037TgtC
TgtG
minus178Agt
GAgt
C(previou
slyminus617[23]
orminus650[22])
Parkinsonrsquos
disease(2010
[26])
C=0980346Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
Postm
enop
ausalvenou
sthrombo
embo
lism
(2011[36])
A=033311
OR=25CI
=370ndash8570
Caucasia
n(161)
Breastcancer
survivalandNRF
2proteinexpressio
n(2012[27])
AA
OR=4656CI
=13
5ndash1606
FinlandKB
CP(452)
Acutelun
ginjury-related
mortality
followingsyste
micinflammatory
respon
sesynd
rome(2012
[37])
GG
OR=97
3CI
=12
7ndash7480
Caucasia
n(750)
Infection-indu
cedasthma(
2012
[38])
AC
OR=0437CI
=028ndash0
80
Hun
garia
nCaucasia
nGypsy
(307)
rs143406266
g178129391178
129393delGGC
Exon
1(467ndash4
69G
CC)
COPD
(2010[32])
GCC
4=053Dagger
hazard
ratio
=095C
I=091ndash0
99
Taiwanese(69)
rs2886161
g178127839Tgt
ClowastIntro
n1
Parkinsonrsquos
disease(2010
[26])
T=0878309Dagger
inhaplotype
OR=09or
04CI
=06ndash
14or
03ndash06
Swedish
Polish
Caucasian
(357)
rs2364723
g178126546Ggt
CIntro
n1
Basaland
smoker
FEV
1(200
9[39])
CI=minus6360simminus1780C
=0525Dagger
Also
ashaplotype
Netherla
nd(2542)
rs2364722
g178124787Agt
GIntro
n1
Ann
ualF
EV1decline
(2011[34])
A=0082in
haplotype
Japanese
(915)
rs13001694
g178118990Agt
GIntro
n1
Basaland
smoker
FEV
1(200
9[39])
G=040
11578Dagger
inhaplotype
Netherla
nd(2542)
Oxidative Medicine and Cellular Longevity 13
Table4Con
tinued
IDMap
onchromosom
e2 (H
GVS
name)
Region
class
Dise
asea
ssociatio
nandreferences
Risk
allelesa
ndstatistics
Ethn
icgrou
p(num
bero
fcase)
Breastcancer
[40]
Twith
NQO1N
OS3H
O1risk
allelesOR=15
6CI
=097ndash251
Caucasia
nandothers(505)
rs1806
649
(rs58745895)
g178118152Cgt
TIntro
n1
Basaland
smoker
FEV
1(200
9[39])
T=02631119Daggerin
haplotype
CI=minus8730ndash
(minus17
0)Netherla
nd(2542)
Parkinsonrsquos
disease(2010
[26])
T=0422148Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
Particulatem
attera
ndasthmaCO
PDadmiss
ion(2012[41])
Cwith
lowvitamin
Clevel(OR=
31CI
=15
0ndash630)
UK(209)
rs4243387
(rs6003846
4)g178117765Cgt
TIntro
n1
Basaland
smoker
FEV
1(200
9[39])
T=00914
25Daggerin
haplotype
Netherla
nd(2542)
rs1962142
(rs58448508)
g178113484Agt
GIntro
n1
Ann
ualF
EV1decline
(2011[34])
A=0082in
haplotype
Japanese
(915)
Breastcancer
NRF
2andARE
expressio
n(2012[27])
A(119875=0036)
FinlandKB
CP(452)
rs6726395
(rs57309289)
g178103229Agt
GIntro
n1
Smok
ing-relatedFE
V1decline
and
annu
alFE
V1decline
(2011[34])
G=0884Dagger
A=0082in
haplotype
Japanese
(915)
Basaland
smoker
FEV
1(200
9[39])
G=046
41764Daggerin
haplotype
Netherla
nd(2542)
rs2001350
(rs17515179
rs60883775)
g178100
425Cgt
TIntro
n1
Ann
ualF
EV1decline
(2011[34])
T=0082in
haplotype
Japanese
(915)
Parkinsonrsquos
disease(2010
[26])
T=0986350Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
rs10183914
(rs58731187
rs61374844
)g17809766
6Cgt
TIntro
n3
Parkinsonrsquos
disease(2010
[26])
T=0536188Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
rs2706110
g178092162Tgt
C31015840Flanking
Breastcancer
(2012[27])
TT
OR=2079CI
=118ndash368
FinlandKB
CP(452)
rs2588882
g178087165Ggt
T31015840Flanking
Infection-indu
cedasthma(
2012
[38])
TG
OR=0290CI
=013ndash0
62
Hun
garia
nCaucasia
nGypsy
(307)
minus686in
reference[22]
=minus653in
reference[23]
=currentlyminus214minus684in
reference[22]
=minus651inreference[23]
=currentlyminus212minus651inreference[22]
=minus617in
reference[23]
=currentlyminus178Ch
romosom
econtig(in
tron
31015840flank
ing)
orreversed
(51015840flank
ingprom
oter)a
llelesinbo
ldhave
been
used
inthetextand
TableOR
odds
ratio
CI95confi
denceinterval
14 Oxidative Medicine and Cellular Longevity
minus212GG (98 rs67006649 previously minus684minus651) minus178Callele (73) and GCC
4(53) was predicted to increase
respiratory failure development (hazard ratio = 095 CI091ndash099) in German COPD patients [32] Significant inter-action was also identified between an intronic SNP Gallele (rs6726395 g178103229AgtG 884 frequency) andsmoking status on FEV
1decline relative to the reference
AA allele in the above Japanese cohort [34] Siedlinskiet al [39] reported that the CC genotype of anotherintronic SNP (rs2364723 g178126546GgtC) was associatedwith a lower FEV
1level compared to the wild-type geno-
type (GG) in two Netherland cohorts (CI minus636minus178frequency = 0525 and pooled cohort size = 2542) ThisSNP alone or as a haplotype with 4 more intronic SNPs(rs13001694Grs1806649Trs4243387Trs6726395G)was alsoassociated with high FEV
1levels in individuals that ever
smoked [39] In a Hungarian population of childhoodasthma SNPs at minus178 (CA) and 31015840 flanking (rs2588882TG)loci were inversely associated with infection-induced asthma(OR 0437 CI 028ndash080 OR 0290 CI 013ndash062 resp) andthese SNPs significantly influenced an asthma-environmentalpollution interaction [38]The intronic SNP rs1806649 (CgtT)was associated but not significantly with an increased risk ofhospitalization during high-level particulate matter (PM
10)
periods in asthma or COPD patients (119899 = 209) of the UnitedKingdom (UK) [41] Asthma andCOPDadmission rateswererelated to the increase in environmental PM
10concentration
Importantly effects of interaction between prenatal stressand NRF2 SNPs on descendant pulmonary health wereinvestigated by the Avalon Longitudinal Study in the UKmaternal smoking during pregnancy was not associated withlung function change determined by maximum mild expi-ratory flow (FEF
25ndash75) or with asthma incidence in school-aged children and this relation was not modified by NRF2SNP genotypes [42]However early gestation acetaminophenexposure significantly influenced the risk of asthma andwheezing at the age of 7 years in gt4000 mothers and gt5000children [33] When maternal copies of the minus212A allele werepresent association with asthma (11374891 OR 173 CI122ndash245) and wheezing (11494949 OR 153 95 CI 106ndash220) was significantly increased [33]
Gastrointestinal Disorders While there was no evidence inlung cancer cases studies in Japanese populations suggesteda potential association of NRF2 variations with gastro-intestinal tumorigenesisHelicobacter pylori (H pylori) causesgastritis which can lead to gastric atrophy and cancer Ingastric epithelium from the Japanese cancer cohorts (39gastric cancers 46 controls) H pylori infection was posi-tively correlated with aberrant CpG island methylation oftumor suppressor genes (eg p14) and minus214Gminus212G orminus214Aminus212G NRF2 haplotype was significantly associatedwith increased (OR 290 95 CI 114ndash736) or decreased(OR 033 95 CI 013ndash088) risk of the CpG methyla-tion respectively in the H pylori-infected patients [29]Further study from the same investigators determined thatminus214Aminus212G allele carriers had significantly (119875 = 0022)reduced risk of gastric cancer inH pylori-negative cases [30]The minus214AGminus212AG genotypes were negatively associated
(OR 045 CI 022ndash093) and the minus214Gminus212G genotypeswere positively associated (chronic continuous phenotypeOR 257 CI 101ndash660) with ulcerative colitis (89 patients 141controls) in a Japanese population [31]
Autoimmune Disorders Systemic lupus erythematosus (SLE)is a long-term autoimmune disease more frequently found infemales than in males It affects organs including skin jointskidneys and brain and nephritis is an aggressive character-istic in some patients Genome-wide association studies inhumans identified a suggestive quantitative trait locus nearNRF2 [43] A study of a Mexican Mestizo population (362patients with childhood-onset SLE 379 controls and 212nephritis diagnosed) determined that lupus with nephritiswas significantly (OR 181 CI 104ndash312) associated with theminus214GA SNP in females [25] The same SNPs were notclosely associated with SLE risk in a Japanese cohort [22]Vitiligo is a skin condition in which there is a loss of browncolor (pigment) from areas of skin resulting in irregularwhite patches It is thought to be an autoimmune diseasecaused by loss of cells (melanocytes) that produce brownpigment A study indicated that theminus178A allele increased therisk of vitiligo dose-dependently (OR 1724 95 CI 135ndash221for CA OR 2902 CI 162ndash519 for AA) [35]
Female Disorders It is well known that estrogen metabolites(eg catechols) cause ROS formation suggesting correla-tion of NRF2 and downstream effectors in postmenopausalmammary cancer In a study of a Finish population (KuopioBreast Cancer Project 119899 = 452 patients 370 controls)the minus178AA homozygous genotype (OR 4656 CI = 135ndash1606) and 31015840 flanking rs2706110 (TT OR 2079 CI 118ndash368) genotype were associated with increased risk of breastcancer while the 51015840 flanking minus3306TT homozygous allelewas significantly associated with lower survival (frequency =71219 OR 1687 CI 1105ndash275) [27] suggesting that NRF2genetic polymorphisms affect susceptibility and outcome ofthe patients The minus178A allele carriers together with intronicrs1962142A allele carriers were associated with lowered tissuelevels of NRF2 proteins [27] In postmenopausal women theminus178A allele (OR 179 95CI 370ndash8570) appeared tomodifythe risk of venous thromboembolism caused by oral estrogentherapy (AA or AC frequency = 333) as demonstratedby the French ESTHER study (161 cases 474 controls) [36]An intronic rs1806649CgtT SNP did not associate with breastcancer risk in postmenopausal women [40] However whenthis SNP and other at-risk alleles of ARE-responsive genes(NQO1 HO-1 NOS3) were combined there was a significantgene-dose effect on the breast cancer risk [40] Althoughcoding region SNPs in NRF2 and KEAP1 were identifiedin the Japanese endometrial adenocarcinoma patients noassociation of NRF2 SNPs with the disease was found [44]
Neurodegenerative Diseases Oxidative stress is known to beinvolved in Parkinsonrsquos disease (PD) presumably due toproduction of ROS fromhigh-dopaminemetabolism and lowlevels of antioxidants in the substantia nigra of the brainInvestigators found a protective NRF2 haplotype consistingof four 51015840 flanking SNPs (minus5238Gminus214Aminus212Gminus178C)
Oxidative Medicine and Cellular Longevity 15
and 4 intronic SNPs (rs2886161Ars1806649Ars2001350Ars10183914A) from Swedish (OR 09 CI 060ndash140) and Polish(OR 04 CI 030ndash060) populations (total 165+192 PD cases190+192 controls) [26]The investigators also suggested thatNRF2 haplotype alleles were associated with 2 years earlierage of Alzheimerrsquos disease (AD) onset 4 years earlier age ofposterior subcapsular cataract surgery and 4 years later ageof cortical cataract surgery while they were not significantlyrelated to AD or age-related cataract risk [45]
34 Genetic Mutations in Mouse Nrf2 Tsang et al [46]compiled 673 SNPs in 55 mouse strains and constructedtheir phylogenetic tree to correlate and clarify the originsof strains based on the assembled mouse genome sequenceand SNP data [20 47 48] Recently using the completegenome sequence of C57BL6J (B6) mouse as a referencehigh-density SNP screening in other laboratory strains orin panels of strains has been published (see [17]) Althoughmillions of mouse SNPs (gt10089892 as of December 2012)and haplotype mappings from more than 120 strains havebeen published as valuable references for dissecting thegenetic basis of complex traits [49ndash51] little attention hasbeen paid to polymorphisms of Nrf2 and their correlationwith disease phenotypes
Figure 3 demonstrates the proximal promoter region(minus1 to minus950)51015840-UTR (exon 1 up to TSS) and proteinsequence of mouse Nrf2 based on GRCm38p1 PrimaryAssembly (75704641ndash75675513 bp) mRNA variant1 and protein (NP 035032 encoded by NM 010902234ndash2027 bp) sequences Genetic variations in the Nrf2genome of inbred strains collected from public databasesare listed in Tables S2 and 5 (See Supplementary TableS2) Overall 968 genetic mutations are compiled forNrf2 gene and 5 kb upstream2 kb downstream regions785 SNPs between B6 and another 16 strains wereacquired from the Mouse Phenome Database (MPDhttpphenomejaxorg dbqrtn=snpret1) and additionalSNPs and other mutations were acquired from NCBI dbSNP(httpwwwncbinlmnihgovsnpterm=mus+musculus20nfe2l2) In total 132 mutations are in the promoter (37in proximal 1 kb) 49 in exons (38 in coding region 19 Cns)727 in introns and 60 in the 31015840 flanking region Excludingmutations in the 51015840 and 31015840 flanking sequences murine Nrf2sequences appear to be more highly variable (1 variationper 375 bp) than much of the mouse genome which has anapproximate frequency of one SNP per every 245 bp (httpwwwinformaticsjaxorgmgihomehomepagesstatsallstatsshtmlallstats snp)
Nrf2 was found to be a susceptibility gene from genome-wide linkage analysis in a murine model of hyperoxia-induced ALI [52] A promoter SNP minus103TgtC (previouslypublished as minus336TgtC) in Nrf2 was found and predicted toadd an additional Sp1 binding site in hyperoxia-susceptibleB6 mice but not in resistant C3HHeJ mice [52] Genotypesfrom the SNP and from simple-sequence length polymor-phism markers of the Nrf2 locus (D2Mit248 and D2Mit94)cosegregated in the B6C3F
2mouse cohort [52] and Nrf2
deficient mice were significantly more susceptible to ALI
sub-phenotypes caused by hyperoxia than similarly exposedwild-type mice supporting Nrf2 as a contributor to thephenotypic traits [53] Although no other functional analyseson Nrf2 SNPs or haplotype association studies have beenconducted in inbred mice strains bearing haplotypes suchas multiple Cns in functional domains (eg F71L L451VH543Q and L575M) may be useful to elucidate the role ofNrf2 in differential susceptibility to oxidative diseases
4 Oncogenic Somatic Mutations in HumanNF-E2-Related Factor 2
Somatic mutation is a change in the DNA of somatic cellsthat affects derived cells but is not inherited by offspringEfforts to discover somatic mutations have provided insightinto mutagenesis and cancer development Lung cancerparticularly non-small cell lung cancer (NSCLC) is theleading cause of cancer death worldwide Somatic mutationsof NRF2 and KEAP1 discovered in lung cancer patients havedetermined the oncogenic potential ofNRF2 [54 55] KEAP1somatic mutations were associated with its reduced proteinlevels in lung cancer tissues and cells [56 57] Investigationsof NSCLC in various ethnic populations as well as cancersin gastrointestine breast and prostate have coordinatelydemonstrated that multiple Cns somatic mutations in KEAP1cause dysfunction of the translated protein and in turnconstitutive activation of NRF2 increasing risk of neoplasiaand chemoresistance [12 55 58 59] Somatic mutations ofNRF2 have been detected in various cancer tissues (largelysquamous cell carcinomas) in Asian populations (Table 6)NRF2 mutations were significantly associated with NSCLCcases (squamous cell lung carcinoma adenocarcinoma) ofthe Japanese (107 [54]) the Chinese (23 [60]) andthe Koreans (8 [61]) as well as with lung cancer celllines Smoking history was also correlated with mutationoccurrence in all of the studies [54 60 61] In addition tolung cancers laryngeal squamous carcinoma (13 in [61])esophageal squamous cancer (ESC 22 in [60] 114 in[61]) head and neck cancers (25 in [54]) skin (117 casein [61]) and oral cancer cell lines had somatic changesin NRF2 In contrast to wide-spread KEAP1 mutationsmutations in NRF2 were clustered in DLGETGE motifs ofthe Neh2 domain which are critical in the ldquohinge and latchrdquomodel of KEAP1 binding [12] Similar to KEAP1 somaticmutations it has been postulated that NRF2 mutations incancer cells lead to NRF2 accumulation by suppressing itsubiquitination or KEAP1 binding which eventually confersmalignant potential and resistance to chemotherapy
Most variable sites in NRF2 included aa residues 29 (AspD) 31 (Gly G) 77 (Asp D) and 79 (Glu E) (Table 6)Residue 33 (Ser S) in the Neh2 domain is mutated by eithergenetic or somatic processes (Figure 4) Cns in the EDGFmotif of NRF2 was experimentally determined to impairrecognition of KEAP1 [54] NRF2 mutations were signif-icantly correlated with increased (25-fold) copy number(31 of mutants versus 3 wild types) in Japanese NSCLCcases [63] Aberrant mutation of NRF2 also led to increasedexpression of downstream effectors includingRagD known to
16 Oxidative Medicine and Cellular Longevity
pL575M
pF71L
pH543QpL451V
pA151P pA151V pN159D pQ198delpV105D
pA204T pS224I225S226del pT253S pS278ApD305G
pM409V pR410HpP388L pA389G pT395I
Genomic DNA (reversed contig GRCm38 p1 C57BL6JNM 0109023)
Protein (NP 035032)1
51101151201251301351401451501551
minus400
minus500
minus50
minus100
minus200
minus300
minus450
minus350
minus250
minus150
minus950
minus900
minus850
minus800
minus750
minus700
151
101151201
2001
g minus470gg minusg minus
c minus41c minus
c
c
c c
gg minus 6C
g minus125insTTC g minus118insC
g minus281delC
g minus63delCg minus1 (75704642 c minus234)
Translation start site (c +1)Termination codon (c +2025 +2027)
c minus136 minus134in-frame stop codonc minus8585
g minus41
246
440
minus381
minus11
minus91 minus90
minus332
minus153
minus98
c minus197c minus209 minus202
gminus942 minus899 (cminus1175 minus1132) CpG islands [71]
g 460CgtT
g minus103CCgtT [51]
CgtTCgtG
g minus320CgtT
CgtT
CgtT
CgtTg minus426GgtA
GgtACgtA
CgtA
GgtA
g minus238GgtTTgtC
TgtC
TgtC
TgtCTgtG
g minus229AgtG
CgtG
CgtG
GgtC
gminus826 minus654 (cminus1059 ) CpG islands [71]minus887
Figure 3 DNA (partial promoter and exons 1 and 5) and protein sequence ofmouseNrf2 SNPs and amino acid residues for non-synonymousSNPs are marked Promoter regions bearing 5CpG islands are underlined
151
201251301351401451501551601
Genetic mutation (Table 3 and Figure 2)Somatic mutation (Table 6)
Neh2
Neh3
Neh4Neh5
Neh6
Neh1
Protein isoform 1 (NP 006155)
101151
Figure 4 Genetic and somatic mutation loci in human NRF2 protein
Oxidative Medicine and Cellular Longevity 17
Table5Mou
seup
stream
andexon
varia
tions
ofNfe2
l2locusin17
inbred
strainsR
eference
sequ
ence
isC5
7BL6J
(B6strain
1)geno
me(GI14933824975547698ndash75513576)SNPalleleand
geno
typesareshow
nas
chromosom
econ
tigsequ
enceStrains
2129S1S
vlmJ3AJ
4AKR
J5BA
LBcJ6C3
HH
eJ7C
57BL
6NJ8CA
STEiJ9CB
AJ
10D
BA2J11FVBNJ12LPJ
13N
ODShiLtJ14N
ZOH
ILtJ
15P
WK
PhJ16SPR
ETEiJ
17W
SBEiJ
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs2566
08517
75705565
GA
(CT)
51015840Flankingminus924
NC
GG
GG
GG
GG
GG
GG
GG
AA
Grs47274959
75705562
AT
(TA
)51015840Flankingminus921
NC
AT
TA
TT
AA
TT
AT
AT
TT
Ars216940398
75705554ndash75705555
Tadd(A
add)
51015840Flankingminus913minus914
NC
CGrs239114134
75705540
CT(G
A)
51015840Flankingminus899
NC
CT
TC
TT
CC
TT
CT
CT
CT
Crs46
461765
75705528
TG(A
C)oA
C
51015840Flankingminus887
NC
TG
GT
GG
TT
GG
TG
TG
GG
Trs5144
9853
75705525
CA(G
T)
51015840Flankingminus884
NC
CA
AC
AA
CC
AA
CA
CA
CC
Crs249093111
75705512
CT(G
A)
51015840Flankingminus871
NC
CT
TC
TT
CC
TT
CT
CT
TT
Crs263745200
75705510
CG(G
C)
51015840Flankingminus869
NC
GG
GG
GG
GG
GG
GG
GG
CG
Grs45651867
75705498
TG(A
C)
51015840Flankingminus857
NC
TG
GT
GG
TT
GG
TG
TG
GG
Trs219997531
75705495
GA
(CT)
51015840Flankingminus854
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs251918379
75705451
TA(A
T)
51015840Flankingminus810
NC
TT
TT
TT
TT
TT
TT
TT
TA
Trs27978306
75705449
TA(A
T)
51015840Flankingminus808
NC
TA
AT
AA
TT
AA
TA
TA
TT
Trs216087412
75705429
TG(A
C)lowast
51015840Flankingminus788
NC
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
GTlowast
Tlowast
rs261229914
75705423ndash75705424
AGdel(CT
del)
51015840Flankingminus783minus784
NC
AGrs27978307
75705400
CT(G
A)
51015840Flankingminus759
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs243167395
75705359
GA
(CT)
51015840Flankingminus718
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs27978308
75705307
GA
(CT)
51015840Flankingminus66
6NC
GG
GG
GG
GG
GG
GG
GG
AG
Grs254744
09875705294
AT
(TA
)51015840Flankingminus653
NC
AA
AA
AA
AA
AA
AA
AA
AT
Ars228133419
75705179
GT
(CA
)51015840Flankingminus538
NC
GG
GG
GG
GG
GG
GG
GG
GT
Grs214719520
75705111
GA
(CT)
51015840Flankingminus470
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs2440
87730
75705101
GA
(CT)
51015840Flankingminus46
0NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs227781699
75705081
CG(G
C)
51015840Flankingminus44
0NC
CC
CC
CC
CC
CC
CC
CC
GC
Crs257870353
75705067
CT(G
A)
51015840Flankingminus426
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs24487744
075705022
AG
(TC)
51015840Flankingminus381
NC
AA
AA
AA
AA
AA
AA
AA
AG
Ars234628138
75704973
AC
(TG
)51015840Flankingminus332
NC
AA
AA
AA
AA
AA
AA
AA
AC
A
18 Oxidative Medicine and Cellular Longevity
Table5Con
tinued
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs247275247
75704961
GA
(CT)
51015840Flankingminus320
NC
GG
GG
GG
GG
GG
GG
AG
GG
Grs247519047
75704922
Gdel(Cdel)
51015840Flankingminus281
NC
Grs27978309
(rs51915758)
75704887
GA
C(C
TG
)51015840Flankingminus246
NC
GA
AG
AA
GG
AA
GA
GA
CC
G
rs218139102
75704879
CA(G
T)
51015840Flankingminus238
NC
CC
CC
CC
CC
CC
CC
CC
AC
Crs251990355
75704870
TC(A
G)
51015840Flankingminus229
NC
TT
TT
TT
TT
TT
TT
TT
TC
Trs238746955
75704794
AG
(TC)
51015840Flankingminus153
NC
AA
AA
AA
AA
AA
AA
AA
GA
Ars221664
405
75704786
AG
(TC)
51015840Flankingminus145
NC
AA
AA
AA
AA
AA
AA
AA
AG
Ars217054035
75704766
ndash75704767
GAAadd(TTC
add)
51015840Flankingminus125minus126
NC
GA
rs248182931
75704759ndash75704760
Gadd(C
add)
51015840Flankingminus118
minus119
NC
AGrs217197904
75704744
GA
(CT)
51015840Flankingminus103
(+)S
p1[23]
GA
AG
AA
GG
AA
GA
AA
GG
Ars2644
9364975704704
Gdel(Cdel)
51015840Flankingminus63
NC
Grs2404
81112
757046
82CT(G
A)
51015840Flankingminus41
NC
CC
CC
CC
CC
CC
CC
CC
CT
Crs27978310
75704617
AG
(TC)
UTR
-525
NC
AA
AA
AA
AA
AA
AA
AA
GG
Ars27978311
75704610
CG(G
C)
UTR
-532
NC
GG
GG
GG
GG
GG
GG
GG
GG
Crs27978312
757046
05GA
(CT)
UTR
-537
NC
GA
AG
AA
GG
AA
GA
GA
GG
Grs214784220
757044
99AG
(TC)
UTR
-5143
NC
AA
AA
AA
AA
AA
AA
AA
GG
Ars244146318
757044
98GT
(CA
)UTR
-5144
NC
GG
GG
GG
GG
GG
GG
GG
GT
Grs27978313
757044
97GC
(CG
)UTR
-5145
NC
GC
CG
CC
GC
CC
GC
CC
CC
Crs215431944
757044
93CT(G
A)
UTR
-5149
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs257015697
757044
49GA
(CT)
UTR
-5193
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs252234782
757044
19GT
(CA
)UTR
-5223
NC
Grs1346
0861
75679262
AG
(TC)
Exon
244
6Cn
sF71L
AG
GA
GG
AG
GG
AG
AG
GG
Grs1346
0859
75679202
GA
(CT)lowast
Exon
2506
CsH91=
GA
AG
AA
GG
AA
GA
GA
GG
Grs27978436
75679187
GA
(CT)lowast
Exon
2521
CsT9
6=G
GG
GG
GG
AG
GG
GG
GA
GG
rs226131070
75679178
GA
(CT)
Exon
2530
CsS99=
GG
GG
GG
GG
GG
GG
GG
GA
Grs227683834
75678576
AT
(TA
)Ex
on3
547
CnsV
105D
AA
AA
AA
AA
AA
AA
AA
AT
Ars257321187
75678506
TC(A
G)
Exon
3617
CsP128=
TT
TT
TT
TC
TT
TT
TT
TT
Trs27978444
75678500
TA(A
T)
Exon
3623
CsV130=
TT
TT
TT
TA
TT
TT
TT
AT
Trs227743136
75677686
GA
(CT)
Exon
4662
CsH143=
GG
GG
GG
GG
GG
GG
GG
GA
Grs215327202
75677664
CG(G
C)
Exon
4684
CnsA
151P
CC
CC
CC
CG
CC
CC
CC
CC
Crs247539755
75677663
GA
(CT)
Exon
4685
CnsA
151V
GG
GG
GG
GA
GG
GG
GG
GG
Grs233164
668
75677640
TC(A
G)
Exon
4708
CnsN
159D
TT
TT
TT
TT
TT
TT
TT
TC
T
rs2340
95816
75677162ndash75677160
GCT
del
(AGCdel)
Exon
5826minus
828
Cds-in
delQ
198
GCT
Oxidative Medicine and Cellular Longevity 19
Table5Con
tinued
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs27978452
75677145
CT(G
A)
Exon
5843
CnsA
204T
CC
CC
CC
CC
CC
CC
CC
CC
T
rs225047937
75677086ndash75677078
GAG
ATCG
ATdel
(ATG
CATC
TCdel)
Exon
5902minus
910
Cds-in
del
(S224I225S226)
GAG
ATC
GAT
rs241469868
75676998
TA(G
T)
Exon
5990
CnT2
53S
TT
TT
TT
TT
TT
TT
TT
TA
Trs221602571
75676923
AC
(TG
)Ex
on5
1065
CnsS
278A
AA
AA
AA
AA
AA
AA
AA
AC
Ars252576472
75676876
GA
(CT)
Exon
51112
CsS293=
GG
GG
GG
GG
GG
GG
GG
GA
Grs250802933
75676841
TC(A
G)
Exon
5114
7Cn
sD305G
TT
TT
TT
TT
TT
TT
TT
TC
Trs225425698
75676792
CT(G
A)
Exon
5119
6Cs
P321=
CC
CC
CC
CC
CC
CC
CC
CT
Crs13459064
75676732
CT(G
A)
Exon
51256
CsT3
41=
CT
TC
TT
CT
TT
CT
CT
TC
Crs27978453
75676717
CT(G
A)
Exon
51271
CsA346=
CC
CC
CC
CC
CC
CC
CC
TC
Crs214335034
75676636
AG
(TC)
Exon
51352
CsD373=
AA
AA
AA
AA
AA
AA
AA
AG
Ars24958364
4756766
03AC
(TG
)Ex
on5
1385
CsP3
84=
AA
AA
AA
AA
AA
AA
AA
AC
Ars27978454
75676592
GA
(CT)
Exon
51396
CnsP
388L
GG
GG
GG
GG
GG
GG
GG
AG
Grs215384328
75676589
GC
(CG
)Ex
on5
1399
CnsA
389G
GG
GG
GG
GG
GG
GG
GG
GC
Grs251728286
75676571
GA
(CT)
Exon
51417
CnsT
395I
Grs247602334
75676567
TC(A
G)
Exon
51421
CsV396=
TT
TT
TT
TT
TT
TT
TT
TC
Trs234216231
75676530
TC(A
G)
Exon
51458
CnsM
409V
TT
TT
TT
TT
TT
TT
TT
TC
Trs212904337
75676526
CT(G
A)
Exon
51462
CnsR
410H
CC
CC
CC
CC
CC
CC
CC
CT
Crs252650779
75676522
TC(A
G)
Exon
51466
CsE4
11=
TT
TT
TT
TT
TT
TT
TT
TC
Trs231273560
75676516
TC(A
G)
Exon
51472
CsQ413=
TT
TT
TT
TT
TT
TT
TT
TC
Trs257886949
756764
13GT
(CA
)Ex
on5
1575
CsR4
48=
GG
GG
GG
GG
GG
GG
GG
GT
Grs241537608
756764
04GC
(CG
)Ex
on5
1584
CnsL
451V
GG
GG
GG
GG
GG
GG
GG
GC
Grs227619071
75676312
TC(A
G)
Exon
51676
CsQ481=
TT
TT
TT
TT
TT
TT
TT
TC
Trs258913831
75676290
AG
(TC)
Exon
51698
CsL4
89=
AA
AA
AA
AA
AA
AA
AA
AG
Ars225593319
75676126
AT
(TA
)Ex
on5
1862
CnsH
543Q
Ars4223233
75676105
GA
(CT)
Exon
51883
CsS550=
GG
GG
GG
GA
GG
GG
GG
GG
Grs4223232
75676032
GT
(CA
)Ex
on5
1956
CnsL
575M
Grs4223231
75675816
CG
UTR
-32172
NC
GG
GG
GG
GG
GG
GG
GG
GC
Grs1346
860
75675682
AT
UTR
-32306
NC
ASequ
ence
varia
tions
inmou
seNr
f2wereob
tained
from
Mou
sePh
enom
eDatabase(http
ph
enom
ejaxorgSNP)
andNCB
ISNPdatabase
(http
wwwncbinlm
nihgovSNP)N
CBIreference
sequ
ence
isMus
Musculusstra
inC5
7BL6J
chromosom
e2GRC
m38p1(NC
0000
687
GI37209910875704641ndash756755192
9123
bp)To
tal968
genetic
mutations
arerepo
rted
inNr
f2gene
and5k
bup
stream
(ge75704
642minus
1)and2k
bdo
wnstre
am(le75675512)sequences
asof
Janu
ary2003A
llSN
Psandallelesa
representedas
geno
miccontig
(reversedsequ
ence
indicated)E
xon175704
641ndash75704364(278
bpT
TS=75704
408)
exon
275679431ndash756791
65(267
bp)exon
375678579ndash75678490(90b
p)exon475677713ndash756775
46(168
bp)andexon
57567718
4ndash75678849
(1666
bp)Proteinam
inoacid
(aa)
resid
uesinNP00
61552
(597
aa)Cs
cod
ing-syno
nymou
sCn
scoding
-non
syno
nymou
sAminoacid
A-alanineD-aspartic
acidE
-glutamicacidF-phenylalanineG
-glycineH
-histidineI-iso
leucineL-leu
cineand
M-m
ethion
ineN-
asparagineP-prolin
eQ-glutamineR-arginineS-serineT-threon
ineandV-valin
elowastErrorsin
databasesfi
xedNC
notcon
firmed
20 Oxidative Medicine and Cellular Longevity
Table 6 NRF2 somatic mutations revealed in various human cancers
Domain Amino acid residues DNA mutation Cancer types (cases) ReferencesLocus Wild type Mutant
DLG motif
24 W (Trp) C (Cys) c72GgtCGgtT NSCLC neck ESC [54 62]K (Lys) c72TgtC ESC [62]
26 Q (Gln) E (Glu) c76CgtG NSCLC ESC [54 62]
27 D (Asp) G (Gly)lowast c80GgtA NSCLC [60]Y (Tyr) c79GgtT ESC [61]
28 I (Ile) T (Thr) c83CgtT NSCLC [54]
29 D (Asp) G (Gly) c86AgtG Head and neck ESC [54 62]H (His) c85GgtC NSCLC laynx [60 61]
30 L (Leu) F (Phe) c88CgtT NSCLC ESC [54 62]
31 G (Gly) A (Ala) c92GgtC NSCLC ESC skin [54 60ndash62]
32 V (Val) T (Thr) c95TgtG NSCLC [54]del c93 95delAGT ESC [61]
33ndash36 S-R-E-V S-R-E-V-S-R-E-Vlowast c97 108dupAGTCGAGCCGTA ESC [61]
34 R (Arg) Q (Gln) c101GgtA NCSCL [54 60 61]P (Pro) c101GgtC NSCLC [63]
ETGF motif
75 Q (Gln) H (His) c225AgtC Head and neck ESC [54 62]
77 D (Asp)
V (Val) c230AgtT NSCLC ESC [54 60 62]G (Gly) c230AgtG ESC [62]A (Ala) c230AgtC NSCLC [61]N (Asn) c229GgtA Larynx [61]
78 E (Glu) K (Lys) c232GgtA NSCLC ESC [54 62]
79 E (Glu)
K (Lys) c235GgtA NSCLC ESC [54 61 62]Q (Gln) c235GgtC NSCLC ESC [54 60ndash62]G (Gly) c236AgtG Larynx [61]E-E c234 236dupAGA ESC [61]
80 T (Thr) K (Lys) c239CgtACgtG NSCLC ESC [54 61 62]
80 T (Thr)P (Phe) c238AgtC ESC [62]I (Ile) c239CgtT Head and neck [54]A (Ala) c238AgtG NSCLC [63]
81 G (Gly) V (Val) c242GgtT ESC [61]D (Asp) c242GgtA NSCLC ESC [60ndash62]
82 E (Glu)
D (Asp) c246AgtT ESC oral cancer cell line [54 62]G (Gly) c245AgtG NSCLC [54]Q (Gln)lowast c244GgtC NSCLC ESC [60 61]V (Val)lowast c245AgtT ESC [61]
83 F (Phe) L (Leu)lowast c247TgtC NSCLC [60]NSCLC non-small cell lung cancer ESC esophageal squamous cancer and lowasterrors in reference fixed Number of cases 82 NSCLC and 10 ESC in [62] 125NSCLC 70 ESC 23 larynx and 17 skin in [61] 103 NSCLC and 12 head and neck in [54] 90 NSCLC in [63] 103 NSCLC in [60]
be involved in squamous lung cancer cell proliferation [64]suggesting that the mutation is functional and overcomesKEAP1 inhibition Singh et al [65] determined in vitrothat RNAi-mediated depletion of NRF2 in lung cancer cellsenhanced ROS production and susceptibility to cell deathby ionizing radiation These studies support the concept thatelevated NRF2 and ARE responsiveness provides cancer cellswith proliferative advantage for malignant transformation
and undue protection from anti-cancer therapy Onco-genic epidermal growth factor receptor (EGFR) signaling isrecently found to be critical in NRF2-mediated proliferationof NSCLC cells [66]
Collectively ldquogain of functionrdquo mutations in NRF2 thatreduce KEAP1 recognition are suggested to be predictivemarkers for poor responsiveness to chemotherapy and radi-ation therapy Although NRF2-mediated cellular defense
Oxidative Medicine and Cellular Longevity 21
processes are essential in the initiation stage enhancedNRF2-ARE activity in advanced stages of cancer developmentmay create a favorable intracellular environment for tumorcell growth and survival [67 68] In this context NRF2may be a potential molecular target for the treatment ofradio-resistance cancers especially those that have ldquoloss offunctionrdquo mutations in EGFR KRAS or KEAP1 as well asldquogain of functionrdquo mutations in NRF2
5 Epigenetic Alterations of NF-E2-RelatedFactor 2
Epigenetic modifications are alterations of molecules inter-acting with genes without changes to the primary DNAsequence They include post-translational modification ofhistones DNA methylation events chromatin conforma-tional changes and alterations to noncoding regulatoryRNAs Epigenetic alterations are stable and often inheritablebut are reversible and may affect expression of the geneDysregulation or defects in epigenetic processes particularlyhypermethylation of tumor suppressor gene promoters (egCpG islands) or histone modifications are thought to beassociated with carcinogenesis Investigators have reportedhypermethylation in CpG islands of KEAP1 which wereassociated with reduced KEAP1 expression in human cancersfrom lung prostate colon and so forth [69ndash72] Similar tosomatic mutations epigenetic changes on KEAP1 impairedthe function of its encoded protein leading to constitutiveNRF2 activation
Supporting the role of ldquopathogenic mutationsrdquo in NRF2expression of Nrf2 and downstream Nqo1 was suppressedin prostate tumors of mice (transgenic adenocarcinomaof mouse prostate TRAMP) Among 15 promoter CpGislands located between minus942 and minus654 (cminus1175 cminus1132and cminus1059 cminus887 gap in cminus1131 cminus1060 see Figure 3)hypermethylation of the first 5 CpG islands (minus942 minus899 andcminus1175 cminus1132) was significantly associated with tumorige-nesis [73] Moreover treatment with inhibitors for DNAmethyltransferase and histone deacetylase restored Nrf2expression in these tumor cells [73] A dietary phytochemicalcurcumin known as a DNA hypomethylation agent restoredepigenetically silent Nrf2 expression through CpG demethy-lation in carcinogen-induced mouse tumor cells [74]
The whole genome epigenetic datasets for 5 species arepublicly accessible at NCBI Epigenomics [75 76]The humanNRF2 epigenome of primary cells (breast penis) and H1stem cell line as well as mouse Nrf2 CpG island methylationdata for sperm blood and cerebellum are currently avail-able (httpwwwncbinlmnihgovepigenomics) Althoughno direct evidence of disease-associated epigenetic modu-lation has been identified in human NRF2 various phyto-chemical NRF2 agonists such as sulforaphane and curcuminhave shown their roles in DNA methylation and histonemodification (see reviews by Lee and colleagues eg [77])Taken together epigenetic modifications of theNRF2KEAP1axle are predicted to cause dysregulation of ARE-mediatedcellular defense leading to deleterious health effects and
phytochemical antioxidants as epigenetic modulators forNRF2 are suggested to be useful in cancer prevention
6 Conclusions
NRF2 is evolutionally conserved with high-sequence homol-ogy in many species However it is a highly mutable geneand numerous genetic variants have been discovered inhuman ethnic groups Importantly certain SNPs or haplo-types have been identified in various diseases as ldquoat-riskrdquoalleles and are related to functional alterations In additionto genetic variations multiple somatic mutations identifiedin the KEAP1 recognition domain of NRF2 in cancer cellshave been found to be oncogenic due to dysregulation ofNRF2 homeostasis by its excess ldquogain of functionrdquo Epigeneticalteration of theNRF2 is under investigation and is predictedto have pathogenic influences as learned from mouse andphytochemical agonist studies Continuous updates of Nrf2allelic variants in inbred mouse strains will provide a usefultool for effective experimental designs formodels of oxidativedisorders to provide insight into the disease mechanisms andintervention strategies
Abbreviations
AD Alzheimerrsquos diseaseALI acute lung injuryARE antioxidant response elementbZIP basic leucine zippercds coding DNA sequencesCI confidence intervalCOPD chronic obstructive pulmonary diseaseESC esophageal squamous cancerFEV1 forced expiratory volume in one second
Gbp giga base pairsGST glutathione S-transferaseGWAS genome-wide association studyHGVS Human Genome Variation SocietyHO-1 heme oxygenase-1119867 119901119910119897119900119903119894 119867119890119897119894119888119900119887119886119888119905119890119903 119901119910119897119900119903119894KEAP1 Kelch-like ECH activating protein 1MPD Mouse Phenome DatabaseMRP multidrug resistance proteinNCBI National Center for Biotechnology InformationNeh NRF2-ECH homologyNfe2l2 nuclear factor (erythroid-derived 2)-like 2Nrf2 NF-E2-related factor 2NSCLC nonsmall cell lung cancerNQO1 NAD(P)Hquinone oxidoreductase 1OR odds ratioPD Parkinsonrsquos diseasePM particulate matterROS reactive oxygen speciesSLE systemic lupus erythematosusSNP single nucleotide polymorphismSOD superoxide dismutaseURT untranslated region
22 Oxidative Medicine and Cellular Longevity
Disclosure
Authorrsquos contribution to the Work was done as part of theAuthorrsquos official duties as a NIH employee and is a Work ofthe United States Government Therefore copyright may notbe established in the United States
Conflict of Interests
The author declares that there is no conflict of interests
Acknowledgments
The research related to this paper was supported by theIntramural Research Program of the National Institutes ofHealth of the National Institute of Environmental HealthSciences (NIEHS) Drs Steven Kleeberger and StephanieLondon at the NIEHS provided excellent critical review ofthis paper The author thanks Mrs Jacqui Marzec for herhelpful comments and English editing
References
[1] J Y Chan M C Cheung P Moi K Chan and Y W KanldquoChromosomal localization of the humanNF-E2 family of bZIPtranscription factors by fluorescence in situ hybridizationrdquoHuman Genetics vol 95 no 3 pp 265ndash269 1995
[2] K Itoh K Igarashi N Hayashi M Nishizawa and MYamamoto ldquoCloning and characterization of a novel erythroidcell-derived CNC family transcription factor heterodimerizingwith the small Maf family proteinsrdquo Molecular and CellularBiology vol 15 no 8 pp 4184ndash4193 1995
[3] W O Osburn and T W Kensler ldquoNRF2 signaling an adaptiveresponse pathway for protection against environmental toxicinsultsrdquoMutation Research vol 659 no 1-2 pp 31ndash39 2008
[4] D Papp K Lenti D Modos D Fazekas Z Dul D Tureiet al ldquoThe NRF2-related interactome and regulome containmultifunctional proteins and fine-tuned autoregulatory loopsrdquoFEBS Letters vol 586 no 13 pp 1795ndash1802 2012
[5] X Wang D J Tomso B N Chorley et al ldquoIdentificationof polymorphic antioxidant response elements in the humangenomerdquo Human Molecular Genetics vol 16 no 10 pp 1188ndash1200 2007
[6] D Malhotra E Portales-Casamar A Singh et al ldquoGlobalmapping of binding sites for NRF2 identifies novel targets incell survival response through ChIP-Seq profiling and networkanalysisrdquo Nucleic Acids Research vol 38 no 17 pp 5718ndash57342010
[7] K Chan R Lu J C Chang and Y W Kan ldquoNRF2 a memberof the NFE2 family of transcription factors is not essential formurine erythropoiesis growth and developmentrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 93 no 24 pp 13943ndash13948 1996
[8] K Itoh T Chiba S Takahashi et al ldquoAn NRF2small Mafheterodimer mediates the induction of phase II detoxifyingenzyme genes through antioxidant response elementsrdquo Bio-chemical and Biophysical Research Communications vol 236no 2 pp 313ndash322 1997
[9] FMartin J M vanDeursen R A Shivdasani CW Jackson AG Troutman and P A Ney ldquoErythroid maturation and globin
gene expression inmicewith combined deficiency ofNF-E2 andNrf-2rdquo Blood vol 91 no 9 pp 3459ndash3466 1998
[10] K Itoh N Wakabayashi Y Katoh et al ldquoKEAP1 repressesnuclear activation of antioxidant responsive elements by NRF2through binding to the amino-terminal Neh2 domainrdquo Genesand Development vol 13 no 1 pp 76ndash86 1999
[11] A Kobayashi M I Kang H Okawa et al ldquoOxidative stresssensor KEAP1 functions as an adaptor for Cul3-based E3 ligaseto regulate proteasomal degradation of NRF2rdquo Molecular andCellular Biology vol 24 no 16 pp 7130ndash7139 2004
[12] K Taguchi H Motohashi and M Yamamoto ldquoMolecularmechanisms of the KEAP1-NRF2 pathway in stress responseand cancer evolutionrdquo Genes to Cells vol 16 no 2 pp 123ndash1402011
[13] K Mukaigasa L T Nguyen L Li H Nakajima M Yamamotoand M Kobayashi ldquoGenetic evidence of an evolutionarilyconserved role for NRF2 in the protection against oxidativestressrdquoMolecular and Cellular Biology vol 32 no 21 pp 4455ndash4461 2012
[14] JMaher andMYamamoto ldquoThe rise of antioxidant signalingmdashthe evolution and hormetic actions of NRF2rdquo Toxicology andApplied Pharmacology vol 244 no 1 pp 4ndash15 2010
[15] S C Lo X Li M T Henzl L J Beamer and M HanninkldquoStructure of the KEAP1NRF2 interface provides mechanisticinsight intoNRF2 signalingrdquoThe EMBO Journal vol 25 no 15pp 3605ndash3617 2006
[16] V A McKusick ldquoMendelian inheritance in man and its onlineversion OMIMrdquo American Journal of Human Genetics vol 80no 4 pp 588ndash604 2007
[17] B Yalcin D J Adams J Flint and T M Keane ldquoNext-generation sequencing of experimental mouse strainsrdquo Mam-malian Genome vol 23 no 9-10 pp 490ndash498 2012
[18] D R Bentley S Balasubramanian H P Swerdlow et alldquoAccurate whole human genome sequencing using reversibleterminator chemistryrdquo Nature vol 456 no 7218 pp 53ndash592008
[19] P S G Chain D V Grafham R S Fulton et al ldquoGenomeproject standards in a new era of sequencingrdquo Science vol 326no 5950 pp 236ndash237 2009
[20] RHWaterstonK Lindblad-Toh E Birney J Rogers J F AbrilP Agarwal et al ldquoInitial sequencing and comparative analysis ofthe mouse genomerdquo Nature vol 420 pp 520ndash562 2002
[21] T M Keane L Goodstadt P Danecek M A White K WongB Yalcin et al ldquoMouse genomic variation and its effect onphenotypes and gene regulationrdquoNature vol 477 pp 289ndash2942011
[22] T Yamamoto K Yoh A Kobayashi et al ldquoIdentification ofpolymorphisms in the promoter region of the human NRF2generdquo Biochemical and Biophysical Research Communicationsvol 321 no 1 pp 72ndash79 2004
[23] J M Marzec J D Christie S P Reddy et al ldquoFunctionalpolymorphisms in the transcription factor NRF2 in humansincrease the risk of acute lung injuryrdquo The FASEB Journal vol21 no 9 pp 2237ndash2246 2007
[24] H Fukushima-Uesaka Y Saito K Maekawa et al ldquoGeneticvariations and haplotype structures of transcriptional factorNRF2 and its cytosolic reservoir protein KEAP1 in JapaneserdquoDrug Metabolism and Pharmacokinetics vol 22 no 3 pp 212ndash219 2007
[25] E J Cordova R Velazquez-Cruz F Centeno V Baca and LOrozco ldquoThe NRF2 gene variant -653GA is associated with
Oxidative Medicine and Cellular Longevity 23
nephritis in childhood-onset systemic lupus erythematosusrdquoLupus vol 19 no 10 pp 1237ndash1242 2010
[26] M von Otter S Landgren S Nilsson et al ldquoAssociation ofNRF2-encoding NFE2L2 haplotypes with Parkinsonrsquos diseaserdquoBMCMedical Genetics vol 11 no 1 article 36 2010
[27] JMHartikainenM TengstromVMKosmaV L Kinnula AMannermaa andY Soini ldquoGenetic polymorphisms and proteinexpression of NRF2 and sulfiredoxin predict survival outcomesin breast cancerrdquo Cancer Research vol 72 pp 5537ndash5546 2012
[28] T Arisawa T Tahara T Shibata et al ldquoThe relationshipbetween Helicobacter pylori infection and promoter polymor-phism of the NRF2 gene in chronic gastritisrdquo InternationalJournal of Molecular Medicine vol 19 no 1 pp 143ndash148 2007
[29] T Arisawa T Tahara T Shibata et al ldquoThe influence ofpromoter polymorphism of nuclear factor-erythroid 2-relatedfactor 2 gene on the aberrant DNA methylation in gastricepitheliumrdquo Oncology Reports vol 19 no 1 pp 211ndash216 2008
[30] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene pro-moter polymorphism and gastric carcinogenesisrdquo Hepato-Gastroenterology vol 55 no 82-83 pp 750ndash754 2008
[31] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene promoterpolymorphism is associated with ulcerative colitis in a Japanesepopulationrdquo Hepato-Gastroenterology vol 55 no 82-83 pp394ndash397 2008
[32] C C Hua L C Chang J C Tseng C M Chu Y C Liu andW B Shieh ldquoFunctional haplotypes in the promoter region oftranscription factor NRF2 in chronic obstructive pulmonarydiseaserdquo Disease Markers vol 28 no 3 pp 185ndash193 2010
[33] S O Shaheen R B Newson S M Ring M J Rose-ZerilliJ W Holloway and A J Henderson ldquoPrenatal and infantacetaminophen exposure antioxidant gene polymorphismsand childhood asthmardquo The Journal of Allergy and ClinicalImmunology vol 126 no 6 pp 1141e7ndash1148e7 2010
[34] H Masuko T Sakamoto Y Kaneko et al ldquoLower FEV1 innon-COPD nonasthmatic subjects association with smokingannual decline in FEV1 total IgE levels and TSLP genotypesrdquoInternational Journal of Chronic Obstructive Pulmonary Diseasevol 6 pp 181ndash189 2011
[35] C P Guan M N Zhou A E Xu et al ldquoThe susceptibilityto vitiligo is associated with NF-E2-related factor 2 (NRF2)gene polymorphisms a study on Chinese Han populationrdquoExperimental Dermatology vol 17 no 12 pp 1059ndash1062 2008
[36] J Bouligand O Cabaret M Canonico et al ldquoEffect of NFE2L2genetic polymorphism on the association between oral estrogentherapy and the risk of venous thromboembolism in post-menopausal womenrdquo Clinical Pharmacology amp Therapeuticsvol 89 no 1 pp 60ndash64 2011
[37] D S OrsquoMahony B J Glavan T D Holden C Fong R A BlackG Rona et al ldquoInflammation and immune-related candidategene associations with acute lung injury susceptibility andseverity a validation studyrdquo PLoS ONE vol 7 no 12 Article IDe51104 2012
[38] I Ungvari E Hadadi V Virag et al ldquoRelationship between airpollutionNFE2L2 gene polymorphisms and childhood asthmain aHungarian populationrdquo Journal of CommunityGenetics vol3 no 1 pp 25ndash33 2012
[39] M SiedlinskiD S Postma JMA Boer et al ldquoLevel and courseof FEV1 in relation to polymorphisms inNFE2L2 andKEAP1 inthe general populationrdquo Respiratory Research vol 10 article 732009
[40] C C Hong C B Ambrosone J Ahn et al ldquoGenetic variabilityin iron-related oxidative stress pathways (NRF2 NQ01 NOS3
and HO-1) iron intake and risk of postmenopausal breastcancerrdquo Cancer Epidemiology Biomarkers and Prevention vol16 no 9 pp 1784ndash1794 2007
[41] C Canova C Dunster F J Kelly C Minelli P L Shah CCaneja et al ldquoPM10-induced hospital admissions for asthmaand chronic obstructive pulmonary disease the modifyingeffect of individual characteristicsrdquo Epidemiology vol 23 no 4pp 607ndash615 2012
[42] A J Henderson R B Newson M Rose-Zerilli S M RingJ W Holloway and S O Shaheen ldquoMaternal NRF2 andgluthathione-S-transferase polymorphisms donotmodify asso-ciations of prenatal tobacco smoke exposure with asthma andlung function in school-aged childrenrdquo Thorax vol 65 no 10pp 897ndash902 2010
[43] C Xing A L Sestak J A Kelly et al ldquoLocalization andreplication of the systemic lupus erythematosus linkage signalat 4p16 interaction with 2p11 12q24 and 19q13 in EuropeanAmericansrdquo Human Genetics vol 120 no 5 pp 623ndash631 2007
[44] T F Wong K Yoshinaga Y Monma K Ito H NiikuraS Nagase et al ldquoAssociation of KEAP1 and NRF2 geneticmutations and polymorphisms with endometrioid endometrialadenocarcinoma survivalrdquo International Journal of Gynecologi-cal Cancer vol 21 no 8 pp 1428ndash1435 2011
[45] M von Otter S Landgren S Nilsson et al ldquoNRF2-encodingNFE2L2 haplotypes influence disease progression but not riskin Alzheimerrsquos disease and age-related cataractrdquoMechanisms ofAgeing and Development vol 131 no 2 pp 105ndash110 2010
[46] S Tsang Z Sun B Luke et al ldquoA comprehensive SNP-basedgenetic analysis of inbred mouse strainsrdquoMammalian Genomevol 16 no 7 pp 476ndash480 2005
[47] C M Wade E J Kulbokas III A W Kirby et al ldquoThe mosaicstructure of variation in the laboratory mouse genomerdquoNaturevol 420 no 6915 pp 574ndash578 2002
[48] T Wiltshire M T Pletcher S Batalov et al ldquoGenome-widesingle-nucleotide polymorphism analysis defines haplotypepatterns in mouserdquo Proceedings of the National Academy ofSciences of the United States of America vol 100 no 6 pp 3380ndash3385 2003
[49] K A Frazer E Eskin H M Kang et al ldquoA sequence-basedvariation map of 827 million SNPs in inbred mouse strainsrdquoNature vol 448 no 7157 pp 1050ndash1053 2007
[50] T P Maddatu S C Grubb C J Bult andM A Bogue ldquoMousephenome database (MPD)rdquo Nucleic Acids Research vol 40 ppD887ndashD894 2007
[51] A Kirby H M Kang C M Wade et al ldquoFine mapping in 94inbred mouse strains using a high-density haplotype resourcerdquoGenetics vol 185 no 3 pp 1081ndash1095 2010
[52] H Y Cho A E Jedlicka S P M Reddy L Y Zhang T WKensler and S R Kleeberger ldquoLinkage analysis of susceptibilityto hyperoxia NRF2 is a candidate generdquo American Journal ofRespiratory Cell and Molecular Biology vol 26 no 1 pp 42ndash512002
[53] H Y Cho A E Jedlicka S P M Reddy et al ldquoRole of NRF2in protection against hyperoxic lung injury in micerdquo AmericanJournal of Respiratory Cell and Molecular Biology vol 26 no 2pp 175ndash182 2002
[54] T Shibata T Ohta K I Tong et al ldquoCancer related mutationsin NRF2 impair its recognition by KEAP1-Cul3 E3 ligase andpromote malignancyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 36 pp13568ndash13573 2008
24 Oxidative Medicine and Cellular Longevity
[55] A Singh V Misra R K Thimmulappa et al ldquoDysfunctionalKEAP1-NRF2 interaction in non-small-cell lung cancerrdquo PLoSMedicine vol 3 no 10 article e420 2006
[56] T Ohta K Iijima M Miyamoto et al ldquoLoss of KEAP1 functionactivates NRF2 and provides advantages for lung cancer cellgrowthrdquo Cancer Research vol 68 no 5 pp 1303ndash1309 2008
[57] B Padmanabhan K I Tong T Ohta et al ldquoStructural basisfor defects of KEAP1 activity provoked by its point mutationsin lung cancerrdquoMolecular Cell vol 21 no 5 pp 689ndash700 2006
[58] T Shibata A Kokubu M Gotoh et al ldquoGenetic alteration ofKEAP1 confers constitutive NRF2 activation and resistance tochemotherapy in gallbladder cancerrdquoGastroenterology vol 135no 4 pp 1358e4ndash1368e4 2008
[59] N J Yoo H R Kim Y R Kim C H An and S H LeeldquoSomatic mutations of the KEAP1 gene in common solidcancersrdquo Histopathology vol 60 no 6 pp 943ndash952 2012
[60] Y Hu Y Ju D Lin Z Wang Y Huang S Zhang et alldquoMutation of the NRF2 gene in non-small cell lung cancerrdquoMolecular Biology Reports vol 39 no 4 pp 4743ndash4747 2012
[61] Y R Kim J EOhM S Kim et al ldquoOncogenicNRF2mutationsin squamous cell carcinomas of oesophagus and skinrdquo TheJournal of Pathology vol 220 no 4 pp 446ndash451 2010
[62] T Shibata A Kokubu S Saito et al ldquoNRF2 mutation confersmalignant potential and resistance to chemoradiation therapyin advanced esophageal squamous cancerrdquo Neoplasia vol 13pp 864ndash873 2011
[63] H Sasaki M Shitara K Yokota Y Hikosaka S MoriyamaM Yano et al ldquoIncreased NRF2 gene (NFE2L2) copy numbercorrelates with mutations in lung squamous cell carcinomasrdquoMolecular Medicine Reports vol 6 no 2 pp 391ndash394 2012
[64] H Sasaki M Shitara K Yokota Y Hikosaka S Moriyama MYano et al ldquoRagD gene expression andNRF2mutations in lungsquamous cell carcinomasrdquo Oncology Letters vol 4 pp 1167ndash1170 2012
[65] A Singh M Bodas N Wakabayashi F Bunz and S BiswalldquoGain of NRF2 function in non-small-cell lung cancer cellsconfers radioresistancerdquo Antioxidants and Redox Signaling vol13 no 11 pp 1627ndash1637 2010
[66] T Yamadori Y Ishii SHommaYMorishimaKKurishimaKItoh et al ldquoMolecular mechanisms for the regulation of NRF2-mediated cell proliferation in non-small-cell lung cancersrdquoOncogene vol 31 pp 4768ndash4777 2012
[67] A K Bauer H Y Cho L Miller-Degraff C Walker K HelmsJ Fostel et al ldquoTargeted deletion of NRF2 reduces urethane-induced lung tumor development in micerdquo PLoS ONE vol 6no 10 Article ID e26590 2011
[68] H Y Cho and S R Kleeberger ldquoNRF2 protects against airwaydisordersrdquo Toxicology and Applied Pharmacology vol 244 no1 pp 43ndash56 2010
[69] R Wang J An F Ji H Jiao H Sun and D Zhou ldquoHyperme-thylation of the KEAP1 gene in human lung cancer cell linesand lung cancer tissuesrdquo Biochemical and Biophysical ResearchCommunications vol 373 no 1 pp 151ndash154 2008
[70] P Zhang A Singh S Yegnasubramanian et al ldquoLoss ofkelch-like ECH-associated protein 1 function in prostate cancercells causes chemoresistance and radioresistance and promotestumor growthrdquoMolecular Cancer Therapeutics vol 9 no 2 pp336ndash346 2010
[71] N Hanada T Takahata Q Zhou et al ldquoMethylation of theKEAP1 gene promoter region in human colorectal cancerrdquoBMCCancer vol 12 article 66 2012
[72] L A Muscarella R Barbano V DrsquoAngelo et al ldquoRegulationof KEAP1 expression by promoter methylation in malignantgliomas and association with patientrsquos outcomerdquo Epigeneticsvol 6 no 3 pp 317ndash325 2011
[73] S Yu T O Khor K L Cheung et al ldquoNRF2 expressionis regulated by epigenetic mechanisms in prostate cancer ofTRAMP micerdquo PLoS ONE vol 5 no 1 Article ID e8579 2010
[74] T O Khor Y Huang T Y Wu L Shu J Lee and A N KongldquoPharmacodynamics of curcumin as DNA hypomethylationagent in restoring the expression of NRF2 via promoter CpGsdemethylationrdquo Biochemical Pharmacology vol 82 no 9 pp1073ndash1078 2011
[75] I M Fingerman L McDaniel X Zhang et al ldquoNCBI epige-nomics a new public resource for exploring epigenomic datasetsrdquo Nucleic Acids Research vol 39 supplemen 1 pp D908ndashD912 2011
[76] I M Fingerman X Zhang W Ratzat N Husain R F Cohenand G D Schuler ldquoNCBI epigenomics whatrsquos new for 2013rdquoNucleic Acids Research vol 41 no 1 pp D221ndashD225 2013
[77] J H Lee T O Khor L Shu Z Y Su F Fuentes andA N Kong ldquoDietary phytochemicals and cancer preventionNRF2 signaling epigenetics and cell death mechanisms inblocking cancer initiation and progressionrdquo Pharmacology ampTherapeutics vol 137 no 2 pp 153ndash171 2012
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Disease Markers
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OncologyJournal of
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Oxidative Medicine and Cellular Longevity
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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
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Evidence-Based Complementary and Alternative Medicine
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Oxidative Medicine and Cellular Longevity 9
Table3Con
tinued
IDMap
onchromosom
e2lowast
(HGVS
name)
Positionin
Nfe2
l2
(HGVS
name)
Region
s(position
from
mRN
A)
Varia
tioncla
ssand
consequences
(HGVS
name)dagger
Minor
allelefrequ
ency
(MAF)
MAcoun
tsDagger(coh
ort
size)
MAFsources
rs199673454
g178096309TgtA
c1022AgtT
Exon
5(1577)
Cns(pAs
p341Va
l)A=000
051
1000
Genom
esrs35007548
g178096299GgtA
c1032CgtT
Exon
5(1587)
Cs(pSer344=
)A=000
092
1000
Genom
esrs200209692
g178096287TgtC
c104
4AgtG
Exon
5(1599)
Cs(pLeu348=
)C=000
051
1000
Genom
es
mdashg178096237CgtA
c1094GgtT
Exon
5(1649)
Cns(pSer365Ile
)A=0125273
A=000
6Dagger(84)
PubM
ed[24]
rs201214197
g178096171CgtT
c1160GgtA
Exon
5(1715)
Cns(pSer387Asn)
T=000
051
1000
Genom
esrs200494292
g178096165TgtC
c1166AgtG
Exon
5(1721)
Cns(pLys389Arg)
nars186171287
g178096115GgtA
c1216CgtT
Exon
5(1771)
Cns(pPro4
06Ser)
A=000
051
1000
Genom
esrs182276775
g178096062CgtA
c1269GgtT
Exon
5(1824)
Cns(pGlu423A
sp)
A=000
051
1000
Genom
esrs201560221
g17809604
8TgtC
c1283AgtG
Exon
5(1838)
Cns(pLys428Arg)
nars189238236
g178096043AgtG
c1288TgtC
Exon
5(1843)
Cs(pLeu430=
)G=000
051
1000
Genom
esrs184287392
g178096022GgtA
c1309CgtT
Exon
5(1864)
Cns(pArg437Trp)
A=000
051
1000
Genom
esrs201871588
g178095986GgtA
c1345CgtT
Exon
5(19
00)
Cns(pAg449Cy
s)na
rs181294188
g178095985TgtC
c1346GgtA
Exon
5(19
01)
Cns(pArg44
9His)
T=000
092
1000
Genom
esrs20169046
6g178095973TgtA
c1358AgtT
Exon
5(19
13)
Cns(pHis4
53Leu)
A=000
051
1000
Genom
esrs105704
4(rs52789869)
g178095781CgtT
c1550GgtA
Exon
5(2105)
Cns(pArg517Lys)
TDagger[23]
rs200750800
g178095603AgtG
c1728TgtC
Exon
5(2283)
Cs(pTyr576=
)na
rs200175942
g178095567AgtG
c1764TgtC
Exon
5(2319)
Cs(pAsp588=
)G=000
051
1000
Genom
esrs77547666
g178095495GgtC
clowast18Cgt
GEx
on5UTR
-31015840(2391)
SNP
C=000
6915
1000
Genom
esrs73031353
g178095425TgtC
clowast88AgtG
Exon
5UTR
-31015840(2461)
SNP
C=000184
1000
Genom
esrs675944
3g178095345TgtC
clowast168AgtG
Exon
5UTR
-31015840(2541)
SNP
C=000
419
1000
Genom
esrs188674558
g178095279CgtA
clowast234GgtT
Exon
5UTR
-31015840(2607)
SNP
A=000
051
1000
Genom
esrs77685897
g178095247AgtG
clowast266TgtC
Exon
5UTR
-31015840(2639)
SNP
G=000
092
1000
Genom
esrs1057092
g178095162TgtG
clowast351AgtC
Exon
5UTR
-31015840(2724)
SNP
nars3197704
g178095162TgtG
clowast351AgtC
Exon
5UTR
-31015840(2724)
SNP
nars184701151
g178095159TgtC
clowast354AgtG
Exon
5UTR
-31015840(2727)
SNP
C=000276
1000
Genom
esrs11543307
g178095153AgtG
clowast360TgtC
Exon
5UTR
-31015840(2733)
SNP
nars111874043
g178095146AgtG
clowast367TgtC
Exon
5UTR
-31015840(2740)
SNP
nars34012004
g178095102AgtC
clowast411TgtG
Exon
5UTR
-31015840(2784)
SNP
C=0071
[24]
10 Oxidative Medicine and Cellular Longevity
Table3Con
tinued
IDMap
onchromosom
e2lowast
(HGVS
name)
Positionin
Nfe2
l2
(HGVS
name)
Region
s(position
from
mRN
A)
Varia
tioncla
ssand
consequences
(HGVS
name)dagger
Minor
allelefrequ
ency
(MAF)
MAcoun
tsDagger(coh
ort
size)
MAFsources
rs201481890
g178095090178095091in
sTclowast
422lowast423insA
Exon
5UTR
-31015840
(2795minus
2796)
Insertion
na
rs3082500
g178095089178095090d
elTT
delTTinsT
clowast423lowast424d
elAAinsA
Exon
5UTR
-31015840
(2796minus
2797)
Dele
tion
insertion
na
rs71792546
(rs71796710)
g178095079d
elT
clowast425delA
Exon
5UTR
-31015840(2798)
Dele
tion
na
rs1057106
g178095078AgtC
AgtT
clowast435TgtA
TgtG
Exon
5UTR
-31015840(2808)
SNP
na
rs34176791
g178095076AgtC
clowast437TgtG
Exon
5UTR
-31015840(2810)
SNP
C=000235
1000
Genom
esrs35911553
g178095045CgtT
clowast46
8GgtA
Exon
5UTR
-31015840(2841)
SNP
T=000
6915
1000
Genom
esSequ
ence
varia
tions
inup
stream
andexon
sofh
uman
NRF
2fro
m655varia
tions
availablein
publicdatabase
asof
Decem
ber2012
(583
activ
esomeSN
Psmergedge14
SNPs
citedin
PubM
ed)lowastNCB
Ireference
sequ
ence
NC
0000
0211
(Hom
osapiens
chromosom
e2G
RCh37p
10prim
arya
ssem
bly)spanning
178095033ndash1781298
59bp
(com
plem
ent34827
bpforexons
andintro
ns)51015840-Flank
ingregions
startat17812
9860
bp(minus1)in
transcrip
tvariant
1HGVS
Hum
anGenom
eVa
riatio
nSo
ciety
Position
sinvaria
nt1(NM
006164
4)2859
bpsectSN
Pscitedin
PubM
edE
xon11781298
59ndash17812
9260
(600
bpT
TS=1781293
04)exon
2178098999ndash
178098733(267
bp)exon
3178098067ndash1780979
78(90b
p)exon41780973
11ndash178097120
(192b
p)and
exon
5178096736ndash
178095033(1704b
p)daggerProteinam
inoacid
(aa)
resid
uesiniso
form
1(N
P00
61552605
aa)Cn
scoding
-non
syno
nymou
sCs
cod
ing-syno
nymou
sDaggerheterozygositydetectednano
tavailable
Oxidative Medicine and Cellular Longevity 11
Table4Hum
anNRF
2SN
Psassociated
with
diseaser
isk
IDMap
onchromosom
e2 (H
GVS
name)
Region
class
Dise
asea
ssociatio
nandreferences
Risk
allelesa
ndstatistics
Ethn
icgrou
p(num
bero
fcase)
rs7557529
g178135097CgtT
minus5238
Ggt
AParkinsonrsquos
disease(2010
[26])
G=0718252Dagger
inhaplotype
OR=090
or040
CI=
060ndash140or
03ndash06
Swedish
Polish
Caucasian
(357)
rs2886162
g178133165AgtG
minus3306
TgtC
Breastcancer
survival(2012[27])
TT=0324
OR=16
87C
I=1105ndash275
FinlandKB
CP(452)
Chronicg
astritisgastric
ulcer(2007
[28])
GDagger
GDaggerin
haplotypefor
ulcer(OR=
252C
I=119ndash
545)
Japanese
(159)
P14methylationin
gastric
cancer
Hpyloriinfectio
n(2008[29])
Gin
haplotype
OR=290C
I=114ndash
736
Japanese
(209)
Gastriccancer
inHpylori-n
egative
cases(2008
[30])
Ain
haplotype
119875=0022
Japanese
(209)
rs35652124
(rs57695243)
g178130073TgtC
minus214Agt
G(previou
slyminus653[23]
orminus686[22])
Ulcerativec
olitis(2008
[31])
AG
(OR=045C
I=022ndash0
93)
G(O
R=257C
I=10
1ndash660)
Japanese
(89)
Lupu
swith
neph
ritisin
female(2010
[25])
GA
OR=18
1CI
=10
4ndash312
Mexican
mestizo(362)
Parkinsonrsquos
disease(2010
[26])
A=0884312Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
COPD
(2010[32])
G=052Dagger
hazard
ratio
=095C
I=091ndash0
99
(Haplotype)
German
(69)
Gastriculcer(2007
[28])
GDaggerin
haplotype
OR=252C
I=119ndash
545
Japanese
(159)
P14methylationin
gastric
cancer
H
pyloriinfection(2008[29])
Gin
haplotype
OR=290C
I=114ndash
736
Japanese
(209)
Gastriccancer
inHpylori-negative
cases(2008
[30])
Gin
haplotype119875=0022
Japanese
(209)
rs6706
649
g178130071CgtT
minus212Ggt
A(previou
slyminus651[23]
orminus684[22])
Ulcerativec
olitis(2008
[31])
AG
(OR=045C
I=022ndash0
93)
G(O
R=257C
I=10
1ndash660)
Japanese
(89)
Maternalacetaminop
henandasthma
(2010[33])
A=02321137Dagger
OR=17
3CI
=12
2245
UKALSPA
C(gt40
00mothersgt
5000
child
ren)
12 Oxidative Medicine and Cellular Longevity
Table4Con
tinued
IDMap
onchromosom
e2 (H
GVS
name)
Region
class
Dise
asea
ssociatio
nandreferences
Risk
allelesa
ndstatistics
Ethn
icgrou
p(num
bero
fcase)
COPD
(2010[32])
G=098Daggerin
haplotype
hazard
ratio
=095C
I=091ndash0
99
German
(69)
Parkinsonrsquos
disease(2010
[26])
G=0972343Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
03ndash06
Swedish
Polish
Caucasian
(357)
Acutelun
ginjury
follo
wingtrauma
(2007[23])
CA=0119Dagger
OR=644
CI=
134ndash
3080
Caucasia
nAfrican-
American
(164
)
Ann
ualF
EV1decline
(2011[34])
A=0082in
haplotype
Japanese
(915)
Vitiligo(2008[35])
CAA
AOR=17
24C
I=13
5ndash221
Chinese(300)
COPD
(2010[32])
G=073Daggerin
haplotype
hazard
ratio
=095C
I=091ndash0
99
German
(69)
rs6721961
(rs117801448)
g178130037TgtC
TgtG
minus178Agt
GAgt
C(previou
slyminus617[23]
orminus650[22])
Parkinsonrsquos
disease(2010
[26])
C=0980346Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
Postm
enop
ausalvenou
sthrombo
embo
lism
(2011[36])
A=033311
OR=25CI
=370ndash8570
Caucasia
n(161)
Breastcancer
survivalandNRF
2proteinexpressio
n(2012[27])
AA
OR=4656CI
=13
5ndash1606
FinlandKB
CP(452)
Acutelun
ginjury-related
mortality
followingsyste
micinflammatory
respon
sesynd
rome(2012
[37])
GG
OR=97
3CI
=12
7ndash7480
Caucasia
n(750)
Infection-indu
cedasthma(
2012
[38])
AC
OR=0437CI
=028ndash0
80
Hun
garia
nCaucasia
nGypsy
(307)
rs143406266
g178129391178
129393delGGC
Exon
1(467ndash4
69G
CC)
COPD
(2010[32])
GCC
4=053Dagger
hazard
ratio
=095C
I=091ndash0
99
Taiwanese(69)
rs2886161
g178127839Tgt
ClowastIntro
n1
Parkinsonrsquos
disease(2010
[26])
T=0878309Dagger
inhaplotype
OR=09or
04CI
=06ndash
14or
03ndash06
Swedish
Polish
Caucasian
(357)
rs2364723
g178126546Ggt
CIntro
n1
Basaland
smoker
FEV
1(200
9[39])
CI=minus6360simminus1780C
=0525Dagger
Also
ashaplotype
Netherla
nd(2542)
rs2364722
g178124787Agt
GIntro
n1
Ann
ualF
EV1decline
(2011[34])
A=0082in
haplotype
Japanese
(915)
rs13001694
g178118990Agt
GIntro
n1
Basaland
smoker
FEV
1(200
9[39])
G=040
11578Dagger
inhaplotype
Netherla
nd(2542)
Oxidative Medicine and Cellular Longevity 13
Table4Con
tinued
IDMap
onchromosom
e2 (H
GVS
name)
Region
class
Dise
asea
ssociatio
nandreferences
Risk
allelesa
ndstatistics
Ethn
icgrou
p(num
bero
fcase)
Breastcancer
[40]
Twith
NQO1N
OS3H
O1risk
allelesOR=15
6CI
=097ndash251
Caucasia
nandothers(505)
rs1806
649
(rs58745895)
g178118152Cgt
TIntro
n1
Basaland
smoker
FEV
1(200
9[39])
T=02631119Daggerin
haplotype
CI=minus8730ndash
(minus17
0)Netherla
nd(2542)
Parkinsonrsquos
disease(2010
[26])
T=0422148Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
Particulatem
attera
ndasthmaCO
PDadmiss
ion(2012[41])
Cwith
lowvitamin
Clevel(OR=
31CI
=15
0ndash630)
UK(209)
rs4243387
(rs6003846
4)g178117765Cgt
TIntro
n1
Basaland
smoker
FEV
1(200
9[39])
T=00914
25Daggerin
haplotype
Netherla
nd(2542)
rs1962142
(rs58448508)
g178113484Agt
GIntro
n1
Ann
ualF
EV1decline
(2011[34])
A=0082in
haplotype
Japanese
(915)
Breastcancer
NRF
2andARE
expressio
n(2012[27])
A(119875=0036)
FinlandKB
CP(452)
rs6726395
(rs57309289)
g178103229Agt
GIntro
n1
Smok
ing-relatedFE
V1decline
and
annu
alFE
V1decline
(2011[34])
G=0884Dagger
A=0082in
haplotype
Japanese
(915)
Basaland
smoker
FEV
1(200
9[39])
G=046
41764Daggerin
haplotype
Netherla
nd(2542)
rs2001350
(rs17515179
rs60883775)
g178100
425Cgt
TIntro
n1
Ann
ualF
EV1decline
(2011[34])
T=0082in
haplotype
Japanese
(915)
Parkinsonrsquos
disease(2010
[26])
T=0986350Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
rs10183914
(rs58731187
rs61374844
)g17809766
6Cgt
TIntro
n3
Parkinsonrsquos
disease(2010
[26])
T=0536188Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
rs2706110
g178092162Tgt
C31015840Flanking
Breastcancer
(2012[27])
TT
OR=2079CI
=118ndash368
FinlandKB
CP(452)
rs2588882
g178087165Ggt
T31015840Flanking
Infection-indu
cedasthma(
2012
[38])
TG
OR=0290CI
=013ndash0
62
Hun
garia
nCaucasia
nGypsy
(307)
minus686in
reference[22]
=minus653in
reference[23]
=currentlyminus214minus684in
reference[22]
=minus651inreference[23]
=currentlyminus212minus651inreference[22]
=minus617in
reference[23]
=currentlyminus178Ch
romosom
econtig(in
tron
31015840flank
ing)
orreversed
(51015840flank
ingprom
oter)a
llelesinbo
ldhave
been
used
inthetextand
TableOR
odds
ratio
CI95confi
denceinterval
14 Oxidative Medicine and Cellular Longevity
minus212GG (98 rs67006649 previously minus684minus651) minus178Callele (73) and GCC
4(53) was predicted to increase
respiratory failure development (hazard ratio = 095 CI091ndash099) in German COPD patients [32] Significant inter-action was also identified between an intronic SNP Gallele (rs6726395 g178103229AgtG 884 frequency) andsmoking status on FEV
1decline relative to the reference
AA allele in the above Japanese cohort [34] Siedlinskiet al [39] reported that the CC genotype of anotherintronic SNP (rs2364723 g178126546GgtC) was associatedwith a lower FEV
1level compared to the wild-type geno-
type (GG) in two Netherland cohorts (CI minus636minus178frequency = 0525 and pooled cohort size = 2542) ThisSNP alone or as a haplotype with 4 more intronic SNPs(rs13001694Grs1806649Trs4243387Trs6726395G)was alsoassociated with high FEV
1levels in individuals that ever
smoked [39] In a Hungarian population of childhoodasthma SNPs at minus178 (CA) and 31015840 flanking (rs2588882TG)loci were inversely associated with infection-induced asthma(OR 0437 CI 028ndash080 OR 0290 CI 013ndash062 resp) andthese SNPs significantly influenced an asthma-environmentalpollution interaction [38]The intronic SNP rs1806649 (CgtT)was associated but not significantly with an increased risk ofhospitalization during high-level particulate matter (PM
10)
periods in asthma or COPD patients (119899 = 209) of the UnitedKingdom (UK) [41] Asthma andCOPDadmission rateswererelated to the increase in environmental PM
10concentration
Importantly effects of interaction between prenatal stressand NRF2 SNPs on descendant pulmonary health wereinvestigated by the Avalon Longitudinal Study in the UKmaternal smoking during pregnancy was not associated withlung function change determined by maximum mild expi-ratory flow (FEF
25ndash75) or with asthma incidence in school-aged children and this relation was not modified by NRF2SNP genotypes [42]However early gestation acetaminophenexposure significantly influenced the risk of asthma andwheezing at the age of 7 years in gt4000 mothers and gt5000children [33] When maternal copies of the minus212A allele werepresent association with asthma (11374891 OR 173 CI122ndash245) and wheezing (11494949 OR 153 95 CI 106ndash220) was significantly increased [33]
Gastrointestinal Disorders While there was no evidence inlung cancer cases studies in Japanese populations suggesteda potential association of NRF2 variations with gastro-intestinal tumorigenesisHelicobacter pylori (H pylori) causesgastritis which can lead to gastric atrophy and cancer Ingastric epithelium from the Japanese cancer cohorts (39gastric cancers 46 controls) H pylori infection was posi-tively correlated with aberrant CpG island methylation oftumor suppressor genes (eg p14) and minus214Gminus212G orminus214Aminus212G NRF2 haplotype was significantly associatedwith increased (OR 290 95 CI 114ndash736) or decreased(OR 033 95 CI 013ndash088) risk of the CpG methyla-tion respectively in the H pylori-infected patients [29]Further study from the same investigators determined thatminus214Aminus212G allele carriers had significantly (119875 = 0022)reduced risk of gastric cancer inH pylori-negative cases [30]The minus214AGminus212AG genotypes were negatively associated
(OR 045 CI 022ndash093) and the minus214Gminus212G genotypeswere positively associated (chronic continuous phenotypeOR 257 CI 101ndash660) with ulcerative colitis (89 patients 141controls) in a Japanese population [31]
Autoimmune Disorders Systemic lupus erythematosus (SLE)is a long-term autoimmune disease more frequently found infemales than in males It affects organs including skin jointskidneys and brain and nephritis is an aggressive character-istic in some patients Genome-wide association studies inhumans identified a suggestive quantitative trait locus nearNRF2 [43] A study of a Mexican Mestizo population (362patients with childhood-onset SLE 379 controls and 212nephritis diagnosed) determined that lupus with nephritiswas significantly (OR 181 CI 104ndash312) associated with theminus214GA SNP in females [25] The same SNPs were notclosely associated with SLE risk in a Japanese cohort [22]Vitiligo is a skin condition in which there is a loss of browncolor (pigment) from areas of skin resulting in irregularwhite patches It is thought to be an autoimmune diseasecaused by loss of cells (melanocytes) that produce brownpigment A study indicated that theminus178A allele increased therisk of vitiligo dose-dependently (OR 1724 95 CI 135ndash221for CA OR 2902 CI 162ndash519 for AA) [35]
Female Disorders It is well known that estrogen metabolites(eg catechols) cause ROS formation suggesting correla-tion of NRF2 and downstream effectors in postmenopausalmammary cancer In a study of a Finish population (KuopioBreast Cancer Project 119899 = 452 patients 370 controls)the minus178AA homozygous genotype (OR 4656 CI = 135ndash1606) and 31015840 flanking rs2706110 (TT OR 2079 CI 118ndash368) genotype were associated with increased risk of breastcancer while the 51015840 flanking minus3306TT homozygous allelewas significantly associated with lower survival (frequency =71219 OR 1687 CI 1105ndash275) [27] suggesting that NRF2genetic polymorphisms affect susceptibility and outcome ofthe patients The minus178A allele carriers together with intronicrs1962142A allele carriers were associated with lowered tissuelevels of NRF2 proteins [27] In postmenopausal women theminus178A allele (OR 179 95CI 370ndash8570) appeared tomodifythe risk of venous thromboembolism caused by oral estrogentherapy (AA or AC frequency = 333) as demonstratedby the French ESTHER study (161 cases 474 controls) [36]An intronic rs1806649CgtT SNP did not associate with breastcancer risk in postmenopausal women [40] However whenthis SNP and other at-risk alleles of ARE-responsive genes(NQO1 HO-1 NOS3) were combined there was a significantgene-dose effect on the breast cancer risk [40] Althoughcoding region SNPs in NRF2 and KEAP1 were identifiedin the Japanese endometrial adenocarcinoma patients noassociation of NRF2 SNPs with the disease was found [44]
Neurodegenerative Diseases Oxidative stress is known to beinvolved in Parkinsonrsquos disease (PD) presumably due toproduction of ROS fromhigh-dopaminemetabolism and lowlevels of antioxidants in the substantia nigra of the brainInvestigators found a protective NRF2 haplotype consistingof four 51015840 flanking SNPs (minus5238Gminus214Aminus212Gminus178C)
Oxidative Medicine and Cellular Longevity 15
and 4 intronic SNPs (rs2886161Ars1806649Ars2001350Ars10183914A) from Swedish (OR 09 CI 060ndash140) and Polish(OR 04 CI 030ndash060) populations (total 165+192 PD cases190+192 controls) [26]The investigators also suggested thatNRF2 haplotype alleles were associated with 2 years earlierage of Alzheimerrsquos disease (AD) onset 4 years earlier age ofposterior subcapsular cataract surgery and 4 years later ageof cortical cataract surgery while they were not significantlyrelated to AD or age-related cataract risk [45]
34 Genetic Mutations in Mouse Nrf2 Tsang et al [46]compiled 673 SNPs in 55 mouse strains and constructedtheir phylogenetic tree to correlate and clarify the originsof strains based on the assembled mouse genome sequenceand SNP data [20 47 48] Recently using the completegenome sequence of C57BL6J (B6) mouse as a referencehigh-density SNP screening in other laboratory strains orin panels of strains has been published (see [17]) Althoughmillions of mouse SNPs (gt10089892 as of December 2012)and haplotype mappings from more than 120 strains havebeen published as valuable references for dissecting thegenetic basis of complex traits [49ndash51] little attention hasbeen paid to polymorphisms of Nrf2 and their correlationwith disease phenotypes
Figure 3 demonstrates the proximal promoter region(minus1 to minus950)51015840-UTR (exon 1 up to TSS) and proteinsequence of mouse Nrf2 based on GRCm38p1 PrimaryAssembly (75704641ndash75675513 bp) mRNA variant1 and protein (NP 035032 encoded by NM 010902234ndash2027 bp) sequences Genetic variations in the Nrf2genome of inbred strains collected from public databasesare listed in Tables S2 and 5 (See Supplementary TableS2) Overall 968 genetic mutations are compiled forNrf2 gene and 5 kb upstream2 kb downstream regions785 SNPs between B6 and another 16 strains wereacquired from the Mouse Phenome Database (MPDhttpphenomejaxorg dbqrtn=snpret1) and additionalSNPs and other mutations were acquired from NCBI dbSNP(httpwwwncbinlmnihgovsnpterm=mus+musculus20nfe2l2) In total 132 mutations are in the promoter (37in proximal 1 kb) 49 in exons (38 in coding region 19 Cns)727 in introns and 60 in the 31015840 flanking region Excludingmutations in the 51015840 and 31015840 flanking sequences murine Nrf2sequences appear to be more highly variable (1 variationper 375 bp) than much of the mouse genome which has anapproximate frequency of one SNP per every 245 bp (httpwwwinformaticsjaxorgmgihomehomepagesstatsallstatsshtmlallstats snp)
Nrf2 was found to be a susceptibility gene from genome-wide linkage analysis in a murine model of hyperoxia-induced ALI [52] A promoter SNP minus103TgtC (previouslypublished as minus336TgtC) in Nrf2 was found and predicted toadd an additional Sp1 binding site in hyperoxia-susceptibleB6 mice but not in resistant C3HHeJ mice [52] Genotypesfrom the SNP and from simple-sequence length polymor-phism markers of the Nrf2 locus (D2Mit248 and D2Mit94)cosegregated in the B6C3F
2mouse cohort [52] and Nrf2
deficient mice were significantly more susceptible to ALI
sub-phenotypes caused by hyperoxia than similarly exposedwild-type mice supporting Nrf2 as a contributor to thephenotypic traits [53] Although no other functional analyseson Nrf2 SNPs or haplotype association studies have beenconducted in inbred mice strains bearing haplotypes suchas multiple Cns in functional domains (eg F71L L451VH543Q and L575M) may be useful to elucidate the role ofNrf2 in differential susceptibility to oxidative diseases
4 Oncogenic Somatic Mutations in HumanNF-E2-Related Factor 2
Somatic mutation is a change in the DNA of somatic cellsthat affects derived cells but is not inherited by offspringEfforts to discover somatic mutations have provided insightinto mutagenesis and cancer development Lung cancerparticularly non-small cell lung cancer (NSCLC) is theleading cause of cancer death worldwide Somatic mutationsof NRF2 and KEAP1 discovered in lung cancer patients havedetermined the oncogenic potential ofNRF2 [54 55] KEAP1somatic mutations were associated with its reduced proteinlevels in lung cancer tissues and cells [56 57] Investigationsof NSCLC in various ethnic populations as well as cancersin gastrointestine breast and prostate have coordinatelydemonstrated that multiple Cns somatic mutations in KEAP1cause dysfunction of the translated protein and in turnconstitutive activation of NRF2 increasing risk of neoplasiaand chemoresistance [12 55 58 59] Somatic mutations ofNRF2 have been detected in various cancer tissues (largelysquamous cell carcinomas) in Asian populations (Table 6)NRF2 mutations were significantly associated with NSCLCcases (squamous cell lung carcinoma adenocarcinoma) ofthe Japanese (107 [54]) the Chinese (23 [60]) andthe Koreans (8 [61]) as well as with lung cancer celllines Smoking history was also correlated with mutationoccurrence in all of the studies [54 60 61] In addition tolung cancers laryngeal squamous carcinoma (13 in [61])esophageal squamous cancer (ESC 22 in [60] 114 in[61]) head and neck cancers (25 in [54]) skin (117 casein [61]) and oral cancer cell lines had somatic changesin NRF2 In contrast to wide-spread KEAP1 mutationsmutations in NRF2 were clustered in DLGETGE motifs ofthe Neh2 domain which are critical in the ldquohinge and latchrdquomodel of KEAP1 binding [12] Similar to KEAP1 somaticmutations it has been postulated that NRF2 mutations incancer cells lead to NRF2 accumulation by suppressing itsubiquitination or KEAP1 binding which eventually confersmalignant potential and resistance to chemotherapy
Most variable sites in NRF2 included aa residues 29 (AspD) 31 (Gly G) 77 (Asp D) and 79 (Glu E) (Table 6)Residue 33 (Ser S) in the Neh2 domain is mutated by eithergenetic or somatic processes (Figure 4) Cns in the EDGFmotif of NRF2 was experimentally determined to impairrecognition of KEAP1 [54] NRF2 mutations were signif-icantly correlated with increased (25-fold) copy number(31 of mutants versus 3 wild types) in Japanese NSCLCcases [63] Aberrant mutation of NRF2 also led to increasedexpression of downstream effectors includingRagD known to
16 Oxidative Medicine and Cellular Longevity
pL575M
pF71L
pH543QpL451V
pA151P pA151V pN159D pQ198delpV105D
pA204T pS224I225S226del pT253S pS278ApD305G
pM409V pR410HpP388L pA389G pT395I
Genomic DNA (reversed contig GRCm38 p1 C57BL6JNM 0109023)
Protein (NP 035032)1
51101151201251301351401451501551
minus400
minus500
minus50
minus100
minus200
minus300
minus450
minus350
minus250
minus150
minus950
minus900
minus850
minus800
minus750
minus700
151
101151201
2001
g minus470gg minusg minus
c minus41c minus
c
c
c c
gg minus 6C
g minus125insTTC g minus118insC
g minus281delC
g minus63delCg minus1 (75704642 c minus234)
Translation start site (c +1)Termination codon (c +2025 +2027)
c minus136 minus134in-frame stop codonc minus8585
g minus41
246
440
minus381
minus11
minus91 minus90
minus332
minus153
minus98
c minus197c minus209 minus202
gminus942 minus899 (cminus1175 minus1132) CpG islands [71]
g 460CgtT
g minus103CCgtT [51]
CgtTCgtG
g minus320CgtT
CgtT
CgtT
CgtTg minus426GgtA
GgtACgtA
CgtA
GgtA
g minus238GgtTTgtC
TgtC
TgtC
TgtCTgtG
g minus229AgtG
CgtG
CgtG
GgtC
gminus826 minus654 (cminus1059 ) CpG islands [71]minus887
Figure 3 DNA (partial promoter and exons 1 and 5) and protein sequence ofmouseNrf2 SNPs and amino acid residues for non-synonymousSNPs are marked Promoter regions bearing 5CpG islands are underlined
151
201251301351401451501551601
Genetic mutation (Table 3 and Figure 2)Somatic mutation (Table 6)
Neh2
Neh3
Neh4Neh5
Neh6
Neh1
Protein isoform 1 (NP 006155)
101151
Figure 4 Genetic and somatic mutation loci in human NRF2 protein
Oxidative Medicine and Cellular Longevity 17
Table5Mou
seup
stream
andexon
varia
tions
ofNfe2
l2locusin17
inbred
strainsR
eference
sequ
ence
isC5
7BL6J
(B6strain
1)geno
me(GI14933824975547698ndash75513576)SNPalleleand
geno
typesareshow
nas
chromosom
econ
tigsequ
enceStrains
2129S1S
vlmJ3AJ
4AKR
J5BA
LBcJ6C3
HH
eJ7C
57BL
6NJ8CA
STEiJ9CB
AJ
10D
BA2J11FVBNJ12LPJ
13N
ODShiLtJ14N
ZOH
ILtJ
15P
WK
PhJ16SPR
ETEiJ
17W
SBEiJ
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs2566
08517
75705565
GA
(CT)
51015840Flankingminus924
NC
GG
GG
GG
GG
GG
GG
GG
AA
Grs47274959
75705562
AT
(TA
)51015840Flankingminus921
NC
AT
TA
TT
AA
TT
AT
AT
TT
Ars216940398
75705554ndash75705555
Tadd(A
add)
51015840Flankingminus913minus914
NC
CGrs239114134
75705540
CT(G
A)
51015840Flankingminus899
NC
CT
TC
TT
CC
TT
CT
CT
CT
Crs46
461765
75705528
TG(A
C)oA
C
51015840Flankingminus887
NC
TG
GT
GG
TT
GG
TG
TG
GG
Trs5144
9853
75705525
CA(G
T)
51015840Flankingminus884
NC
CA
AC
AA
CC
AA
CA
CA
CC
Crs249093111
75705512
CT(G
A)
51015840Flankingminus871
NC
CT
TC
TT
CC
TT
CT
CT
TT
Crs263745200
75705510
CG(G
C)
51015840Flankingminus869
NC
GG
GG
GG
GG
GG
GG
GG
CG
Grs45651867
75705498
TG(A
C)
51015840Flankingminus857
NC
TG
GT
GG
TT
GG
TG
TG
GG
Trs219997531
75705495
GA
(CT)
51015840Flankingminus854
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs251918379
75705451
TA(A
T)
51015840Flankingminus810
NC
TT
TT
TT
TT
TT
TT
TT
TA
Trs27978306
75705449
TA(A
T)
51015840Flankingminus808
NC
TA
AT
AA
TT
AA
TA
TA
TT
Trs216087412
75705429
TG(A
C)lowast
51015840Flankingminus788
NC
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
GTlowast
Tlowast
rs261229914
75705423ndash75705424
AGdel(CT
del)
51015840Flankingminus783minus784
NC
AGrs27978307
75705400
CT(G
A)
51015840Flankingminus759
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs243167395
75705359
GA
(CT)
51015840Flankingminus718
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs27978308
75705307
GA
(CT)
51015840Flankingminus66
6NC
GG
GG
GG
GG
GG
GG
GG
AG
Grs254744
09875705294
AT
(TA
)51015840Flankingminus653
NC
AA
AA
AA
AA
AA
AA
AA
AT
Ars228133419
75705179
GT
(CA
)51015840Flankingminus538
NC
GG
GG
GG
GG
GG
GG
GG
GT
Grs214719520
75705111
GA
(CT)
51015840Flankingminus470
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs2440
87730
75705101
GA
(CT)
51015840Flankingminus46
0NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs227781699
75705081
CG(G
C)
51015840Flankingminus44
0NC
CC
CC
CC
CC
CC
CC
CC
GC
Crs257870353
75705067
CT(G
A)
51015840Flankingminus426
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs24487744
075705022
AG
(TC)
51015840Flankingminus381
NC
AA
AA
AA
AA
AA
AA
AA
AG
Ars234628138
75704973
AC
(TG
)51015840Flankingminus332
NC
AA
AA
AA
AA
AA
AA
AA
AC
A
18 Oxidative Medicine and Cellular Longevity
Table5Con
tinued
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs247275247
75704961
GA
(CT)
51015840Flankingminus320
NC
GG
GG
GG
GG
GG
GG
AG
GG
Grs247519047
75704922
Gdel(Cdel)
51015840Flankingminus281
NC
Grs27978309
(rs51915758)
75704887
GA
C(C
TG
)51015840Flankingminus246
NC
GA
AG
AA
GG
AA
GA
GA
CC
G
rs218139102
75704879
CA(G
T)
51015840Flankingminus238
NC
CC
CC
CC
CC
CC
CC
CC
AC
Crs251990355
75704870
TC(A
G)
51015840Flankingminus229
NC
TT
TT
TT
TT
TT
TT
TT
TC
Trs238746955
75704794
AG
(TC)
51015840Flankingminus153
NC
AA
AA
AA
AA
AA
AA
AA
GA
Ars221664
405
75704786
AG
(TC)
51015840Flankingminus145
NC
AA
AA
AA
AA
AA
AA
AA
AG
Ars217054035
75704766
ndash75704767
GAAadd(TTC
add)
51015840Flankingminus125minus126
NC
GA
rs248182931
75704759ndash75704760
Gadd(C
add)
51015840Flankingminus118
minus119
NC
AGrs217197904
75704744
GA
(CT)
51015840Flankingminus103
(+)S
p1[23]
GA
AG
AA
GG
AA
GA
AA
GG
Ars2644
9364975704704
Gdel(Cdel)
51015840Flankingminus63
NC
Grs2404
81112
757046
82CT(G
A)
51015840Flankingminus41
NC
CC
CC
CC
CC
CC
CC
CC
CT
Crs27978310
75704617
AG
(TC)
UTR
-525
NC
AA
AA
AA
AA
AA
AA
AA
GG
Ars27978311
75704610
CG(G
C)
UTR
-532
NC
GG
GG
GG
GG
GG
GG
GG
GG
Crs27978312
757046
05GA
(CT)
UTR
-537
NC
GA
AG
AA
GG
AA
GA
GA
GG
Grs214784220
757044
99AG
(TC)
UTR
-5143
NC
AA
AA
AA
AA
AA
AA
AA
GG
Ars244146318
757044
98GT
(CA
)UTR
-5144
NC
GG
GG
GG
GG
GG
GG
GG
GT
Grs27978313
757044
97GC
(CG
)UTR
-5145
NC
GC
CG
CC
GC
CC
GC
CC
CC
Crs215431944
757044
93CT(G
A)
UTR
-5149
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs257015697
757044
49GA
(CT)
UTR
-5193
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs252234782
757044
19GT
(CA
)UTR
-5223
NC
Grs1346
0861
75679262
AG
(TC)
Exon
244
6Cn
sF71L
AG
GA
GG
AG
GG
AG
AG
GG
Grs1346
0859
75679202
GA
(CT)lowast
Exon
2506
CsH91=
GA
AG
AA
GG
AA
GA
GA
GG
Grs27978436
75679187
GA
(CT)lowast
Exon
2521
CsT9
6=G
GG
GG
GG
AG
GG
GG
GA
GG
rs226131070
75679178
GA
(CT)
Exon
2530
CsS99=
GG
GG
GG
GG
GG
GG
GG
GA
Grs227683834
75678576
AT
(TA
)Ex
on3
547
CnsV
105D
AA
AA
AA
AA
AA
AA
AA
AT
Ars257321187
75678506
TC(A
G)
Exon
3617
CsP128=
TT
TT
TT
TC
TT
TT
TT
TT
Trs27978444
75678500
TA(A
T)
Exon
3623
CsV130=
TT
TT
TT
TA
TT
TT
TT
AT
Trs227743136
75677686
GA
(CT)
Exon
4662
CsH143=
GG
GG
GG
GG
GG
GG
GG
GA
Grs215327202
75677664
CG(G
C)
Exon
4684
CnsA
151P
CC
CC
CC
CG
CC
CC
CC
CC
Crs247539755
75677663
GA
(CT)
Exon
4685
CnsA
151V
GG
GG
GG
GA
GG
GG
GG
GG
Grs233164
668
75677640
TC(A
G)
Exon
4708
CnsN
159D
TT
TT
TT
TT
TT
TT
TT
TC
T
rs2340
95816
75677162ndash75677160
GCT
del
(AGCdel)
Exon
5826minus
828
Cds-in
delQ
198
GCT
Oxidative Medicine and Cellular Longevity 19
Table5Con
tinued
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs27978452
75677145
CT(G
A)
Exon
5843
CnsA
204T
CC
CC
CC
CC
CC
CC
CC
CC
T
rs225047937
75677086ndash75677078
GAG
ATCG
ATdel
(ATG
CATC
TCdel)
Exon
5902minus
910
Cds-in
del
(S224I225S226)
GAG
ATC
GAT
rs241469868
75676998
TA(G
T)
Exon
5990
CnT2
53S
TT
TT
TT
TT
TT
TT
TT
TA
Trs221602571
75676923
AC
(TG
)Ex
on5
1065
CnsS
278A
AA
AA
AA
AA
AA
AA
AA
AC
Ars252576472
75676876
GA
(CT)
Exon
51112
CsS293=
GG
GG
GG
GG
GG
GG
GG
GA
Grs250802933
75676841
TC(A
G)
Exon
5114
7Cn
sD305G
TT
TT
TT
TT
TT
TT
TT
TC
Trs225425698
75676792
CT(G
A)
Exon
5119
6Cs
P321=
CC
CC
CC
CC
CC
CC
CC
CT
Crs13459064
75676732
CT(G
A)
Exon
51256
CsT3
41=
CT
TC
TT
CT
TT
CT
CT
TC
Crs27978453
75676717
CT(G
A)
Exon
51271
CsA346=
CC
CC
CC
CC
CC
CC
CC
TC
Crs214335034
75676636
AG
(TC)
Exon
51352
CsD373=
AA
AA
AA
AA
AA
AA
AA
AG
Ars24958364
4756766
03AC
(TG
)Ex
on5
1385
CsP3
84=
AA
AA
AA
AA
AA
AA
AA
AC
Ars27978454
75676592
GA
(CT)
Exon
51396
CnsP
388L
GG
GG
GG
GG
GG
GG
GG
AG
Grs215384328
75676589
GC
(CG
)Ex
on5
1399
CnsA
389G
GG
GG
GG
GG
GG
GG
GG
GC
Grs251728286
75676571
GA
(CT)
Exon
51417
CnsT
395I
Grs247602334
75676567
TC(A
G)
Exon
51421
CsV396=
TT
TT
TT
TT
TT
TT
TT
TC
Trs234216231
75676530
TC(A
G)
Exon
51458
CnsM
409V
TT
TT
TT
TT
TT
TT
TT
TC
Trs212904337
75676526
CT(G
A)
Exon
51462
CnsR
410H
CC
CC
CC
CC
CC
CC
CC
CT
Crs252650779
75676522
TC(A
G)
Exon
51466
CsE4
11=
TT
TT
TT
TT
TT
TT
TT
TC
Trs231273560
75676516
TC(A
G)
Exon
51472
CsQ413=
TT
TT
TT
TT
TT
TT
TT
TC
Trs257886949
756764
13GT
(CA
)Ex
on5
1575
CsR4
48=
GG
GG
GG
GG
GG
GG
GG
GT
Grs241537608
756764
04GC
(CG
)Ex
on5
1584
CnsL
451V
GG
GG
GG
GG
GG
GG
GG
GC
Grs227619071
75676312
TC(A
G)
Exon
51676
CsQ481=
TT
TT
TT
TT
TT
TT
TT
TC
Trs258913831
75676290
AG
(TC)
Exon
51698
CsL4
89=
AA
AA
AA
AA
AA
AA
AA
AG
Ars225593319
75676126
AT
(TA
)Ex
on5
1862
CnsH
543Q
Ars4223233
75676105
GA
(CT)
Exon
51883
CsS550=
GG
GG
GG
GA
GG
GG
GG
GG
Grs4223232
75676032
GT
(CA
)Ex
on5
1956
CnsL
575M
Grs4223231
75675816
CG
UTR
-32172
NC
GG
GG
GG
GG
GG
GG
GG
GC
Grs1346
860
75675682
AT
UTR
-32306
NC
ASequ
ence
varia
tions
inmou
seNr
f2wereob
tained
from
Mou
sePh
enom
eDatabase(http
ph
enom
ejaxorgSNP)
andNCB
ISNPdatabase
(http
wwwncbinlm
nihgovSNP)N
CBIreference
sequ
ence
isMus
Musculusstra
inC5
7BL6J
chromosom
e2GRC
m38p1(NC
0000
687
GI37209910875704641ndash756755192
9123
bp)To
tal968
genetic
mutations
arerepo
rted
inNr
f2gene
and5k
bup
stream
(ge75704
642minus
1)and2k
bdo
wnstre
am(le75675512)sequences
asof
Janu
ary2003A
llSN
Psandallelesa
representedas
geno
miccontig
(reversedsequ
ence
indicated)E
xon175704
641ndash75704364(278
bpT
TS=75704
408)
exon
275679431ndash756791
65(267
bp)exon
375678579ndash75678490(90b
p)exon475677713ndash756775
46(168
bp)andexon
57567718
4ndash75678849
(1666
bp)Proteinam
inoacid
(aa)
resid
uesinNP00
61552
(597
aa)Cs
cod
ing-syno
nymou
sCn
scoding
-non
syno
nymou
sAminoacid
A-alanineD-aspartic
acidE
-glutamicacidF-phenylalanineG
-glycineH
-histidineI-iso
leucineL-leu
cineand
M-m
ethion
ineN-
asparagineP-prolin
eQ-glutamineR-arginineS-serineT-threon
ineandV-valin
elowastErrorsin
databasesfi
xedNC
notcon
firmed
20 Oxidative Medicine and Cellular Longevity
Table 6 NRF2 somatic mutations revealed in various human cancers
Domain Amino acid residues DNA mutation Cancer types (cases) ReferencesLocus Wild type Mutant
DLG motif
24 W (Trp) C (Cys) c72GgtCGgtT NSCLC neck ESC [54 62]K (Lys) c72TgtC ESC [62]
26 Q (Gln) E (Glu) c76CgtG NSCLC ESC [54 62]
27 D (Asp) G (Gly)lowast c80GgtA NSCLC [60]Y (Tyr) c79GgtT ESC [61]
28 I (Ile) T (Thr) c83CgtT NSCLC [54]
29 D (Asp) G (Gly) c86AgtG Head and neck ESC [54 62]H (His) c85GgtC NSCLC laynx [60 61]
30 L (Leu) F (Phe) c88CgtT NSCLC ESC [54 62]
31 G (Gly) A (Ala) c92GgtC NSCLC ESC skin [54 60ndash62]
32 V (Val) T (Thr) c95TgtG NSCLC [54]del c93 95delAGT ESC [61]
33ndash36 S-R-E-V S-R-E-V-S-R-E-Vlowast c97 108dupAGTCGAGCCGTA ESC [61]
34 R (Arg) Q (Gln) c101GgtA NCSCL [54 60 61]P (Pro) c101GgtC NSCLC [63]
ETGF motif
75 Q (Gln) H (His) c225AgtC Head and neck ESC [54 62]
77 D (Asp)
V (Val) c230AgtT NSCLC ESC [54 60 62]G (Gly) c230AgtG ESC [62]A (Ala) c230AgtC NSCLC [61]N (Asn) c229GgtA Larynx [61]
78 E (Glu) K (Lys) c232GgtA NSCLC ESC [54 62]
79 E (Glu)
K (Lys) c235GgtA NSCLC ESC [54 61 62]Q (Gln) c235GgtC NSCLC ESC [54 60ndash62]G (Gly) c236AgtG Larynx [61]E-E c234 236dupAGA ESC [61]
80 T (Thr) K (Lys) c239CgtACgtG NSCLC ESC [54 61 62]
80 T (Thr)P (Phe) c238AgtC ESC [62]I (Ile) c239CgtT Head and neck [54]A (Ala) c238AgtG NSCLC [63]
81 G (Gly) V (Val) c242GgtT ESC [61]D (Asp) c242GgtA NSCLC ESC [60ndash62]
82 E (Glu)
D (Asp) c246AgtT ESC oral cancer cell line [54 62]G (Gly) c245AgtG NSCLC [54]Q (Gln)lowast c244GgtC NSCLC ESC [60 61]V (Val)lowast c245AgtT ESC [61]
83 F (Phe) L (Leu)lowast c247TgtC NSCLC [60]NSCLC non-small cell lung cancer ESC esophageal squamous cancer and lowasterrors in reference fixed Number of cases 82 NSCLC and 10 ESC in [62] 125NSCLC 70 ESC 23 larynx and 17 skin in [61] 103 NSCLC and 12 head and neck in [54] 90 NSCLC in [63] 103 NSCLC in [60]
be involved in squamous lung cancer cell proliferation [64]suggesting that the mutation is functional and overcomesKEAP1 inhibition Singh et al [65] determined in vitrothat RNAi-mediated depletion of NRF2 in lung cancer cellsenhanced ROS production and susceptibility to cell deathby ionizing radiation These studies support the concept thatelevated NRF2 and ARE responsiveness provides cancer cellswith proliferative advantage for malignant transformation
and undue protection from anti-cancer therapy Onco-genic epidermal growth factor receptor (EGFR) signaling isrecently found to be critical in NRF2-mediated proliferationof NSCLC cells [66]
Collectively ldquogain of functionrdquo mutations in NRF2 thatreduce KEAP1 recognition are suggested to be predictivemarkers for poor responsiveness to chemotherapy and radi-ation therapy Although NRF2-mediated cellular defense
Oxidative Medicine and Cellular Longevity 21
processes are essential in the initiation stage enhancedNRF2-ARE activity in advanced stages of cancer developmentmay create a favorable intracellular environment for tumorcell growth and survival [67 68] In this context NRF2may be a potential molecular target for the treatment ofradio-resistance cancers especially those that have ldquoloss offunctionrdquo mutations in EGFR KRAS or KEAP1 as well asldquogain of functionrdquo mutations in NRF2
5 Epigenetic Alterations of NF-E2-RelatedFactor 2
Epigenetic modifications are alterations of molecules inter-acting with genes without changes to the primary DNAsequence They include post-translational modification ofhistones DNA methylation events chromatin conforma-tional changes and alterations to noncoding regulatoryRNAs Epigenetic alterations are stable and often inheritablebut are reversible and may affect expression of the geneDysregulation or defects in epigenetic processes particularlyhypermethylation of tumor suppressor gene promoters (egCpG islands) or histone modifications are thought to beassociated with carcinogenesis Investigators have reportedhypermethylation in CpG islands of KEAP1 which wereassociated with reduced KEAP1 expression in human cancersfrom lung prostate colon and so forth [69ndash72] Similar tosomatic mutations epigenetic changes on KEAP1 impairedthe function of its encoded protein leading to constitutiveNRF2 activation
Supporting the role of ldquopathogenic mutationsrdquo in NRF2expression of Nrf2 and downstream Nqo1 was suppressedin prostate tumors of mice (transgenic adenocarcinomaof mouse prostate TRAMP) Among 15 promoter CpGislands located between minus942 and minus654 (cminus1175 cminus1132and cminus1059 cminus887 gap in cminus1131 cminus1060 see Figure 3)hypermethylation of the first 5 CpG islands (minus942 minus899 andcminus1175 cminus1132) was significantly associated with tumorige-nesis [73] Moreover treatment with inhibitors for DNAmethyltransferase and histone deacetylase restored Nrf2expression in these tumor cells [73] A dietary phytochemicalcurcumin known as a DNA hypomethylation agent restoredepigenetically silent Nrf2 expression through CpG demethy-lation in carcinogen-induced mouse tumor cells [74]
The whole genome epigenetic datasets for 5 species arepublicly accessible at NCBI Epigenomics [75 76]The humanNRF2 epigenome of primary cells (breast penis) and H1stem cell line as well as mouse Nrf2 CpG island methylationdata for sperm blood and cerebellum are currently avail-able (httpwwwncbinlmnihgovepigenomics) Althoughno direct evidence of disease-associated epigenetic modu-lation has been identified in human NRF2 various phyto-chemical NRF2 agonists such as sulforaphane and curcuminhave shown their roles in DNA methylation and histonemodification (see reviews by Lee and colleagues eg [77])Taken together epigenetic modifications of theNRF2KEAP1axle are predicted to cause dysregulation of ARE-mediatedcellular defense leading to deleterious health effects and
phytochemical antioxidants as epigenetic modulators forNRF2 are suggested to be useful in cancer prevention
6 Conclusions
NRF2 is evolutionally conserved with high-sequence homol-ogy in many species However it is a highly mutable geneand numerous genetic variants have been discovered inhuman ethnic groups Importantly certain SNPs or haplo-types have been identified in various diseases as ldquoat-riskrdquoalleles and are related to functional alterations In additionto genetic variations multiple somatic mutations identifiedin the KEAP1 recognition domain of NRF2 in cancer cellshave been found to be oncogenic due to dysregulation ofNRF2 homeostasis by its excess ldquogain of functionrdquo Epigeneticalteration of theNRF2 is under investigation and is predictedto have pathogenic influences as learned from mouse andphytochemical agonist studies Continuous updates of Nrf2allelic variants in inbred mouse strains will provide a usefultool for effective experimental designs formodels of oxidativedisorders to provide insight into the disease mechanisms andintervention strategies
Abbreviations
AD Alzheimerrsquos diseaseALI acute lung injuryARE antioxidant response elementbZIP basic leucine zippercds coding DNA sequencesCI confidence intervalCOPD chronic obstructive pulmonary diseaseESC esophageal squamous cancerFEV1 forced expiratory volume in one second
Gbp giga base pairsGST glutathione S-transferaseGWAS genome-wide association studyHGVS Human Genome Variation SocietyHO-1 heme oxygenase-1119867 119901119910119897119900119903119894 119867119890119897119894119888119900119887119886119888119905119890119903 119901119910119897119900119903119894KEAP1 Kelch-like ECH activating protein 1MPD Mouse Phenome DatabaseMRP multidrug resistance proteinNCBI National Center for Biotechnology InformationNeh NRF2-ECH homologyNfe2l2 nuclear factor (erythroid-derived 2)-like 2Nrf2 NF-E2-related factor 2NSCLC nonsmall cell lung cancerNQO1 NAD(P)Hquinone oxidoreductase 1OR odds ratioPD Parkinsonrsquos diseasePM particulate matterROS reactive oxygen speciesSLE systemic lupus erythematosusSNP single nucleotide polymorphismSOD superoxide dismutaseURT untranslated region
22 Oxidative Medicine and Cellular Longevity
Disclosure
Authorrsquos contribution to the Work was done as part of theAuthorrsquos official duties as a NIH employee and is a Work ofthe United States Government Therefore copyright may notbe established in the United States
Conflict of Interests
The author declares that there is no conflict of interests
Acknowledgments
The research related to this paper was supported by theIntramural Research Program of the National Institutes ofHealth of the National Institute of Environmental HealthSciences (NIEHS) Drs Steven Kleeberger and StephanieLondon at the NIEHS provided excellent critical review ofthis paper The author thanks Mrs Jacqui Marzec for herhelpful comments and English editing
References
[1] J Y Chan M C Cheung P Moi K Chan and Y W KanldquoChromosomal localization of the humanNF-E2 family of bZIPtranscription factors by fluorescence in situ hybridizationrdquoHuman Genetics vol 95 no 3 pp 265ndash269 1995
[2] K Itoh K Igarashi N Hayashi M Nishizawa and MYamamoto ldquoCloning and characterization of a novel erythroidcell-derived CNC family transcription factor heterodimerizingwith the small Maf family proteinsrdquo Molecular and CellularBiology vol 15 no 8 pp 4184ndash4193 1995
[3] W O Osburn and T W Kensler ldquoNRF2 signaling an adaptiveresponse pathway for protection against environmental toxicinsultsrdquoMutation Research vol 659 no 1-2 pp 31ndash39 2008
[4] D Papp K Lenti D Modos D Fazekas Z Dul D Tureiet al ldquoThe NRF2-related interactome and regulome containmultifunctional proteins and fine-tuned autoregulatory loopsrdquoFEBS Letters vol 586 no 13 pp 1795ndash1802 2012
[5] X Wang D J Tomso B N Chorley et al ldquoIdentificationof polymorphic antioxidant response elements in the humangenomerdquo Human Molecular Genetics vol 16 no 10 pp 1188ndash1200 2007
[6] D Malhotra E Portales-Casamar A Singh et al ldquoGlobalmapping of binding sites for NRF2 identifies novel targets incell survival response through ChIP-Seq profiling and networkanalysisrdquo Nucleic Acids Research vol 38 no 17 pp 5718ndash57342010
[7] K Chan R Lu J C Chang and Y W Kan ldquoNRF2 a memberof the NFE2 family of transcription factors is not essential formurine erythropoiesis growth and developmentrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 93 no 24 pp 13943ndash13948 1996
[8] K Itoh T Chiba S Takahashi et al ldquoAn NRF2small Mafheterodimer mediates the induction of phase II detoxifyingenzyme genes through antioxidant response elementsrdquo Bio-chemical and Biophysical Research Communications vol 236no 2 pp 313ndash322 1997
[9] FMartin J M vanDeursen R A Shivdasani CW Jackson AG Troutman and P A Ney ldquoErythroid maturation and globin
gene expression inmicewith combined deficiency ofNF-E2 andNrf-2rdquo Blood vol 91 no 9 pp 3459ndash3466 1998
[10] K Itoh N Wakabayashi Y Katoh et al ldquoKEAP1 repressesnuclear activation of antioxidant responsive elements by NRF2through binding to the amino-terminal Neh2 domainrdquo Genesand Development vol 13 no 1 pp 76ndash86 1999
[11] A Kobayashi M I Kang H Okawa et al ldquoOxidative stresssensor KEAP1 functions as an adaptor for Cul3-based E3 ligaseto regulate proteasomal degradation of NRF2rdquo Molecular andCellular Biology vol 24 no 16 pp 7130ndash7139 2004
[12] K Taguchi H Motohashi and M Yamamoto ldquoMolecularmechanisms of the KEAP1-NRF2 pathway in stress responseand cancer evolutionrdquo Genes to Cells vol 16 no 2 pp 123ndash1402011
[13] K Mukaigasa L T Nguyen L Li H Nakajima M Yamamotoand M Kobayashi ldquoGenetic evidence of an evolutionarilyconserved role for NRF2 in the protection against oxidativestressrdquoMolecular and Cellular Biology vol 32 no 21 pp 4455ndash4461 2012
[14] JMaher andMYamamoto ldquoThe rise of antioxidant signalingmdashthe evolution and hormetic actions of NRF2rdquo Toxicology andApplied Pharmacology vol 244 no 1 pp 4ndash15 2010
[15] S C Lo X Li M T Henzl L J Beamer and M HanninkldquoStructure of the KEAP1NRF2 interface provides mechanisticinsight intoNRF2 signalingrdquoThe EMBO Journal vol 25 no 15pp 3605ndash3617 2006
[16] V A McKusick ldquoMendelian inheritance in man and its onlineversion OMIMrdquo American Journal of Human Genetics vol 80no 4 pp 588ndash604 2007
[17] B Yalcin D J Adams J Flint and T M Keane ldquoNext-generation sequencing of experimental mouse strainsrdquo Mam-malian Genome vol 23 no 9-10 pp 490ndash498 2012
[18] D R Bentley S Balasubramanian H P Swerdlow et alldquoAccurate whole human genome sequencing using reversibleterminator chemistryrdquo Nature vol 456 no 7218 pp 53ndash592008
[19] P S G Chain D V Grafham R S Fulton et al ldquoGenomeproject standards in a new era of sequencingrdquo Science vol 326no 5950 pp 236ndash237 2009
[20] RHWaterstonK Lindblad-Toh E Birney J Rogers J F AbrilP Agarwal et al ldquoInitial sequencing and comparative analysis ofthe mouse genomerdquo Nature vol 420 pp 520ndash562 2002
[21] T M Keane L Goodstadt P Danecek M A White K WongB Yalcin et al ldquoMouse genomic variation and its effect onphenotypes and gene regulationrdquoNature vol 477 pp 289ndash2942011
[22] T Yamamoto K Yoh A Kobayashi et al ldquoIdentification ofpolymorphisms in the promoter region of the human NRF2generdquo Biochemical and Biophysical Research Communicationsvol 321 no 1 pp 72ndash79 2004
[23] J M Marzec J D Christie S P Reddy et al ldquoFunctionalpolymorphisms in the transcription factor NRF2 in humansincrease the risk of acute lung injuryrdquo The FASEB Journal vol21 no 9 pp 2237ndash2246 2007
[24] H Fukushima-Uesaka Y Saito K Maekawa et al ldquoGeneticvariations and haplotype structures of transcriptional factorNRF2 and its cytosolic reservoir protein KEAP1 in JapaneserdquoDrug Metabolism and Pharmacokinetics vol 22 no 3 pp 212ndash219 2007
[25] E J Cordova R Velazquez-Cruz F Centeno V Baca and LOrozco ldquoThe NRF2 gene variant -653GA is associated with
Oxidative Medicine and Cellular Longevity 23
nephritis in childhood-onset systemic lupus erythematosusrdquoLupus vol 19 no 10 pp 1237ndash1242 2010
[26] M von Otter S Landgren S Nilsson et al ldquoAssociation ofNRF2-encoding NFE2L2 haplotypes with Parkinsonrsquos diseaserdquoBMCMedical Genetics vol 11 no 1 article 36 2010
[27] JMHartikainenM TengstromVMKosmaV L Kinnula AMannermaa andY Soini ldquoGenetic polymorphisms and proteinexpression of NRF2 and sulfiredoxin predict survival outcomesin breast cancerrdquo Cancer Research vol 72 pp 5537ndash5546 2012
[28] T Arisawa T Tahara T Shibata et al ldquoThe relationshipbetween Helicobacter pylori infection and promoter polymor-phism of the NRF2 gene in chronic gastritisrdquo InternationalJournal of Molecular Medicine vol 19 no 1 pp 143ndash148 2007
[29] T Arisawa T Tahara T Shibata et al ldquoThe influence ofpromoter polymorphism of nuclear factor-erythroid 2-relatedfactor 2 gene on the aberrant DNA methylation in gastricepitheliumrdquo Oncology Reports vol 19 no 1 pp 211ndash216 2008
[30] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene pro-moter polymorphism and gastric carcinogenesisrdquo Hepato-Gastroenterology vol 55 no 82-83 pp 750ndash754 2008
[31] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene promoterpolymorphism is associated with ulcerative colitis in a Japanesepopulationrdquo Hepato-Gastroenterology vol 55 no 82-83 pp394ndash397 2008
[32] C C Hua L C Chang J C Tseng C M Chu Y C Liu andW B Shieh ldquoFunctional haplotypes in the promoter region oftranscription factor NRF2 in chronic obstructive pulmonarydiseaserdquo Disease Markers vol 28 no 3 pp 185ndash193 2010
[33] S O Shaheen R B Newson S M Ring M J Rose-ZerilliJ W Holloway and A J Henderson ldquoPrenatal and infantacetaminophen exposure antioxidant gene polymorphismsand childhood asthmardquo The Journal of Allergy and ClinicalImmunology vol 126 no 6 pp 1141e7ndash1148e7 2010
[34] H Masuko T Sakamoto Y Kaneko et al ldquoLower FEV1 innon-COPD nonasthmatic subjects association with smokingannual decline in FEV1 total IgE levels and TSLP genotypesrdquoInternational Journal of Chronic Obstructive Pulmonary Diseasevol 6 pp 181ndash189 2011
[35] C P Guan M N Zhou A E Xu et al ldquoThe susceptibilityto vitiligo is associated with NF-E2-related factor 2 (NRF2)gene polymorphisms a study on Chinese Han populationrdquoExperimental Dermatology vol 17 no 12 pp 1059ndash1062 2008
[36] J Bouligand O Cabaret M Canonico et al ldquoEffect of NFE2L2genetic polymorphism on the association between oral estrogentherapy and the risk of venous thromboembolism in post-menopausal womenrdquo Clinical Pharmacology amp Therapeuticsvol 89 no 1 pp 60ndash64 2011
[37] D S OrsquoMahony B J Glavan T D Holden C Fong R A BlackG Rona et al ldquoInflammation and immune-related candidategene associations with acute lung injury susceptibility andseverity a validation studyrdquo PLoS ONE vol 7 no 12 Article IDe51104 2012
[38] I Ungvari E Hadadi V Virag et al ldquoRelationship between airpollutionNFE2L2 gene polymorphisms and childhood asthmain aHungarian populationrdquo Journal of CommunityGenetics vol3 no 1 pp 25ndash33 2012
[39] M SiedlinskiD S Postma JMA Boer et al ldquoLevel and courseof FEV1 in relation to polymorphisms inNFE2L2 andKEAP1 inthe general populationrdquo Respiratory Research vol 10 article 732009
[40] C C Hong C B Ambrosone J Ahn et al ldquoGenetic variabilityin iron-related oxidative stress pathways (NRF2 NQ01 NOS3
and HO-1) iron intake and risk of postmenopausal breastcancerrdquo Cancer Epidemiology Biomarkers and Prevention vol16 no 9 pp 1784ndash1794 2007
[41] C Canova C Dunster F J Kelly C Minelli P L Shah CCaneja et al ldquoPM10-induced hospital admissions for asthmaand chronic obstructive pulmonary disease the modifyingeffect of individual characteristicsrdquo Epidemiology vol 23 no 4pp 607ndash615 2012
[42] A J Henderson R B Newson M Rose-Zerilli S M RingJ W Holloway and S O Shaheen ldquoMaternal NRF2 andgluthathione-S-transferase polymorphisms donotmodify asso-ciations of prenatal tobacco smoke exposure with asthma andlung function in school-aged childrenrdquo Thorax vol 65 no 10pp 897ndash902 2010
[43] C Xing A L Sestak J A Kelly et al ldquoLocalization andreplication of the systemic lupus erythematosus linkage signalat 4p16 interaction with 2p11 12q24 and 19q13 in EuropeanAmericansrdquo Human Genetics vol 120 no 5 pp 623ndash631 2007
[44] T F Wong K Yoshinaga Y Monma K Ito H NiikuraS Nagase et al ldquoAssociation of KEAP1 and NRF2 geneticmutations and polymorphisms with endometrioid endometrialadenocarcinoma survivalrdquo International Journal of Gynecologi-cal Cancer vol 21 no 8 pp 1428ndash1435 2011
[45] M von Otter S Landgren S Nilsson et al ldquoNRF2-encodingNFE2L2 haplotypes influence disease progression but not riskin Alzheimerrsquos disease and age-related cataractrdquoMechanisms ofAgeing and Development vol 131 no 2 pp 105ndash110 2010
[46] S Tsang Z Sun B Luke et al ldquoA comprehensive SNP-basedgenetic analysis of inbred mouse strainsrdquoMammalian Genomevol 16 no 7 pp 476ndash480 2005
[47] C M Wade E J Kulbokas III A W Kirby et al ldquoThe mosaicstructure of variation in the laboratory mouse genomerdquoNaturevol 420 no 6915 pp 574ndash578 2002
[48] T Wiltshire M T Pletcher S Batalov et al ldquoGenome-widesingle-nucleotide polymorphism analysis defines haplotypepatterns in mouserdquo Proceedings of the National Academy ofSciences of the United States of America vol 100 no 6 pp 3380ndash3385 2003
[49] K A Frazer E Eskin H M Kang et al ldquoA sequence-basedvariation map of 827 million SNPs in inbred mouse strainsrdquoNature vol 448 no 7157 pp 1050ndash1053 2007
[50] T P Maddatu S C Grubb C J Bult andM A Bogue ldquoMousephenome database (MPD)rdquo Nucleic Acids Research vol 40 ppD887ndashD894 2007
[51] A Kirby H M Kang C M Wade et al ldquoFine mapping in 94inbred mouse strains using a high-density haplotype resourcerdquoGenetics vol 185 no 3 pp 1081ndash1095 2010
[52] H Y Cho A E Jedlicka S P M Reddy L Y Zhang T WKensler and S R Kleeberger ldquoLinkage analysis of susceptibilityto hyperoxia NRF2 is a candidate generdquo American Journal ofRespiratory Cell and Molecular Biology vol 26 no 1 pp 42ndash512002
[53] H Y Cho A E Jedlicka S P M Reddy et al ldquoRole of NRF2in protection against hyperoxic lung injury in micerdquo AmericanJournal of Respiratory Cell and Molecular Biology vol 26 no 2pp 175ndash182 2002
[54] T Shibata T Ohta K I Tong et al ldquoCancer related mutationsin NRF2 impair its recognition by KEAP1-Cul3 E3 ligase andpromote malignancyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 36 pp13568ndash13573 2008
24 Oxidative Medicine and Cellular Longevity
[55] A Singh V Misra R K Thimmulappa et al ldquoDysfunctionalKEAP1-NRF2 interaction in non-small-cell lung cancerrdquo PLoSMedicine vol 3 no 10 article e420 2006
[56] T Ohta K Iijima M Miyamoto et al ldquoLoss of KEAP1 functionactivates NRF2 and provides advantages for lung cancer cellgrowthrdquo Cancer Research vol 68 no 5 pp 1303ndash1309 2008
[57] B Padmanabhan K I Tong T Ohta et al ldquoStructural basisfor defects of KEAP1 activity provoked by its point mutationsin lung cancerrdquoMolecular Cell vol 21 no 5 pp 689ndash700 2006
[58] T Shibata A Kokubu M Gotoh et al ldquoGenetic alteration ofKEAP1 confers constitutive NRF2 activation and resistance tochemotherapy in gallbladder cancerrdquoGastroenterology vol 135no 4 pp 1358e4ndash1368e4 2008
[59] N J Yoo H R Kim Y R Kim C H An and S H LeeldquoSomatic mutations of the KEAP1 gene in common solidcancersrdquo Histopathology vol 60 no 6 pp 943ndash952 2012
[60] Y Hu Y Ju D Lin Z Wang Y Huang S Zhang et alldquoMutation of the NRF2 gene in non-small cell lung cancerrdquoMolecular Biology Reports vol 39 no 4 pp 4743ndash4747 2012
[61] Y R Kim J EOhM S Kim et al ldquoOncogenicNRF2mutationsin squamous cell carcinomas of oesophagus and skinrdquo TheJournal of Pathology vol 220 no 4 pp 446ndash451 2010
[62] T Shibata A Kokubu S Saito et al ldquoNRF2 mutation confersmalignant potential and resistance to chemoradiation therapyin advanced esophageal squamous cancerrdquo Neoplasia vol 13pp 864ndash873 2011
[63] H Sasaki M Shitara K Yokota Y Hikosaka S MoriyamaM Yano et al ldquoIncreased NRF2 gene (NFE2L2) copy numbercorrelates with mutations in lung squamous cell carcinomasrdquoMolecular Medicine Reports vol 6 no 2 pp 391ndash394 2012
[64] H Sasaki M Shitara K Yokota Y Hikosaka S Moriyama MYano et al ldquoRagD gene expression andNRF2mutations in lungsquamous cell carcinomasrdquo Oncology Letters vol 4 pp 1167ndash1170 2012
[65] A Singh M Bodas N Wakabayashi F Bunz and S BiswalldquoGain of NRF2 function in non-small-cell lung cancer cellsconfers radioresistancerdquo Antioxidants and Redox Signaling vol13 no 11 pp 1627ndash1637 2010
[66] T Yamadori Y Ishii SHommaYMorishimaKKurishimaKItoh et al ldquoMolecular mechanisms for the regulation of NRF2-mediated cell proliferation in non-small-cell lung cancersrdquoOncogene vol 31 pp 4768ndash4777 2012
[67] A K Bauer H Y Cho L Miller-Degraff C Walker K HelmsJ Fostel et al ldquoTargeted deletion of NRF2 reduces urethane-induced lung tumor development in micerdquo PLoS ONE vol 6no 10 Article ID e26590 2011
[68] H Y Cho and S R Kleeberger ldquoNRF2 protects against airwaydisordersrdquo Toxicology and Applied Pharmacology vol 244 no1 pp 43ndash56 2010
[69] R Wang J An F Ji H Jiao H Sun and D Zhou ldquoHyperme-thylation of the KEAP1 gene in human lung cancer cell linesand lung cancer tissuesrdquo Biochemical and Biophysical ResearchCommunications vol 373 no 1 pp 151ndash154 2008
[70] P Zhang A Singh S Yegnasubramanian et al ldquoLoss ofkelch-like ECH-associated protein 1 function in prostate cancercells causes chemoresistance and radioresistance and promotestumor growthrdquoMolecular Cancer Therapeutics vol 9 no 2 pp336ndash346 2010
[71] N Hanada T Takahata Q Zhou et al ldquoMethylation of theKEAP1 gene promoter region in human colorectal cancerrdquoBMCCancer vol 12 article 66 2012
[72] L A Muscarella R Barbano V DrsquoAngelo et al ldquoRegulationof KEAP1 expression by promoter methylation in malignantgliomas and association with patientrsquos outcomerdquo Epigeneticsvol 6 no 3 pp 317ndash325 2011
[73] S Yu T O Khor K L Cheung et al ldquoNRF2 expressionis regulated by epigenetic mechanisms in prostate cancer ofTRAMP micerdquo PLoS ONE vol 5 no 1 Article ID e8579 2010
[74] T O Khor Y Huang T Y Wu L Shu J Lee and A N KongldquoPharmacodynamics of curcumin as DNA hypomethylationagent in restoring the expression of NRF2 via promoter CpGsdemethylationrdquo Biochemical Pharmacology vol 82 no 9 pp1073ndash1078 2011
[75] I M Fingerman L McDaniel X Zhang et al ldquoNCBI epige-nomics a new public resource for exploring epigenomic datasetsrdquo Nucleic Acids Research vol 39 supplemen 1 pp D908ndashD912 2011
[76] I M Fingerman X Zhang W Ratzat N Husain R F Cohenand G D Schuler ldquoNCBI epigenomics whatrsquos new for 2013rdquoNucleic Acids Research vol 41 no 1 pp D221ndashD225 2013
[77] J H Lee T O Khor L Shu Z Y Su F Fuentes andA N Kong ldquoDietary phytochemicals and cancer preventionNRF2 signaling epigenetics and cell death mechanisms inblocking cancer initiation and progressionrdquo Pharmacology ampTherapeutics vol 137 no 2 pp 153ndash171 2012
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Disease Markers
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OncologyJournal of
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Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
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Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
10 Oxidative Medicine and Cellular Longevity
Table3Con
tinued
IDMap
onchromosom
e2lowast
(HGVS
name)
Positionin
Nfe2
l2
(HGVS
name)
Region
s(position
from
mRN
A)
Varia
tioncla
ssand
consequences
(HGVS
name)dagger
Minor
allelefrequ
ency
(MAF)
MAcoun
tsDagger(coh
ort
size)
MAFsources
rs201481890
g178095090178095091in
sTclowast
422lowast423insA
Exon
5UTR
-31015840
(2795minus
2796)
Insertion
na
rs3082500
g178095089178095090d
elTT
delTTinsT
clowast423lowast424d
elAAinsA
Exon
5UTR
-31015840
(2796minus
2797)
Dele
tion
insertion
na
rs71792546
(rs71796710)
g178095079d
elT
clowast425delA
Exon
5UTR
-31015840(2798)
Dele
tion
na
rs1057106
g178095078AgtC
AgtT
clowast435TgtA
TgtG
Exon
5UTR
-31015840(2808)
SNP
na
rs34176791
g178095076AgtC
clowast437TgtG
Exon
5UTR
-31015840(2810)
SNP
C=000235
1000
Genom
esrs35911553
g178095045CgtT
clowast46
8GgtA
Exon
5UTR
-31015840(2841)
SNP
T=000
6915
1000
Genom
esSequ
ence
varia
tions
inup
stream
andexon
sofh
uman
NRF
2fro
m655varia
tions
availablein
publicdatabase
asof
Decem
ber2012
(583
activ
esomeSN
Psmergedge14
SNPs
citedin
PubM
ed)lowastNCB
Ireference
sequ
ence
NC
0000
0211
(Hom
osapiens
chromosom
e2G
RCh37p
10prim
arya
ssem
bly)spanning
178095033ndash1781298
59bp
(com
plem
ent34827
bpforexons
andintro
ns)51015840-Flank
ingregions
startat17812
9860
bp(minus1)in
transcrip
tvariant
1HGVS
Hum
anGenom
eVa
riatio
nSo
ciety
Position
sinvaria
nt1(NM
006164
4)2859
bpsectSN
Pscitedin
PubM
edE
xon11781298
59ndash17812
9260
(600
bpT
TS=1781293
04)exon
2178098999ndash
178098733(267
bp)exon
3178098067ndash1780979
78(90b
p)exon41780973
11ndash178097120
(192b
p)and
exon
5178096736ndash
178095033(1704b
p)daggerProteinam
inoacid
(aa)
resid
uesiniso
form
1(N
P00
61552605
aa)Cn
scoding
-non
syno
nymou
sCs
cod
ing-syno
nymou
sDaggerheterozygositydetectednano
tavailable
Oxidative Medicine and Cellular Longevity 11
Table4Hum
anNRF
2SN
Psassociated
with
diseaser
isk
IDMap
onchromosom
e2 (H
GVS
name)
Region
class
Dise
asea
ssociatio
nandreferences
Risk
allelesa
ndstatistics
Ethn
icgrou
p(num
bero
fcase)
rs7557529
g178135097CgtT
minus5238
Ggt
AParkinsonrsquos
disease(2010
[26])
G=0718252Dagger
inhaplotype
OR=090
or040
CI=
060ndash140or
03ndash06
Swedish
Polish
Caucasian
(357)
rs2886162
g178133165AgtG
minus3306
TgtC
Breastcancer
survival(2012[27])
TT=0324
OR=16
87C
I=1105ndash275
FinlandKB
CP(452)
Chronicg
astritisgastric
ulcer(2007
[28])
GDagger
GDaggerin
haplotypefor
ulcer(OR=
252C
I=119ndash
545)
Japanese
(159)
P14methylationin
gastric
cancer
Hpyloriinfectio
n(2008[29])
Gin
haplotype
OR=290C
I=114ndash
736
Japanese
(209)
Gastriccancer
inHpylori-n
egative
cases(2008
[30])
Ain
haplotype
119875=0022
Japanese
(209)
rs35652124
(rs57695243)
g178130073TgtC
minus214Agt
G(previou
slyminus653[23]
orminus686[22])
Ulcerativec
olitis(2008
[31])
AG
(OR=045C
I=022ndash0
93)
G(O
R=257C
I=10
1ndash660)
Japanese
(89)
Lupu
swith
neph
ritisin
female(2010
[25])
GA
OR=18
1CI
=10
4ndash312
Mexican
mestizo(362)
Parkinsonrsquos
disease(2010
[26])
A=0884312Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
COPD
(2010[32])
G=052Dagger
hazard
ratio
=095C
I=091ndash0
99
(Haplotype)
German
(69)
Gastriculcer(2007
[28])
GDaggerin
haplotype
OR=252C
I=119ndash
545
Japanese
(159)
P14methylationin
gastric
cancer
H
pyloriinfection(2008[29])
Gin
haplotype
OR=290C
I=114ndash
736
Japanese
(209)
Gastriccancer
inHpylori-negative
cases(2008
[30])
Gin
haplotype119875=0022
Japanese
(209)
rs6706
649
g178130071CgtT
minus212Ggt
A(previou
slyminus651[23]
orminus684[22])
Ulcerativec
olitis(2008
[31])
AG
(OR=045C
I=022ndash0
93)
G(O
R=257C
I=10
1ndash660)
Japanese
(89)
Maternalacetaminop
henandasthma
(2010[33])
A=02321137Dagger
OR=17
3CI
=12
2245
UKALSPA
C(gt40
00mothersgt
5000
child
ren)
12 Oxidative Medicine and Cellular Longevity
Table4Con
tinued
IDMap
onchromosom
e2 (H
GVS
name)
Region
class
Dise
asea
ssociatio
nandreferences
Risk
allelesa
ndstatistics
Ethn
icgrou
p(num
bero
fcase)
COPD
(2010[32])
G=098Daggerin
haplotype
hazard
ratio
=095C
I=091ndash0
99
German
(69)
Parkinsonrsquos
disease(2010
[26])
G=0972343Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
03ndash06
Swedish
Polish
Caucasian
(357)
Acutelun
ginjury
follo
wingtrauma
(2007[23])
CA=0119Dagger
OR=644
CI=
134ndash
3080
Caucasia
nAfrican-
American
(164
)
Ann
ualF
EV1decline
(2011[34])
A=0082in
haplotype
Japanese
(915)
Vitiligo(2008[35])
CAA
AOR=17
24C
I=13
5ndash221
Chinese(300)
COPD
(2010[32])
G=073Daggerin
haplotype
hazard
ratio
=095C
I=091ndash0
99
German
(69)
rs6721961
(rs117801448)
g178130037TgtC
TgtG
minus178Agt
GAgt
C(previou
slyminus617[23]
orminus650[22])
Parkinsonrsquos
disease(2010
[26])
C=0980346Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
Postm
enop
ausalvenou
sthrombo
embo
lism
(2011[36])
A=033311
OR=25CI
=370ndash8570
Caucasia
n(161)
Breastcancer
survivalandNRF
2proteinexpressio
n(2012[27])
AA
OR=4656CI
=13
5ndash1606
FinlandKB
CP(452)
Acutelun
ginjury-related
mortality
followingsyste
micinflammatory
respon
sesynd
rome(2012
[37])
GG
OR=97
3CI
=12
7ndash7480
Caucasia
n(750)
Infection-indu
cedasthma(
2012
[38])
AC
OR=0437CI
=028ndash0
80
Hun
garia
nCaucasia
nGypsy
(307)
rs143406266
g178129391178
129393delGGC
Exon
1(467ndash4
69G
CC)
COPD
(2010[32])
GCC
4=053Dagger
hazard
ratio
=095C
I=091ndash0
99
Taiwanese(69)
rs2886161
g178127839Tgt
ClowastIntro
n1
Parkinsonrsquos
disease(2010
[26])
T=0878309Dagger
inhaplotype
OR=09or
04CI
=06ndash
14or
03ndash06
Swedish
Polish
Caucasian
(357)
rs2364723
g178126546Ggt
CIntro
n1
Basaland
smoker
FEV
1(200
9[39])
CI=minus6360simminus1780C
=0525Dagger
Also
ashaplotype
Netherla
nd(2542)
rs2364722
g178124787Agt
GIntro
n1
Ann
ualF
EV1decline
(2011[34])
A=0082in
haplotype
Japanese
(915)
rs13001694
g178118990Agt
GIntro
n1
Basaland
smoker
FEV
1(200
9[39])
G=040
11578Dagger
inhaplotype
Netherla
nd(2542)
Oxidative Medicine and Cellular Longevity 13
Table4Con
tinued
IDMap
onchromosom
e2 (H
GVS
name)
Region
class
Dise
asea
ssociatio
nandreferences
Risk
allelesa
ndstatistics
Ethn
icgrou
p(num
bero
fcase)
Breastcancer
[40]
Twith
NQO1N
OS3H
O1risk
allelesOR=15
6CI
=097ndash251
Caucasia
nandothers(505)
rs1806
649
(rs58745895)
g178118152Cgt
TIntro
n1
Basaland
smoker
FEV
1(200
9[39])
T=02631119Daggerin
haplotype
CI=minus8730ndash
(minus17
0)Netherla
nd(2542)
Parkinsonrsquos
disease(2010
[26])
T=0422148Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
Particulatem
attera
ndasthmaCO
PDadmiss
ion(2012[41])
Cwith
lowvitamin
Clevel(OR=
31CI
=15
0ndash630)
UK(209)
rs4243387
(rs6003846
4)g178117765Cgt
TIntro
n1
Basaland
smoker
FEV
1(200
9[39])
T=00914
25Daggerin
haplotype
Netherla
nd(2542)
rs1962142
(rs58448508)
g178113484Agt
GIntro
n1
Ann
ualF
EV1decline
(2011[34])
A=0082in
haplotype
Japanese
(915)
Breastcancer
NRF
2andARE
expressio
n(2012[27])
A(119875=0036)
FinlandKB
CP(452)
rs6726395
(rs57309289)
g178103229Agt
GIntro
n1
Smok
ing-relatedFE
V1decline
and
annu
alFE
V1decline
(2011[34])
G=0884Dagger
A=0082in
haplotype
Japanese
(915)
Basaland
smoker
FEV
1(200
9[39])
G=046
41764Daggerin
haplotype
Netherla
nd(2542)
rs2001350
(rs17515179
rs60883775)
g178100
425Cgt
TIntro
n1
Ann
ualF
EV1decline
(2011[34])
T=0082in
haplotype
Japanese
(915)
Parkinsonrsquos
disease(2010
[26])
T=0986350Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
rs10183914
(rs58731187
rs61374844
)g17809766
6Cgt
TIntro
n3
Parkinsonrsquos
disease(2010
[26])
T=0536188Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
rs2706110
g178092162Tgt
C31015840Flanking
Breastcancer
(2012[27])
TT
OR=2079CI
=118ndash368
FinlandKB
CP(452)
rs2588882
g178087165Ggt
T31015840Flanking
Infection-indu
cedasthma(
2012
[38])
TG
OR=0290CI
=013ndash0
62
Hun
garia
nCaucasia
nGypsy
(307)
minus686in
reference[22]
=minus653in
reference[23]
=currentlyminus214minus684in
reference[22]
=minus651inreference[23]
=currentlyminus212minus651inreference[22]
=minus617in
reference[23]
=currentlyminus178Ch
romosom
econtig(in
tron
31015840flank
ing)
orreversed
(51015840flank
ingprom
oter)a
llelesinbo
ldhave
been
used
inthetextand
TableOR
odds
ratio
CI95confi
denceinterval
14 Oxidative Medicine and Cellular Longevity
minus212GG (98 rs67006649 previously minus684minus651) minus178Callele (73) and GCC
4(53) was predicted to increase
respiratory failure development (hazard ratio = 095 CI091ndash099) in German COPD patients [32] Significant inter-action was also identified between an intronic SNP Gallele (rs6726395 g178103229AgtG 884 frequency) andsmoking status on FEV
1decline relative to the reference
AA allele in the above Japanese cohort [34] Siedlinskiet al [39] reported that the CC genotype of anotherintronic SNP (rs2364723 g178126546GgtC) was associatedwith a lower FEV
1level compared to the wild-type geno-
type (GG) in two Netherland cohorts (CI minus636minus178frequency = 0525 and pooled cohort size = 2542) ThisSNP alone or as a haplotype with 4 more intronic SNPs(rs13001694Grs1806649Trs4243387Trs6726395G)was alsoassociated with high FEV
1levels in individuals that ever
smoked [39] In a Hungarian population of childhoodasthma SNPs at minus178 (CA) and 31015840 flanking (rs2588882TG)loci were inversely associated with infection-induced asthma(OR 0437 CI 028ndash080 OR 0290 CI 013ndash062 resp) andthese SNPs significantly influenced an asthma-environmentalpollution interaction [38]The intronic SNP rs1806649 (CgtT)was associated but not significantly with an increased risk ofhospitalization during high-level particulate matter (PM
10)
periods in asthma or COPD patients (119899 = 209) of the UnitedKingdom (UK) [41] Asthma andCOPDadmission rateswererelated to the increase in environmental PM
10concentration
Importantly effects of interaction between prenatal stressand NRF2 SNPs on descendant pulmonary health wereinvestigated by the Avalon Longitudinal Study in the UKmaternal smoking during pregnancy was not associated withlung function change determined by maximum mild expi-ratory flow (FEF
25ndash75) or with asthma incidence in school-aged children and this relation was not modified by NRF2SNP genotypes [42]However early gestation acetaminophenexposure significantly influenced the risk of asthma andwheezing at the age of 7 years in gt4000 mothers and gt5000children [33] When maternal copies of the minus212A allele werepresent association with asthma (11374891 OR 173 CI122ndash245) and wheezing (11494949 OR 153 95 CI 106ndash220) was significantly increased [33]
Gastrointestinal Disorders While there was no evidence inlung cancer cases studies in Japanese populations suggesteda potential association of NRF2 variations with gastro-intestinal tumorigenesisHelicobacter pylori (H pylori) causesgastritis which can lead to gastric atrophy and cancer Ingastric epithelium from the Japanese cancer cohorts (39gastric cancers 46 controls) H pylori infection was posi-tively correlated with aberrant CpG island methylation oftumor suppressor genes (eg p14) and minus214Gminus212G orminus214Aminus212G NRF2 haplotype was significantly associatedwith increased (OR 290 95 CI 114ndash736) or decreased(OR 033 95 CI 013ndash088) risk of the CpG methyla-tion respectively in the H pylori-infected patients [29]Further study from the same investigators determined thatminus214Aminus212G allele carriers had significantly (119875 = 0022)reduced risk of gastric cancer inH pylori-negative cases [30]The minus214AGminus212AG genotypes were negatively associated
(OR 045 CI 022ndash093) and the minus214Gminus212G genotypeswere positively associated (chronic continuous phenotypeOR 257 CI 101ndash660) with ulcerative colitis (89 patients 141controls) in a Japanese population [31]
Autoimmune Disorders Systemic lupus erythematosus (SLE)is a long-term autoimmune disease more frequently found infemales than in males It affects organs including skin jointskidneys and brain and nephritis is an aggressive character-istic in some patients Genome-wide association studies inhumans identified a suggestive quantitative trait locus nearNRF2 [43] A study of a Mexican Mestizo population (362patients with childhood-onset SLE 379 controls and 212nephritis diagnosed) determined that lupus with nephritiswas significantly (OR 181 CI 104ndash312) associated with theminus214GA SNP in females [25] The same SNPs were notclosely associated with SLE risk in a Japanese cohort [22]Vitiligo is a skin condition in which there is a loss of browncolor (pigment) from areas of skin resulting in irregularwhite patches It is thought to be an autoimmune diseasecaused by loss of cells (melanocytes) that produce brownpigment A study indicated that theminus178A allele increased therisk of vitiligo dose-dependently (OR 1724 95 CI 135ndash221for CA OR 2902 CI 162ndash519 for AA) [35]
Female Disorders It is well known that estrogen metabolites(eg catechols) cause ROS formation suggesting correla-tion of NRF2 and downstream effectors in postmenopausalmammary cancer In a study of a Finish population (KuopioBreast Cancer Project 119899 = 452 patients 370 controls)the minus178AA homozygous genotype (OR 4656 CI = 135ndash1606) and 31015840 flanking rs2706110 (TT OR 2079 CI 118ndash368) genotype were associated with increased risk of breastcancer while the 51015840 flanking minus3306TT homozygous allelewas significantly associated with lower survival (frequency =71219 OR 1687 CI 1105ndash275) [27] suggesting that NRF2genetic polymorphisms affect susceptibility and outcome ofthe patients The minus178A allele carriers together with intronicrs1962142A allele carriers were associated with lowered tissuelevels of NRF2 proteins [27] In postmenopausal women theminus178A allele (OR 179 95CI 370ndash8570) appeared tomodifythe risk of venous thromboembolism caused by oral estrogentherapy (AA or AC frequency = 333) as demonstratedby the French ESTHER study (161 cases 474 controls) [36]An intronic rs1806649CgtT SNP did not associate with breastcancer risk in postmenopausal women [40] However whenthis SNP and other at-risk alleles of ARE-responsive genes(NQO1 HO-1 NOS3) were combined there was a significantgene-dose effect on the breast cancer risk [40] Althoughcoding region SNPs in NRF2 and KEAP1 were identifiedin the Japanese endometrial adenocarcinoma patients noassociation of NRF2 SNPs with the disease was found [44]
Neurodegenerative Diseases Oxidative stress is known to beinvolved in Parkinsonrsquos disease (PD) presumably due toproduction of ROS fromhigh-dopaminemetabolism and lowlevels of antioxidants in the substantia nigra of the brainInvestigators found a protective NRF2 haplotype consistingof four 51015840 flanking SNPs (minus5238Gminus214Aminus212Gminus178C)
Oxidative Medicine and Cellular Longevity 15
and 4 intronic SNPs (rs2886161Ars1806649Ars2001350Ars10183914A) from Swedish (OR 09 CI 060ndash140) and Polish(OR 04 CI 030ndash060) populations (total 165+192 PD cases190+192 controls) [26]The investigators also suggested thatNRF2 haplotype alleles were associated with 2 years earlierage of Alzheimerrsquos disease (AD) onset 4 years earlier age ofposterior subcapsular cataract surgery and 4 years later ageof cortical cataract surgery while they were not significantlyrelated to AD or age-related cataract risk [45]
34 Genetic Mutations in Mouse Nrf2 Tsang et al [46]compiled 673 SNPs in 55 mouse strains and constructedtheir phylogenetic tree to correlate and clarify the originsof strains based on the assembled mouse genome sequenceand SNP data [20 47 48] Recently using the completegenome sequence of C57BL6J (B6) mouse as a referencehigh-density SNP screening in other laboratory strains orin panels of strains has been published (see [17]) Althoughmillions of mouse SNPs (gt10089892 as of December 2012)and haplotype mappings from more than 120 strains havebeen published as valuable references for dissecting thegenetic basis of complex traits [49ndash51] little attention hasbeen paid to polymorphisms of Nrf2 and their correlationwith disease phenotypes
Figure 3 demonstrates the proximal promoter region(minus1 to minus950)51015840-UTR (exon 1 up to TSS) and proteinsequence of mouse Nrf2 based on GRCm38p1 PrimaryAssembly (75704641ndash75675513 bp) mRNA variant1 and protein (NP 035032 encoded by NM 010902234ndash2027 bp) sequences Genetic variations in the Nrf2genome of inbred strains collected from public databasesare listed in Tables S2 and 5 (See Supplementary TableS2) Overall 968 genetic mutations are compiled forNrf2 gene and 5 kb upstream2 kb downstream regions785 SNPs between B6 and another 16 strains wereacquired from the Mouse Phenome Database (MPDhttpphenomejaxorg dbqrtn=snpret1) and additionalSNPs and other mutations were acquired from NCBI dbSNP(httpwwwncbinlmnihgovsnpterm=mus+musculus20nfe2l2) In total 132 mutations are in the promoter (37in proximal 1 kb) 49 in exons (38 in coding region 19 Cns)727 in introns and 60 in the 31015840 flanking region Excludingmutations in the 51015840 and 31015840 flanking sequences murine Nrf2sequences appear to be more highly variable (1 variationper 375 bp) than much of the mouse genome which has anapproximate frequency of one SNP per every 245 bp (httpwwwinformaticsjaxorgmgihomehomepagesstatsallstatsshtmlallstats snp)
Nrf2 was found to be a susceptibility gene from genome-wide linkage analysis in a murine model of hyperoxia-induced ALI [52] A promoter SNP minus103TgtC (previouslypublished as minus336TgtC) in Nrf2 was found and predicted toadd an additional Sp1 binding site in hyperoxia-susceptibleB6 mice but not in resistant C3HHeJ mice [52] Genotypesfrom the SNP and from simple-sequence length polymor-phism markers of the Nrf2 locus (D2Mit248 and D2Mit94)cosegregated in the B6C3F
2mouse cohort [52] and Nrf2
deficient mice were significantly more susceptible to ALI
sub-phenotypes caused by hyperoxia than similarly exposedwild-type mice supporting Nrf2 as a contributor to thephenotypic traits [53] Although no other functional analyseson Nrf2 SNPs or haplotype association studies have beenconducted in inbred mice strains bearing haplotypes suchas multiple Cns in functional domains (eg F71L L451VH543Q and L575M) may be useful to elucidate the role ofNrf2 in differential susceptibility to oxidative diseases
4 Oncogenic Somatic Mutations in HumanNF-E2-Related Factor 2
Somatic mutation is a change in the DNA of somatic cellsthat affects derived cells but is not inherited by offspringEfforts to discover somatic mutations have provided insightinto mutagenesis and cancer development Lung cancerparticularly non-small cell lung cancer (NSCLC) is theleading cause of cancer death worldwide Somatic mutationsof NRF2 and KEAP1 discovered in lung cancer patients havedetermined the oncogenic potential ofNRF2 [54 55] KEAP1somatic mutations were associated with its reduced proteinlevels in lung cancer tissues and cells [56 57] Investigationsof NSCLC in various ethnic populations as well as cancersin gastrointestine breast and prostate have coordinatelydemonstrated that multiple Cns somatic mutations in KEAP1cause dysfunction of the translated protein and in turnconstitutive activation of NRF2 increasing risk of neoplasiaand chemoresistance [12 55 58 59] Somatic mutations ofNRF2 have been detected in various cancer tissues (largelysquamous cell carcinomas) in Asian populations (Table 6)NRF2 mutations were significantly associated with NSCLCcases (squamous cell lung carcinoma adenocarcinoma) ofthe Japanese (107 [54]) the Chinese (23 [60]) andthe Koreans (8 [61]) as well as with lung cancer celllines Smoking history was also correlated with mutationoccurrence in all of the studies [54 60 61] In addition tolung cancers laryngeal squamous carcinoma (13 in [61])esophageal squamous cancer (ESC 22 in [60] 114 in[61]) head and neck cancers (25 in [54]) skin (117 casein [61]) and oral cancer cell lines had somatic changesin NRF2 In contrast to wide-spread KEAP1 mutationsmutations in NRF2 were clustered in DLGETGE motifs ofthe Neh2 domain which are critical in the ldquohinge and latchrdquomodel of KEAP1 binding [12] Similar to KEAP1 somaticmutations it has been postulated that NRF2 mutations incancer cells lead to NRF2 accumulation by suppressing itsubiquitination or KEAP1 binding which eventually confersmalignant potential and resistance to chemotherapy
Most variable sites in NRF2 included aa residues 29 (AspD) 31 (Gly G) 77 (Asp D) and 79 (Glu E) (Table 6)Residue 33 (Ser S) in the Neh2 domain is mutated by eithergenetic or somatic processes (Figure 4) Cns in the EDGFmotif of NRF2 was experimentally determined to impairrecognition of KEAP1 [54] NRF2 mutations were signif-icantly correlated with increased (25-fold) copy number(31 of mutants versus 3 wild types) in Japanese NSCLCcases [63] Aberrant mutation of NRF2 also led to increasedexpression of downstream effectors includingRagD known to
16 Oxidative Medicine and Cellular Longevity
pL575M
pF71L
pH543QpL451V
pA151P pA151V pN159D pQ198delpV105D
pA204T pS224I225S226del pT253S pS278ApD305G
pM409V pR410HpP388L pA389G pT395I
Genomic DNA (reversed contig GRCm38 p1 C57BL6JNM 0109023)
Protein (NP 035032)1
51101151201251301351401451501551
minus400
minus500
minus50
minus100
minus200
minus300
minus450
minus350
minus250
minus150
minus950
minus900
minus850
minus800
minus750
minus700
151
101151201
2001
g minus470gg minusg minus
c minus41c minus
c
c
c c
gg minus 6C
g minus125insTTC g minus118insC
g minus281delC
g minus63delCg minus1 (75704642 c minus234)
Translation start site (c +1)Termination codon (c +2025 +2027)
c minus136 minus134in-frame stop codonc minus8585
g minus41
246
440
minus381
minus11
minus91 minus90
minus332
minus153
minus98
c minus197c minus209 minus202
gminus942 minus899 (cminus1175 minus1132) CpG islands [71]
g 460CgtT
g minus103CCgtT [51]
CgtTCgtG
g minus320CgtT
CgtT
CgtT
CgtTg minus426GgtA
GgtACgtA
CgtA
GgtA
g minus238GgtTTgtC
TgtC
TgtC
TgtCTgtG
g minus229AgtG
CgtG
CgtG
GgtC
gminus826 minus654 (cminus1059 ) CpG islands [71]minus887
Figure 3 DNA (partial promoter and exons 1 and 5) and protein sequence ofmouseNrf2 SNPs and amino acid residues for non-synonymousSNPs are marked Promoter regions bearing 5CpG islands are underlined
151
201251301351401451501551601
Genetic mutation (Table 3 and Figure 2)Somatic mutation (Table 6)
Neh2
Neh3
Neh4Neh5
Neh6
Neh1
Protein isoform 1 (NP 006155)
101151
Figure 4 Genetic and somatic mutation loci in human NRF2 protein
Oxidative Medicine and Cellular Longevity 17
Table5Mou
seup
stream
andexon
varia
tions
ofNfe2
l2locusin17
inbred
strainsR
eference
sequ
ence
isC5
7BL6J
(B6strain
1)geno
me(GI14933824975547698ndash75513576)SNPalleleand
geno
typesareshow
nas
chromosom
econ
tigsequ
enceStrains
2129S1S
vlmJ3AJ
4AKR
J5BA
LBcJ6C3
HH
eJ7C
57BL
6NJ8CA
STEiJ9CB
AJ
10D
BA2J11FVBNJ12LPJ
13N
ODShiLtJ14N
ZOH
ILtJ
15P
WK
PhJ16SPR
ETEiJ
17W
SBEiJ
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs2566
08517
75705565
GA
(CT)
51015840Flankingminus924
NC
GG
GG
GG
GG
GG
GG
GG
AA
Grs47274959
75705562
AT
(TA
)51015840Flankingminus921
NC
AT
TA
TT
AA
TT
AT
AT
TT
Ars216940398
75705554ndash75705555
Tadd(A
add)
51015840Flankingminus913minus914
NC
CGrs239114134
75705540
CT(G
A)
51015840Flankingminus899
NC
CT
TC
TT
CC
TT
CT
CT
CT
Crs46
461765
75705528
TG(A
C)oA
C
51015840Flankingminus887
NC
TG
GT
GG
TT
GG
TG
TG
GG
Trs5144
9853
75705525
CA(G
T)
51015840Flankingminus884
NC
CA
AC
AA
CC
AA
CA
CA
CC
Crs249093111
75705512
CT(G
A)
51015840Flankingminus871
NC
CT
TC
TT
CC
TT
CT
CT
TT
Crs263745200
75705510
CG(G
C)
51015840Flankingminus869
NC
GG
GG
GG
GG
GG
GG
GG
CG
Grs45651867
75705498
TG(A
C)
51015840Flankingminus857
NC
TG
GT
GG
TT
GG
TG
TG
GG
Trs219997531
75705495
GA
(CT)
51015840Flankingminus854
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs251918379
75705451
TA(A
T)
51015840Flankingminus810
NC
TT
TT
TT
TT
TT
TT
TT
TA
Trs27978306
75705449
TA(A
T)
51015840Flankingminus808
NC
TA
AT
AA
TT
AA
TA
TA
TT
Trs216087412
75705429
TG(A
C)lowast
51015840Flankingminus788
NC
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
GTlowast
Tlowast
rs261229914
75705423ndash75705424
AGdel(CT
del)
51015840Flankingminus783minus784
NC
AGrs27978307
75705400
CT(G
A)
51015840Flankingminus759
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs243167395
75705359
GA
(CT)
51015840Flankingminus718
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs27978308
75705307
GA
(CT)
51015840Flankingminus66
6NC
GG
GG
GG
GG
GG
GG
GG
AG
Grs254744
09875705294
AT
(TA
)51015840Flankingminus653
NC
AA
AA
AA
AA
AA
AA
AA
AT
Ars228133419
75705179
GT
(CA
)51015840Flankingminus538
NC
GG
GG
GG
GG
GG
GG
GG
GT
Grs214719520
75705111
GA
(CT)
51015840Flankingminus470
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs2440
87730
75705101
GA
(CT)
51015840Flankingminus46
0NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs227781699
75705081
CG(G
C)
51015840Flankingminus44
0NC
CC
CC
CC
CC
CC
CC
CC
GC
Crs257870353
75705067
CT(G
A)
51015840Flankingminus426
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs24487744
075705022
AG
(TC)
51015840Flankingminus381
NC
AA
AA
AA
AA
AA
AA
AA
AG
Ars234628138
75704973
AC
(TG
)51015840Flankingminus332
NC
AA
AA
AA
AA
AA
AA
AA
AC
A
18 Oxidative Medicine and Cellular Longevity
Table5Con
tinued
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs247275247
75704961
GA
(CT)
51015840Flankingminus320
NC
GG
GG
GG
GG
GG
GG
AG
GG
Grs247519047
75704922
Gdel(Cdel)
51015840Flankingminus281
NC
Grs27978309
(rs51915758)
75704887
GA
C(C
TG
)51015840Flankingminus246
NC
GA
AG
AA
GG
AA
GA
GA
CC
G
rs218139102
75704879
CA(G
T)
51015840Flankingminus238
NC
CC
CC
CC
CC
CC
CC
CC
AC
Crs251990355
75704870
TC(A
G)
51015840Flankingminus229
NC
TT
TT
TT
TT
TT
TT
TT
TC
Trs238746955
75704794
AG
(TC)
51015840Flankingminus153
NC
AA
AA
AA
AA
AA
AA
AA
GA
Ars221664
405
75704786
AG
(TC)
51015840Flankingminus145
NC
AA
AA
AA
AA
AA
AA
AA
AG
Ars217054035
75704766
ndash75704767
GAAadd(TTC
add)
51015840Flankingminus125minus126
NC
GA
rs248182931
75704759ndash75704760
Gadd(C
add)
51015840Flankingminus118
minus119
NC
AGrs217197904
75704744
GA
(CT)
51015840Flankingminus103
(+)S
p1[23]
GA
AG
AA
GG
AA
GA
AA
GG
Ars2644
9364975704704
Gdel(Cdel)
51015840Flankingminus63
NC
Grs2404
81112
757046
82CT(G
A)
51015840Flankingminus41
NC
CC
CC
CC
CC
CC
CC
CC
CT
Crs27978310
75704617
AG
(TC)
UTR
-525
NC
AA
AA
AA
AA
AA
AA
AA
GG
Ars27978311
75704610
CG(G
C)
UTR
-532
NC
GG
GG
GG
GG
GG
GG
GG
GG
Crs27978312
757046
05GA
(CT)
UTR
-537
NC
GA
AG
AA
GG
AA
GA
GA
GG
Grs214784220
757044
99AG
(TC)
UTR
-5143
NC
AA
AA
AA
AA
AA
AA
AA
GG
Ars244146318
757044
98GT
(CA
)UTR
-5144
NC
GG
GG
GG
GG
GG
GG
GG
GT
Grs27978313
757044
97GC
(CG
)UTR
-5145
NC
GC
CG
CC
GC
CC
GC
CC
CC
Crs215431944
757044
93CT(G
A)
UTR
-5149
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs257015697
757044
49GA
(CT)
UTR
-5193
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs252234782
757044
19GT
(CA
)UTR
-5223
NC
Grs1346
0861
75679262
AG
(TC)
Exon
244
6Cn
sF71L
AG
GA
GG
AG
GG
AG
AG
GG
Grs1346
0859
75679202
GA
(CT)lowast
Exon
2506
CsH91=
GA
AG
AA
GG
AA
GA
GA
GG
Grs27978436
75679187
GA
(CT)lowast
Exon
2521
CsT9
6=G
GG
GG
GG
AG
GG
GG
GA
GG
rs226131070
75679178
GA
(CT)
Exon
2530
CsS99=
GG
GG
GG
GG
GG
GG
GG
GA
Grs227683834
75678576
AT
(TA
)Ex
on3
547
CnsV
105D
AA
AA
AA
AA
AA
AA
AA
AT
Ars257321187
75678506
TC(A
G)
Exon
3617
CsP128=
TT
TT
TT
TC
TT
TT
TT
TT
Trs27978444
75678500
TA(A
T)
Exon
3623
CsV130=
TT
TT
TT
TA
TT
TT
TT
AT
Trs227743136
75677686
GA
(CT)
Exon
4662
CsH143=
GG
GG
GG
GG
GG
GG
GG
GA
Grs215327202
75677664
CG(G
C)
Exon
4684
CnsA
151P
CC
CC
CC
CG
CC
CC
CC
CC
Crs247539755
75677663
GA
(CT)
Exon
4685
CnsA
151V
GG
GG
GG
GA
GG
GG
GG
GG
Grs233164
668
75677640
TC(A
G)
Exon
4708
CnsN
159D
TT
TT
TT
TT
TT
TT
TT
TC
T
rs2340
95816
75677162ndash75677160
GCT
del
(AGCdel)
Exon
5826minus
828
Cds-in
delQ
198
GCT
Oxidative Medicine and Cellular Longevity 19
Table5Con
tinued
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs27978452
75677145
CT(G
A)
Exon
5843
CnsA
204T
CC
CC
CC
CC
CC
CC
CC
CC
T
rs225047937
75677086ndash75677078
GAG
ATCG
ATdel
(ATG
CATC
TCdel)
Exon
5902minus
910
Cds-in
del
(S224I225S226)
GAG
ATC
GAT
rs241469868
75676998
TA(G
T)
Exon
5990
CnT2
53S
TT
TT
TT
TT
TT
TT
TT
TA
Trs221602571
75676923
AC
(TG
)Ex
on5
1065
CnsS
278A
AA
AA
AA
AA
AA
AA
AA
AC
Ars252576472
75676876
GA
(CT)
Exon
51112
CsS293=
GG
GG
GG
GG
GG
GG
GG
GA
Grs250802933
75676841
TC(A
G)
Exon
5114
7Cn
sD305G
TT
TT
TT
TT
TT
TT
TT
TC
Trs225425698
75676792
CT(G
A)
Exon
5119
6Cs
P321=
CC
CC
CC
CC
CC
CC
CC
CT
Crs13459064
75676732
CT(G
A)
Exon
51256
CsT3
41=
CT
TC
TT
CT
TT
CT
CT
TC
Crs27978453
75676717
CT(G
A)
Exon
51271
CsA346=
CC
CC
CC
CC
CC
CC
CC
TC
Crs214335034
75676636
AG
(TC)
Exon
51352
CsD373=
AA
AA
AA
AA
AA
AA
AA
AG
Ars24958364
4756766
03AC
(TG
)Ex
on5
1385
CsP3
84=
AA
AA
AA
AA
AA
AA
AA
AC
Ars27978454
75676592
GA
(CT)
Exon
51396
CnsP
388L
GG
GG
GG
GG
GG
GG
GG
AG
Grs215384328
75676589
GC
(CG
)Ex
on5
1399
CnsA
389G
GG
GG
GG
GG
GG
GG
GG
GC
Grs251728286
75676571
GA
(CT)
Exon
51417
CnsT
395I
Grs247602334
75676567
TC(A
G)
Exon
51421
CsV396=
TT
TT
TT
TT
TT
TT
TT
TC
Trs234216231
75676530
TC(A
G)
Exon
51458
CnsM
409V
TT
TT
TT
TT
TT
TT
TT
TC
Trs212904337
75676526
CT(G
A)
Exon
51462
CnsR
410H
CC
CC
CC
CC
CC
CC
CC
CT
Crs252650779
75676522
TC(A
G)
Exon
51466
CsE4
11=
TT
TT
TT
TT
TT
TT
TT
TC
Trs231273560
75676516
TC(A
G)
Exon
51472
CsQ413=
TT
TT
TT
TT
TT
TT
TT
TC
Trs257886949
756764
13GT
(CA
)Ex
on5
1575
CsR4
48=
GG
GG
GG
GG
GG
GG
GG
GT
Grs241537608
756764
04GC
(CG
)Ex
on5
1584
CnsL
451V
GG
GG
GG
GG
GG
GG
GG
GC
Grs227619071
75676312
TC(A
G)
Exon
51676
CsQ481=
TT
TT
TT
TT
TT
TT
TT
TC
Trs258913831
75676290
AG
(TC)
Exon
51698
CsL4
89=
AA
AA
AA
AA
AA
AA
AA
AG
Ars225593319
75676126
AT
(TA
)Ex
on5
1862
CnsH
543Q
Ars4223233
75676105
GA
(CT)
Exon
51883
CsS550=
GG
GG
GG
GA
GG
GG
GG
GG
Grs4223232
75676032
GT
(CA
)Ex
on5
1956
CnsL
575M
Grs4223231
75675816
CG
UTR
-32172
NC
GG
GG
GG
GG
GG
GG
GG
GC
Grs1346
860
75675682
AT
UTR
-32306
NC
ASequ
ence
varia
tions
inmou
seNr
f2wereob
tained
from
Mou
sePh
enom
eDatabase(http
ph
enom
ejaxorgSNP)
andNCB
ISNPdatabase
(http
wwwncbinlm
nihgovSNP)N
CBIreference
sequ
ence
isMus
Musculusstra
inC5
7BL6J
chromosom
e2GRC
m38p1(NC
0000
687
GI37209910875704641ndash756755192
9123
bp)To
tal968
genetic
mutations
arerepo
rted
inNr
f2gene
and5k
bup
stream
(ge75704
642minus
1)and2k
bdo
wnstre
am(le75675512)sequences
asof
Janu
ary2003A
llSN
Psandallelesa
representedas
geno
miccontig
(reversedsequ
ence
indicated)E
xon175704
641ndash75704364(278
bpT
TS=75704
408)
exon
275679431ndash756791
65(267
bp)exon
375678579ndash75678490(90b
p)exon475677713ndash756775
46(168
bp)andexon
57567718
4ndash75678849
(1666
bp)Proteinam
inoacid
(aa)
resid
uesinNP00
61552
(597
aa)Cs
cod
ing-syno
nymou
sCn
scoding
-non
syno
nymou
sAminoacid
A-alanineD-aspartic
acidE
-glutamicacidF-phenylalanineG
-glycineH
-histidineI-iso
leucineL-leu
cineand
M-m
ethion
ineN-
asparagineP-prolin
eQ-glutamineR-arginineS-serineT-threon
ineandV-valin
elowastErrorsin
databasesfi
xedNC
notcon
firmed
20 Oxidative Medicine and Cellular Longevity
Table 6 NRF2 somatic mutations revealed in various human cancers
Domain Amino acid residues DNA mutation Cancer types (cases) ReferencesLocus Wild type Mutant
DLG motif
24 W (Trp) C (Cys) c72GgtCGgtT NSCLC neck ESC [54 62]K (Lys) c72TgtC ESC [62]
26 Q (Gln) E (Glu) c76CgtG NSCLC ESC [54 62]
27 D (Asp) G (Gly)lowast c80GgtA NSCLC [60]Y (Tyr) c79GgtT ESC [61]
28 I (Ile) T (Thr) c83CgtT NSCLC [54]
29 D (Asp) G (Gly) c86AgtG Head and neck ESC [54 62]H (His) c85GgtC NSCLC laynx [60 61]
30 L (Leu) F (Phe) c88CgtT NSCLC ESC [54 62]
31 G (Gly) A (Ala) c92GgtC NSCLC ESC skin [54 60ndash62]
32 V (Val) T (Thr) c95TgtG NSCLC [54]del c93 95delAGT ESC [61]
33ndash36 S-R-E-V S-R-E-V-S-R-E-Vlowast c97 108dupAGTCGAGCCGTA ESC [61]
34 R (Arg) Q (Gln) c101GgtA NCSCL [54 60 61]P (Pro) c101GgtC NSCLC [63]
ETGF motif
75 Q (Gln) H (His) c225AgtC Head and neck ESC [54 62]
77 D (Asp)
V (Val) c230AgtT NSCLC ESC [54 60 62]G (Gly) c230AgtG ESC [62]A (Ala) c230AgtC NSCLC [61]N (Asn) c229GgtA Larynx [61]
78 E (Glu) K (Lys) c232GgtA NSCLC ESC [54 62]
79 E (Glu)
K (Lys) c235GgtA NSCLC ESC [54 61 62]Q (Gln) c235GgtC NSCLC ESC [54 60ndash62]G (Gly) c236AgtG Larynx [61]E-E c234 236dupAGA ESC [61]
80 T (Thr) K (Lys) c239CgtACgtG NSCLC ESC [54 61 62]
80 T (Thr)P (Phe) c238AgtC ESC [62]I (Ile) c239CgtT Head and neck [54]A (Ala) c238AgtG NSCLC [63]
81 G (Gly) V (Val) c242GgtT ESC [61]D (Asp) c242GgtA NSCLC ESC [60ndash62]
82 E (Glu)
D (Asp) c246AgtT ESC oral cancer cell line [54 62]G (Gly) c245AgtG NSCLC [54]Q (Gln)lowast c244GgtC NSCLC ESC [60 61]V (Val)lowast c245AgtT ESC [61]
83 F (Phe) L (Leu)lowast c247TgtC NSCLC [60]NSCLC non-small cell lung cancer ESC esophageal squamous cancer and lowasterrors in reference fixed Number of cases 82 NSCLC and 10 ESC in [62] 125NSCLC 70 ESC 23 larynx and 17 skin in [61] 103 NSCLC and 12 head and neck in [54] 90 NSCLC in [63] 103 NSCLC in [60]
be involved in squamous lung cancer cell proliferation [64]suggesting that the mutation is functional and overcomesKEAP1 inhibition Singh et al [65] determined in vitrothat RNAi-mediated depletion of NRF2 in lung cancer cellsenhanced ROS production and susceptibility to cell deathby ionizing radiation These studies support the concept thatelevated NRF2 and ARE responsiveness provides cancer cellswith proliferative advantage for malignant transformation
and undue protection from anti-cancer therapy Onco-genic epidermal growth factor receptor (EGFR) signaling isrecently found to be critical in NRF2-mediated proliferationof NSCLC cells [66]
Collectively ldquogain of functionrdquo mutations in NRF2 thatreduce KEAP1 recognition are suggested to be predictivemarkers for poor responsiveness to chemotherapy and radi-ation therapy Although NRF2-mediated cellular defense
Oxidative Medicine and Cellular Longevity 21
processes are essential in the initiation stage enhancedNRF2-ARE activity in advanced stages of cancer developmentmay create a favorable intracellular environment for tumorcell growth and survival [67 68] In this context NRF2may be a potential molecular target for the treatment ofradio-resistance cancers especially those that have ldquoloss offunctionrdquo mutations in EGFR KRAS or KEAP1 as well asldquogain of functionrdquo mutations in NRF2
5 Epigenetic Alterations of NF-E2-RelatedFactor 2
Epigenetic modifications are alterations of molecules inter-acting with genes without changes to the primary DNAsequence They include post-translational modification ofhistones DNA methylation events chromatin conforma-tional changes and alterations to noncoding regulatoryRNAs Epigenetic alterations are stable and often inheritablebut are reversible and may affect expression of the geneDysregulation or defects in epigenetic processes particularlyhypermethylation of tumor suppressor gene promoters (egCpG islands) or histone modifications are thought to beassociated with carcinogenesis Investigators have reportedhypermethylation in CpG islands of KEAP1 which wereassociated with reduced KEAP1 expression in human cancersfrom lung prostate colon and so forth [69ndash72] Similar tosomatic mutations epigenetic changes on KEAP1 impairedthe function of its encoded protein leading to constitutiveNRF2 activation
Supporting the role of ldquopathogenic mutationsrdquo in NRF2expression of Nrf2 and downstream Nqo1 was suppressedin prostate tumors of mice (transgenic adenocarcinomaof mouse prostate TRAMP) Among 15 promoter CpGislands located between minus942 and minus654 (cminus1175 cminus1132and cminus1059 cminus887 gap in cminus1131 cminus1060 see Figure 3)hypermethylation of the first 5 CpG islands (minus942 minus899 andcminus1175 cminus1132) was significantly associated with tumorige-nesis [73] Moreover treatment with inhibitors for DNAmethyltransferase and histone deacetylase restored Nrf2expression in these tumor cells [73] A dietary phytochemicalcurcumin known as a DNA hypomethylation agent restoredepigenetically silent Nrf2 expression through CpG demethy-lation in carcinogen-induced mouse tumor cells [74]
The whole genome epigenetic datasets for 5 species arepublicly accessible at NCBI Epigenomics [75 76]The humanNRF2 epigenome of primary cells (breast penis) and H1stem cell line as well as mouse Nrf2 CpG island methylationdata for sperm blood and cerebellum are currently avail-able (httpwwwncbinlmnihgovepigenomics) Althoughno direct evidence of disease-associated epigenetic modu-lation has been identified in human NRF2 various phyto-chemical NRF2 agonists such as sulforaphane and curcuminhave shown their roles in DNA methylation and histonemodification (see reviews by Lee and colleagues eg [77])Taken together epigenetic modifications of theNRF2KEAP1axle are predicted to cause dysregulation of ARE-mediatedcellular defense leading to deleterious health effects and
phytochemical antioxidants as epigenetic modulators forNRF2 are suggested to be useful in cancer prevention
6 Conclusions
NRF2 is evolutionally conserved with high-sequence homol-ogy in many species However it is a highly mutable geneand numerous genetic variants have been discovered inhuman ethnic groups Importantly certain SNPs or haplo-types have been identified in various diseases as ldquoat-riskrdquoalleles and are related to functional alterations In additionto genetic variations multiple somatic mutations identifiedin the KEAP1 recognition domain of NRF2 in cancer cellshave been found to be oncogenic due to dysregulation ofNRF2 homeostasis by its excess ldquogain of functionrdquo Epigeneticalteration of theNRF2 is under investigation and is predictedto have pathogenic influences as learned from mouse andphytochemical agonist studies Continuous updates of Nrf2allelic variants in inbred mouse strains will provide a usefultool for effective experimental designs formodels of oxidativedisorders to provide insight into the disease mechanisms andintervention strategies
Abbreviations
AD Alzheimerrsquos diseaseALI acute lung injuryARE antioxidant response elementbZIP basic leucine zippercds coding DNA sequencesCI confidence intervalCOPD chronic obstructive pulmonary diseaseESC esophageal squamous cancerFEV1 forced expiratory volume in one second
Gbp giga base pairsGST glutathione S-transferaseGWAS genome-wide association studyHGVS Human Genome Variation SocietyHO-1 heme oxygenase-1119867 119901119910119897119900119903119894 119867119890119897119894119888119900119887119886119888119905119890119903 119901119910119897119900119903119894KEAP1 Kelch-like ECH activating protein 1MPD Mouse Phenome DatabaseMRP multidrug resistance proteinNCBI National Center for Biotechnology InformationNeh NRF2-ECH homologyNfe2l2 nuclear factor (erythroid-derived 2)-like 2Nrf2 NF-E2-related factor 2NSCLC nonsmall cell lung cancerNQO1 NAD(P)Hquinone oxidoreductase 1OR odds ratioPD Parkinsonrsquos diseasePM particulate matterROS reactive oxygen speciesSLE systemic lupus erythematosusSNP single nucleotide polymorphismSOD superoxide dismutaseURT untranslated region
22 Oxidative Medicine and Cellular Longevity
Disclosure
Authorrsquos contribution to the Work was done as part of theAuthorrsquos official duties as a NIH employee and is a Work ofthe United States Government Therefore copyright may notbe established in the United States
Conflict of Interests
The author declares that there is no conflict of interests
Acknowledgments
The research related to this paper was supported by theIntramural Research Program of the National Institutes ofHealth of the National Institute of Environmental HealthSciences (NIEHS) Drs Steven Kleeberger and StephanieLondon at the NIEHS provided excellent critical review ofthis paper The author thanks Mrs Jacqui Marzec for herhelpful comments and English editing
References
[1] J Y Chan M C Cheung P Moi K Chan and Y W KanldquoChromosomal localization of the humanNF-E2 family of bZIPtranscription factors by fluorescence in situ hybridizationrdquoHuman Genetics vol 95 no 3 pp 265ndash269 1995
[2] K Itoh K Igarashi N Hayashi M Nishizawa and MYamamoto ldquoCloning and characterization of a novel erythroidcell-derived CNC family transcription factor heterodimerizingwith the small Maf family proteinsrdquo Molecular and CellularBiology vol 15 no 8 pp 4184ndash4193 1995
[3] W O Osburn and T W Kensler ldquoNRF2 signaling an adaptiveresponse pathway for protection against environmental toxicinsultsrdquoMutation Research vol 659 no 1-2 pp 31ndash39 2008
[4] D Papp K Lenti D Modos D Fazekas Z Dul D Tureiet al ldquoThe NRF2-related interactome and regulome containmultifunctional proteins and fine-tuned autoregulatory loopsrdquoFEBS Letters vol 586 no 13 pp 1795ndash1802 2012
[5] X Wang D J Tomso B N Chorley et al ldquoIdentificationof polymorphic antioxidant response elements in the humangenomerdquo Human Molecular Genetics vol 16 no 10 pp 1188ndash1200 2007
[6] D Malhotra E Portales-Casamar A Singh et al ldquoGlobalmapping of binding sites for NRF2 identifies novel targets incell survival response through ChIP-Seq profiling and networkanalysisrdquo Nucleic Acids Research vol 38 no 17 pp 5718ndash57342010
[7] K Chan R Lu J C Chang and Y W Kan ldquoNRF2 a memberof the NFE2 family of transcription factors is not essential formurine erythropoiesis growth and developmentrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 93 no 24 pp 13943ndash13948 1996
[8] K Itoh T Chiba S Takahashi et al ldquoAn NRF2small Mafheterodimer mediates the induction of phase II detoxifyingenzyme genes through antioxidant response elementsrdquo Bio-chemical and Biophysical Research Communications vol 236no 2 pp 313ndash322 1997
[9] FMartin J M vanDeursen R A Shivdasani CW Jackson AG Troutman and P A Ney ldquoErythroid maturation and globin
gene expression inmicewith combined deficiency ofNF-E2 andNrf-2rdquo Blood vol 91 no 9 pp 3459ndash3466 1998
[10] K Itoh N Wakabayashi Y Katoh et al ldquoKEAP1 repressesnuclear activation of antioxidant responsive elements by NRF2through binding to the amino-terminal Neh2 domainrdquo Genesand Development vol 13 no 1 pp 76ndash86 1999
[11] A Kobayashi M I Kang H Okawa et al ldquoOxidative stresssensor KEAP1 functions as an adaptor for Cul3-based E3 ligaseto regulate proteasomal degradation of NRF2rdquo Molecular andCellular Biology vol 24 no 16 pp 7130ndash7139 2004
[12] K Taguchi H Motohashi and M Yamamoto ldquoMolecularmechanisms of the KEAP1-NRF2 pathway in stress responseand cancer evolutionrdquo Genes to Cells vol 16 no 2 pp 123ndash1402011
[13] K Mukaigasa L T Nguyen L Li H Nakajima M Yamamotoand M Kobayashi ldquoGenetic evidence of an evolutionarilyconserved role for NRF2 in the protection against oxidativestressrdquoMolecular and Cellular Biology vol 32 no 21 pp 4455ndash4461 2012
[14] JMaher andMYamamoto ldquoThe rise of antioxidant signalingmdashthe evolution and hormetic actions of NRF2rdquo Toxicology andApplied Pharmacology vol 244 no 1 pp 4ndash15 2010
[15] S C Lo X Li M T Henzl L J Beamer and M HanninkldquoStructure of the KEAP1NRF2 interface provides mechanisticinsight intoNRF2 signalingrdquoThe EMBO Journal vol 25 no 15pp 3605ndash3617 2006
[16] V A McKusick ldquoMendelian inheritance in man and its onlineversion OMIMrdquo American Journal of Human Genetics vol 80no 4 pp 588ndash604 2007
[17] B Yalcin D J Adams J Flint and T M Keane ldquoNext-generation sequencing of experimental mouse strainsrdquo Mam-malian Genome vol 23 no 9-10 pp 490ndash498 2012
[18] D R Bentley S Balasubramanian H P Swerdlow et alldquoAccurate whole human genome sequencing using reversibleterminator chemistryrdquo Nature vol 456 no 7218 pp 53ndash592008
[19] P S G Chain D V Grafham R S Fulton et al ldquoGenomeproject standards in a new era of sequencingrdquo Science vol 326no 5950 pp 236ndash237 2009
[20] RHWaterstonK Lindblad-Toh E Birney J Rogers J F AbrilP Agarwal et al ldquoInitial sequencing and comparative analysis ofthe mouse genomerdquo Nature vol 420 pp 520ndash562 2002
[21] T M Keane L Goodstadt P Danecek M A White K WongB Yalcin et al ldquoMouse genomic variation and its effect onphenotypes and gene regulationrdquoNature vol 477 pp 289ndash2942011
[22] T Yamamoto K Yoh A Kobayashi et al ldquoIdentification ofpolymorphisms in the promoter region of the human NRF2generdquo Biochemical and Biophysical Research Communicationsvol 321 no 1 pp 72ndash79 2004
[23] J M Marzec J D Christie S P Reddy et al ldquoFunctionalpolymorphisms in the transcription factor NRF2 in humansincrease the risk of acute lung injuryrdquo The FASEB Journal vol21 no 9 pp 2237ndash2246 2007
[24] H Fukushima-Uesaka Y Saito K Maekawa et al ldquoGeneticvariations and haplotype structures of transcriptional factorNRF2 and its cytosolic reservoir protein KEAP1 in JapaneserdquoDrug Metabolism and Pharmacokinetics vol 22 no 3 pp 212ndash219 2007
[25] E J Cordova R Velazquez-Cruz F Centeno V Baca and LOrozco ldquoThe NRF2 gene variant -653GA is associated with
Oxidative Medicine and Cellular Longevity 23
nephritis in childhood-onset systemic lupus erythematosusrdquoLupus vol 19 no 10 pp 1237ndash1242 2010
[26] M von Otter S Landgren S Nilsson et al ldquoAssociation ofNRF2-encoding NFE2L2 haplotypes with Parkinsonrsquos diseaserdquoBMCMedical Genetics vol 11 no 1 article 36 2010
[27] JMHartikainenM TengstromVMKosmaV L Kinnula AMannermaa andY Soini ldquoGenetic polymorphisms and proteinexpression of NRF2 and sulfiredoxin predict survival outcomesin breast cancerrdquo Cancer Research vol 72 pp 5537ndash5546 2012
[28] T Arisawa T Tahara T Shibata et al ldquoThe relationshipbetween Helicobacter pylori infection and promoter polymor-phism of the NRF2 gene in chronic gastritisrdquo InternationalJournal of Molecular Medicine vol 19 no 1 pp 143ndash148 2007
[29] T Arisawa T Tahara T Shibata et al ldquoThe influence ofpromoter polymorphism of nuclear factor-erythroid 2-relatedfactor 2 gene on the aberrant DNA methylation in gastricepitheliumrdquo Oncology Reports vol 19 no 1 pp 211ndash216 2008
[30] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene pro-moter polymorphism and gastric carcinogenesisrdquo Hepato-Gastroenterology vol 55 no 82-83 pp 750ndash754 2008
[31] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene promoterpolymorphism is associated with ulcerative colitis in a Japanesepopulationrdquo Hepato-Gastroenterology vol 55 no 82-83 pp394ndash397 2008
[32] C C Hua L C Chang J C Tseng C M Chu Y C Liu andW B Shieh ldquoFunctional haplotypes in the promoter region oftranscription factor NRF2 in chronic obstructive pulmonarydiseaserdquo Disease Markers vol 28 no 3 pp 185ndash193 2010
[33] S O Shaheen R B Newson S M Ring M J Rose-ZerilliJ W Holloway and A J Henderson ldquoPrenatal and infantacetaminophen exposure antioxidant gene polymorphismsand childhood asthmardquo The Journal of Allergy and ClinicalImmunology vol 126 no 6 pp 1141e7ndash1148e7 2010
[34] H Masuko T Sakamoto Y Kaneko et al ldquoLower FEV1 innon-COPD nonasthmatic subjects association with smokingannual decline in FEV1 total IgE levels and TSLP genotypesrdquoInternational Journal of Chronic Obstructive Pulmonary Diseasevol 6 pp 181ndash189 2011
[35] C P Guan M N Zhou A E Xu et al ldquoThe susceptibilityto vitiligo is associated with NF-E2-related factor 2 (NRF2)gene polymorphisms a study on Chinese Han populationrdquoExperimental Dermatology vol 17 no 12 pp 1059ndash1062 2008
[36] J Bouligand O Cabaret M Canonico et al ldquoEffect of NFE2L2genetic polymorphism on the association between oral estrogentherapy and the risk of venous thromboembolism in post-menopausal womenrdquo Clinical Pharmacology amp Therapeuticsvol 89 no 1 pp 60ndash64 2011
[37] D S OrsquoMahony B J Glavan T D Holden C Fong R A BlackG Rona et al ldquoInflammation and immune-related candidategene associations with acute lung injury susceptibility andseverity a validation studyrdquo PLoS ONE vol 7 no 12 Article IDe51104 2012
[38] I Ungvari E Hadadi V Virag et al ldquoRelationship between airpollutionNFE2L2 gene polymorphisms and childhood asthmain aHungarian populationrdquo Journal of CommunityGenetics vol3 no 1 pp 25ndash33 2012
[39] M SiedlinskiD S Postma JMA Boer et al ldquoLevel and courseof FEV1 in relation to polymorphisms inNFE2L2 andKEAP1 inthe general populationrdquo Respiratory Research vol 10 article 732009
[40] C C Hong C B Ambrosone J Ahn et al ldquoGenetic variabilityin iron-related oxidative stress pathways (NRF2 NQ01 NOS3
and HO-1) iron intake and risk of postmenopausal breastcancerrdquo Cancer Epidemiology Biomarkers and Prevention vol16 no 9 pp 1784ndash1794 2007
[41] C Canova C Dunster F J Kelly C Minelli P L Shah CCaneja et al ldquoPM10-induced hospital admissions for asthmaand chronic obstructive pulmonary disease the modifyingeffect of individual characteristicsrdquo Epidemiology vol 23 no 4pp 607ndash615 2012
[42] A J Henderson R B Newson M Rose-Zerilli S M RingJ W Holloway and S O Shaheen ldquoMaternal NRF2 andgluthathione-S-transferase polymorphisms donotmodify asso-ciations of prenatal tobacco smoke exposure with asthma andlung function in school-aged childrenrdquo Thorax vol 65 no 10pp 897ndash902 2010
[43] C Xing A L Sestak J A Kelly et al ldquoLocalization andreplication of the systemic lupus erythematosus linkage signalat 4p16 interaction with 2p11 12q24 and 19q13 in EuropeanAmericansrdquo Human Genetics vol 120 no 5 pp 623ndash631 2007
[44] T F Wong K Yoshinaga Y Monma K Ito H NiikuraS Nagase et al ldquoAssociation of KEAP1 and NRF2 geneticmutations and polymorphisms with endometrioid endometrialadenocarcinoma survivalrdquo International Journal of Gynecologi-cal Cancer vol 21 no 8 pp 1428ndash1435 2011
[45] M von Otter S Landgren S Nilsson et al ldquoNRF2-encodingNFE2L2 haplotypes influence disease progression but not riskin Alzheimerrsquos disease and age-related cataractrdquoMechanisms ofAgeing and Development vol 131 no 2 pp 105ndash110 2010
[46] S Tsang Z Sun B Luke et al ldquoA comprehensive SNP-basedgenetic analysis of inbred mouse strainsrdquoMammalian Genomevol 16 no 7 pp 476ndash480 2005
[47] C M Wade E J Kulbokas III A W Kirby et al ldquoThe mosaicstructure of variation in the laboratory mouse genomerdquoNaturevol 420 no 6915 pp 574ndash578 2002
[48] T Wiltshire M T Pletcher S Batalov et al ldquoGenome-widesingle-nucleotide polymorphism analysis defines haplotypepatterns in mouserdquo Proceedings of the National Academy ofSciences of the United States of America vol 100 no 6 pp 3380ndash3385 2003
[49] K A Frazer E Eskin H M Kang et al ldquoA sequence-basedvariation map of 827 million SNPs in inbred mouse strainsrdquoNature vol 448 no 7157 pp 1050ndash1053 2007
[50] T P Maddatu S C Grubb C J Bult andM A Bogue ldquoMousephenome database (MPD)rdquo Nucleic Acids Research vol 40 ppD887ndashD894 2007
[51] A Kirby H M Kang C M Wade et al ldquoFine mapping in 94inbred mouse strains using a high-density haplotype resourcerdquoGenetics vol 185 no 3 pp 1081ndash1095 2010
[52] H Y Cho A E Jedlicka S P M Reddy L Y Zhang T WKensler and S R Kleeberger ldquoLinkage analysis of susceptibilityto hyperoxia NRF2 is a candidate generdquo American Journal ofRespiratory Cell and Molecular Biology vol 26 no 1 pp 42ndash512002
[53] H Y Cho A E Jedlicka S P M Reddy et al ldquoRole of NRF2in protection against hyperoxic lung injury in micerdquo AmericanJournal of Respiratory Cell and Molecular Biology vol 26 no 2pp 175ndash182 2002
[54] T Shibata T Ohta K I Tong et al ldquoCancer related mutationsin NRF2 impair its recognition by KEAP1-Cul3 E3 ligase andpromote malignancyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 36 pp13568ndash13573 2008
24 Oxidative Medicine and Cellular Longevity
[55] A Singh V Misra R K Thimmulappa et al ldquoDysfunctionalKEAP1-NRF2 interaction in non-small-cell lung cancerrdquo PLoSMedicine vol 3 no 10 article e420 2006
[56] T Ohta K Iijima M Miyamoto et al ldquoLoss of KEAP1 functionactivates NRF2 and provides advantages for lung cancer cellgrowthrdquo Cancer Research vol 68 no 5 pp 1303ndash1309 2008
[57] B Padmanabhan K I Tong T Ohta et al ldquoStructural basisfor defects of KEAP1 activity provoked by its point mutationsin lung cancerrdquoMolecular Cell vol 21 no 5 pp 689ndash700 2006
[58] T Shibata A Kokubu M Gotoh et al ldquoGenetic alteration ofKEAP1 confers constitutive NRF2 activation and resistance tochemotherapy in gallbladder cancerrdquoGastroenterology vol 135no 4 pp 1358e4ndash1368e4 2008
[59] N J Yoo H R Kim Y R Kim C H An and S H LeeldquoSomatic mutations of the KEAP1 gene in common solidcancersrdquo Histopathology vol 60 no 6 pp 943ndash952 2012
[60] Y Hu Y Ju D Lin Z Wang Y Huang S Zhang et alldquoMutation of the NRF2 gene in non-small cell lung cancerrdquoMolecular Biology Reports vol 39 no 4 pp 4743ndash4747 2012
[61] Y R Kim J EOhM S Kim et al ldquoOncogenicNRF2mutationsin squamous cell carcinomas of oesophagus and skinrdquo TheJournal of Pathology vol 220 no 4 pp 446ndash451 2010
[62] T Shibata A Kokubu S Saito et al ldquoNRF2 mutation confersmalignant potential and resistance to chemoradiation therapyin advanced esophageal squamous cancerrdquo Neoplasia vol 13pp 864ndash873 2011
[63] H Sasaki M Shitara K Yokota Y Hikosaka S MoriyamaM Yano et al ldquoIncreased NRF2 gene (NFE2L2) copy numbercorrelates with mutations in lung squamous cell carcinomasrdquoMolecular Medicine Reports vol 6 no 2 pp 391ndash394 2012
[64] H Sasaki M Shitara K Yokota Y Hikosaka S Moriyama MYano et al ldquoRagD gene expression andNRF2mutations in lungsquamous cell carcinomasrdquo Oncology Letters vol 4 pp 1167ndash1170 2012
[65] A Singh M Bodas N Wakabayashi F Bunz and S BiswalldquoGain of NRF2 function in non-small-cell lung cancer cellsconfers radioresistancerdquo Antioxidants and Redox Signaling vol13 no 11 pp 1627ndash1637 2010
[66] T Yamadori Y Ishii SHommaYMorishimaKKurishimaKItoh et al ldquoMolecular mechanisms for the regulation of NRF2-mediated cell proliferation in non-small-cell lung cancersrdquoOncogene vol 31 pp 4768ndash4777 2012
[67] A K Bauer H Y Cho L Miller-Degraff C Walker K HelmsJ Fostel et al ldquoTargeted deletion of NRF2 reduces urethane-induced lung tumor development in micerdquo PLoS ONE vol 6no 10 Article ID e26590 2011
[68] H Y Cho and S R Kleeberger ldquoNRF2 protects against airwaydisordersrdquo Toxicology and Applied Pharmacology vol 244 no1 pp 43ndash56 2010
[69] R Wang J An F Ji H Jiao H Sun and D Zhou ldquoHyperme-thylation of the KEAP1 gene in human lung cancer cell linesand lung cancer tissuesrdquo Biochemical and Biophysical ResearchCommunications vol 373 no 1 pp 151ndash154 2008
[70] P Zhang A Singh S Yegnasubramanian et al ldquoLoss ofkelch-like ECH-associated protein 1 function in prostate cancercells causes chemoresistance and radioresistance and promotestumor growthrdquoMolecular Cancer Therapeutics vol 9 no 2 pp336ndash346 2010
[71] N Hanada T Takahata Q Zhou et al ldquoMethylation of theKEAP1 gene promoter region in human colorectal cancerrdquoBMCCancer vol 12 article 66 2012
[72] L A Muscarella R Barbano V DrsquoAngelo et al ldquoRegulationof KEAP1 expression by promoter methylation in malignantgliomas and association with patientrsquos outcomerdquo Epigeneticsvol 6 no 3 pp 317ndash325 2011
[73] S Yu T O Khor K L Cheung et al ldquoNRF2 expressionis regulated by epigenetic mechanisms in prostate cancer ofTRAMP micerdquo PLoS ONE vol 5 no 1 Article ID e8579 2010
[74] T O Khor Y Huang T Y Wu L Shu J Lee and A N KongldquoPharmacodynamics of curcumin as DNA hypomethylationagent in restoring the expression of NRF2 via promoter CpGsdemethylationrdquo Biochemical Pharmacology vol 82 no 9 pp1073ndash1078 2011
[75] I M Fingerman L McDaniel X Zhang et al ldquoNCBI epige-nomics a new public resource for exploring epigenomic datasetsrdquo Nucleic Acids Research vol 39 supplemen 1 pp D908ndashD912 2011
[76] I M Fingerman X Zhang W Ratzat N Husain R F Cohenand G D Schuler ldquoNCBI epigenomics whatrsquos new for 2013rdquoNucleic Acids Research vol 41 no 1 pp D221ndashD225 2013
[77] J H Lee T O Khor L Shu Z Y Su F Fuentes andA N Kong ldquoDietary phytochemicals and cancer preventionNRF2 signaling epigenetics and cell death mechanisms inblocking cancer initiation and progressionrdquo Pharmacology ampTherapeutics vol 137 no 2 pp 153ndash171 2012
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
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Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Oxidative Medicine and Cellular Longevity 11
Table4Hum
anNRF
2SN
Psassociated
with
diseaser
isk
IDMap
onchromosom
e2 (H
GVS
name)
Region
class
Dise
asea
ssociatio
nandreferences
Risk
allelesa
ndstatistics
Ethn
icgrou
p(num
bero
fcase)
rs7557529
g178135097CgtT
minus5238
Ggt
AParkinsonrsquos
disease(2010
[26])
G=0718252Dagger
inhaplotype
OR=090
or040
CI=
060ndash140or
03ndash06
Swedish
Polish
Caucasian
(357)
rs2886162
g178133165AgtG
minus3306
TgtC
Breastcancer
survival(2012[27])
TT=0324
OR=16
87C
I=1105ndash275
FinlandKB
CP(452)
Chronicg
astritisgastric
ulcer(2007
[28])
GDagger
GDaggerin
haplotypefor
ulcer(OR=
252C
I=119ndash
545)
Japanese
(159)
P14methylationin
gastric
cancer
Hpyloriinfectio
n(2008[29])
Gin
haplotype
OR=290C
I=114ndash
736
Japanese
(209)
Gastriccancer
inHpylori-n
egative
cases(2008
[30])
Ain
haplotype
119875=0022
Japanese
(209)
rs35652124
(rs57695243)
g178130073TgtC
minus214Agt
G(previou
slyminus653[23]
orminus686[22])
Ulcerativec
olitis(2008
[31])
AG
(OR=045C
I=022ndash0
93)
G(O
R=257C
I=10
1ndash660)
Japanese
(89)
Lupu
swith
neph
ritisin
female(2010
[25])
GA
OR=18
1CI
=10
4ndash312
Mexican
mestizo(362)
Parkinsonrsquos
disease(2010
[26])
A=0884312Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
COPD
(2010[32])
G=052Dagger
hazard
ratio
=095C
I=091ndash0
99
(Haplotype)
German
(69)
Gastriculcer(2007
[28])
GDaggerin
haplotype
OR=252C
I=119ndash
545
Japanese
(159)
P14methylationin
gastric
cancer
H
pyloriinfection(2008[29])
Gin
haplotype
OR=290C
I=114ndash
736
Japanese
(209)
Gastriccancer
inHpylori-negative
cases(2008
[30])
Gin
haplotype119875=0022
Japanese
(209)
rs6706
649
g178130071CgtT
minus212Ggt
A(previou
slyminus651[23]
orminus684[22])
Ulcerativec
olitis(2008
[31])
AG
(OR=045C
I=022ndash0
93)
G(O
R=257C
I=10
1ndash660)
Japanese
(89)
Maternalacetaminop
henandasthma
(2010[33])
A=02321137Dagger
OR=17
3CI
=12
2245
UKALSPA
C(gt40
00mothersgt
5000
child
ren)
12 Oxidative Medicine and Cellular Longevity
Table4Con
tinued
IDMap
onchromosom
e2 (H
GVS
name)
Region
class
Dise
asea
ssociatio
nandreferences
Risk
allelesa
ndstatistics
Ethn
icgrou
p(num
bero
fcase)
COPD
(2010[32])
G=098Daggerin
haplotype
hazard
ratio
=095C
I=091ndash0
99
German
(69)
Parkinsonrsquos
disease(2010
[26])
G=0972343Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
03ndash06
Swedish
Polish
Caucasian
(357)
Acutelun
ginjury
follo
wingtrauma
(2007[23])
CA=0119Dagger
OR=644
CI=
134ndash
3080
Caucasia
nAfrican-
American
(164
)
Ann
ualF
EV1decline
(2011[34])
A=0082in
haplotype
Japanese
(915)
Vitiligo(2008[35])
CAA
AOR=17
24C
I=13
5ndash221
Chinese(300)
COPD
(2010[32])
G=073Daggerin
haplotype
hazard
ratio
=095C
I=091ndash0
99
German
(69)
rs6721961
(rs117801448)
g178130037TgtC
TgtG
minus178Agt
GAgt
C(previou
slyminus617[23]
orminus650[22])
Parkinsonrsquos
disease(2010
[26])
C=0980346Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
Postm
enop
ausalvenou
sthrombo
embo
lism
(2011[36])
A=033311
OR=25CI
=370ndash8570
Caucasia
n(161)
Breastcancer
survivalandNRF
2proteinexpressio
n(2012[27])
AA
OR=4656CI
=13
5ndash1606
FinlandKB
CP(452)
Acutelun
ginjury-related
mortality
followingsyste
micinflammatory
respon
sesynd
rome(2012
[37])
GG
OR=97
3CI
=12
7ndash7480
Caucasia
n(750)
Infection-indu
cedasthma(
2012
[38])
AC
OR=0437CI
=028ndash0
80
Hun
garia
nCaucasia
nGypsy
(307)
rs143406266
g178129391178
129393delGGC
Exon
1(467ndash4
69G
CC)
COPD
(2010[32])
GCC
4=053Dagger
hazard
ratio
=095C
I=091ndash0
99
Taiwanese(69)
rs2886161
g178127839Tgt
ClowastIntro
n1
Parkinsonrsquos
disease(2010
[26])
T=0878309Dagger
inhaplotype
OR=09or
04CI
=06ndash
14or
03ndash06
Swedish
Polish
Caucasian
(357)
rs2364723
g178126546Ggt
CIntro
n1
Basaland
smoker
FEV
1(200
9[39])
CI=minus6360simminus1780C
=0525Dagger
Also
ashaplotype
Netherla
nd(2542)
rs2364722
g178124787Agt
GIntro
n1
Ann
ualF
EV1decline
(2011[34])
A=0082in
haplotype
Japanese
(915)
rs13001694
g178118990Agt
GIntro
n1
Basaland
smoker
FEV
1(200
9[39])
G=040
11578Dagger
inhaplotype
Netherla
nd(2542)
Oxidative Medicine and Cellular Longevity 13
Table4Con
tinued
IDMap
onchromosom
e2 (H
GVS
name)
Region
class
Dise
asea
ssociatio
nandreferences
Risk
allelesa
ndstatistics
Ethn
icgrou
p(num
bero
fcase)
Breastcancer
[40]
Twith
NQO1N
OS3H
O1risk
allelesOR=15
6CI
=097ndash251
Caucasia
nandothers(505)
rs1806
649
(rs58745895)
g178118152Cgt
TIntro
n1
Basaland
smoker
FEV
1(200
9[39])
T=02631119Daggerin
haplotype
CI=minus8730ndash
(minus17
0)Netherla
nd(2542)
Parkinsonrsquos
disease(2010
[26])
T=0422148Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
Particulatem
attera
ndasthmaCO
PDadmiss
ion(2012[41])
Cwith
lowvitamin
Clevel(OR=
31CI
=15
0ndash630)
UK(209)
rs4243387
(rs6003846
4)g178117765Cgt
TIntro
n1
Basaland
smoker
FEV
1(200
9[39])
T=00914
25Daggerin
haplotype
Netherla
nd(2542)
rs1962142
(rs58448508)
g178113484Agt
GIntro
n1
Ann
ualF
EV1decline
(2011[34])
A=0082in
haplotype
Japanese
(915)
Breastcancer
NRF
2andARE
expressio
n(2012[27])
A(119875=0036)
FinlandKB
CP(452)
rs6726395
(rs57309289)
g178103229Agt
GIntro
n1
Smok
ing-relatedFE
V1decline
and
annu
alFE
V1decline
(2011[34])
G=0884Dagger
A=0082in
haplotype
Japanese
(915)
Basaland
smoker
FEV
1(200
9[39])
G=046
41764Daggerin
haplotype
Netherla
nd(2542)
rs2001350
(rs17515179
rs60883775)
g178100
425Cgt
TIntro
n1
Ann
ualF
EV1decline
(2011[34])
T=0082in
haplotype
Japanese
(915)
Parkinsonrsquos
disease(2010
[26])
T=0986350Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
rs10183914
(rs58731187
rs61374844
)g17809766
6Cgt
TIntro
n3
Parkinsonrsquos
disease(2010
[26])
T=0536188Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
rs2706110
g178092162Tgt
C31015840Flanking
Breastcancer
(2012[27])
TT
OR=2079CI
=118ndash368
FinlandKB
CP(452)
rs2588882
g178087165Ggt
T31015840Flanking
Infection-indu
cedasthma(
2012
[38])
TG
OR=0290CI
=013ndash0
62
Hun
garia
nCaucasia
nGypsy
(307)
minus686in
reference[22]
=minus653in
reference[23]
=currentlyminus214minus684in
reference[22]
=minus651inreference[23]
=currentlyminus212minus651inreference[22]
=minus617in
reference[23]
=currentlyminus178Ch
romosom
econtig(in
tron
31015840flank
ing)
orreversed
(51015840flank
ingprom
oter)a
llelesinbo
ldhave
been
used
inthetextand
TableOR
odds
ratio
CI95confi
denceinterval
14 Oxidative Medicine and Cellular Longevity
minus212GG (98 rs67006649 previously minus684minus651) minus178Callele (73) and GCC
4(53) was predicted to increase
respiratory failure development (hazard ratio = 095 CI091ndash099) in German COPD patients [32] Significant inter-action was also identified between an intronic SNP Gallele (rs6726395 g178103229AgtG 884 frequency) andsmoking status on FEV
1decline relative to the reference
AA allele in the above Japanese cohort [34] Siedlinskiet al [39] reported that the CC genotype of anotherintronic SNP (rs2364723 g178126546GgtC) was associatedwith a lower FEV
1level compared to the wild-type geno-
type (GG) in two Netherland cohorts (CI minus636minus178frequency = 0525 and pooled cohort size = 2542) ThisSNP alone or as a haplotype with 4 more intronic SNPs(rs13001694Grs1806649Trs4243387Trs6726395G)was alsoassociated with high FEV
1levels in individuals that ever
smoked [39] In a Hungarian population of childhoodasthma SNPs at minus178 (CA) and 31015840 flanking (rs2588882TG)loci were inversely associated with infection-induced asthma(OR 0437 CI 028ndash080 OR 0290 CI 013ndash062 resp) andthese SNPs significantly influenced an asthma-environmentalpollution interaction [38]The intronic SNP rs1806649 (CgtT)was associated but not significantly with an increased risk ofhospitalization during high-level particulate matter (PM
10)
periods in asthma or COPD patients (119899 = 209) of the UnitedKingdom (UK) [41] Asthma andCOPDadmission rateswererelated to the increase in environmental PM
10concentration
Importantly effects of interaction between prenatal stressand NRF2 SNPs on descendant pulmonary health wereinvestigated by the Avalon Longitudinal Study in the UKmaternal smoking during pregnancy was not associated withlung function change determined by maximum mild expi-ratory flow (FEF
25ndash75) or with asthma incidence in school-aged children and this relation was not modified by NRF2SNP genotypes [42]However early gestation acetaminophenexposure significantly influenced the risk of asthma andwheezing at the age of 7 years in gt4000 mothers and gt5000children [33] When maternal copies of the minus212A allele werepresent association with asthma (11374891 OR 173 CI122ndash245) and wheezing (11494949 OR 153 95 CI 106ndash220) was significantly increased [33]
Gastrointestinal Disorders While there was no evidence inlung cancer cases studies in Japanese populations suggesteda potential association of NRF2 variations with gastro-intestinal tumorigenesisHelicobacter pylori (H pylori) causesgastritis which can lead to gastric atrophy and cancer Ingastric epithelium from the Japanese cancer cohorts (39gastric cancers 46 controls) H pylori infection was posi-tively correlated with aberrant CpG island methylation oftumor suppressor genes (eg p14) and minus214Gminus212G orminus214Aminus212G NRF2 haplotype was significantly associatedwith increased (OR 290 95 CI 114ndash736) or decreased(OR 033 95 CI 013ndash088) risk of the CpG methyla-tion respectively in the H pylori-infected patients [29]Further study from the same investigators determined thatminus214Aminus212G allele carriers had significantly (119875 = 0022)reduced risk of gastric cancer inH pylori-negative cases [30]The minus214AGminus212AG genotypes were negatively associated
(OR 045 CI 022ndash093) and the minus214Gminus212G genotypeswere positively associated (chronic continuous phenotypeOR 257 CI 101ndash660) with ulcerative colitis (89 patients 141controls) in a Japanese population [31]
Autoimmune Disorders Systemic lupus erythematosus (SLE)is a long-term autoimmune disease more frequently found infemales than in males It affects organs including skin jointskidneys and brain and nephritis is an aggressive character-istic in some patients Genome-wide association studies inhumans identified a suggestive quantitative trait locus nearNRF2 [43] A study of a Mexican Mestizo population (362patients with childhood-onset SLE 379 controls and 212nephritis diagnosed) determined that lupus with nephritiswas significantly (OR 181 CI 104ndash312) associated with theminus214GA SNP in females [25] The same SNPs were notclosely associated with SLE risk in a Japanese cohort [22]Vitiligo is a skin condition in which there is a loss of browncolor (pigment) from areas of skin resulting in irregularwhite patches It is thought to be an autoimmune diseasecaused by loss of cells (melanocytes) that produce brownpigment A study indicated that theminus178A allele increased therisk of vitiligo dose-dependently (OR 1724 95 CI 135ndash221for CA OR 2902 CI 162ndash519 for AA) [35]
Female Disorders It is well known that estrogen metabolites(eg catechols) cause ROS formation suggesting correla-tion of NRF2 and downstream effectors in postmenopausalmammary cancer In a study of a Finish population (KuopioBreast Cancer Project 119899 = 452 patients 370 controls)the minus178AA homozygous genotype (OR 4656 CI = 135ndash1606) and 31015840 flanking rs2706110 (TT OR 2079 CI 118ndash368) genotype were associated with increased risk of breastcancer while the 51015840 flanking minus3306TT homozygous allelewas significantly associated with lower survival (frequency =71219 OR 1687 CI 1105ndash275) [27] suggesting that NRF2genetic polymorphisms affect susceptibility and outcome ofthe patients The minus178A allele carriers together with intronicrs1962142A allele carriers were associated with lowered tissuelevels of NRF2 proteins [27] In postmenopausal women theminus178A allele (OR 179 95CI 370ndash8570) appeared tomodifythe risk of venous thromboembolism caused by oral estrogentherapy (AA or AC frequency = 333) as demonstratedby the French ESTHER study (161 cases 474 controls) [36]An intronic rs1806649CgtT SNP did not associate with breastcancer risk in postmenopausal women [40] However whenthis SNP and other at-risk alleles of ARE-responsive genes(NQO1 HO-1 NOS3) were combined there was a significantgene-dose effect on the breast cancer risk [40] Althoughcoding region SNPs in NRF2 and KEAP1 were identifiedin the Japanese endometrial adenocarcinoma patients noassociation of NRF2 SNPs with the disease was found [44]
Neurodegenerative Diseases Oxidative stress is known to beinvolved in Parkinsonrsquos disease (PD) presumably due toproduction of ROS fromhigh-dopaminemetabolism and lowlevels of antioxidants in the substantia nigra of the brainInvestigators found a protective NRF2 haplotype consistingof four 51015840 flanking SNPs (minus5238Gminus214Aminus212Gminus178C)
Oxidative Medicine and Cellular Longevity 15
and 4 intronic SNPs (rs2886161Ars1806649Ars2001350Ars10183914A) from Swedish (OR 09 CI 060ndash140) and Polish(OR 04 CI 030ndash060) populations (total 165+192 PD cases190+192 controls) [26]The investigators also suggested thatNRF2 haplotype alleles were associated with 2 years earlierage of Alzheimerrsquos disease (AD) onset 4 years earlier age ofposterior subcapsular cataract surgery and 4 years later ageof cortical cataract surgery while they were not significantlyrelated to AD or age-related cataract risk [45]
34 Genetic Mutations in Mouse Nrf2 Tsang et al [46]compiled 673 SNPs in 55 mouse strains and constructedtheir phylogenetic tree to correlate and clarify the originsof strains based on the assembled mouse genome sequenceand SNP data [20 47 48] Recently using the completegenome sequence of C57BL6J (B6) mouse as a referencehigh-density SNP screening in other laboratory strains orin panels of strains has been published (see [17]) Althoughmillions of mouse SNPs (gt10089892 as of December 2012)and haplotype mappings from more than 120 strains havebeen published as valuable references for dissecting thegenetic basis of complex traits [49ndash51] little attention hasbeen paid to polymorphisms of Nrf2 and their correlationwith disease phenotypes
Figure 3 demonstrates the proximal promoter region(minus1 to minus950)51015840-UTR (exon 1 up to TSS) and proteinsequence of mouse Nrf2 based on GRCm38p1 PrimaryAssembly (75704641ndash75675513 bp) mRNA variant1 and protein (NP 035032 encoded by NM 010902234ndash2027 bp) sequences Genetic variations in the Nrf2genome of inbred strains collected from public databasesare listed in Tables S2 and 5 (See Supplementary TableS2) Overall 968 genetic mutations are compiled forNrf2 gene and 5 kb upstream2 kb downstream regions785 SNPs between B6 and another 16 strains wereacquired from the Mouse Phenome Database (MPDhttpphenomejaxorg dbqrtn=snpret1) and additionalSNPs and other mutations were acquired from NCBI dbSNP(httpwwwncbinlmnihgovsnpterm=mus+musculus20nfe2l2) In total 132 mutations are in the promoter (37in proximal 1 kb) 49 in exons (38 in coding region 19 Cns)727 in introns and 60 in the 31015840 flanking region Excludingmutations in the 51015840 and 31015840 flanking sequences murine Nrf2sequences appear to be more highly variable (1 variationper 375 bp) than much of the mouse genome which has anapproximate frequency of one SNP per every 245 bp (httpwwwinformaticsjaxorgmgihomehomepagesstatsallstatsshtmlallstats snp)
Nrf2 was found to be a susceptibility gene from genome-wide linkage analysis in a murine model of hyperoxia-induced ALI [52] A promoter SNP minus103TgtC (previouslypublished as minus336TgtC) in Nrf2 was found and predicted toadd an additional Sp1 binding site in hyperoxia-susceptibleB6 mice but not in resistant C3HHeJ mice [52] Genotypesfrom the SNP and from simple-sequence length polymor-phism markers of the Nrf2 locus (D2Mit248 and D2Mit94)cosegregated in the B6C3F
2mouse cohort [52] and Nrf2
deficient mice were significantly more susceptible to ALI
sub-phenotypes caused by hyperoxia than similarly exposedwild-type mice supporting Nrf2 as a contributor to thephenotypic traits [53] Although no other functional analyseson Nrf2 SNPs or haplotype association studies have beenconducted in inbred mice strains bearing haplotypes suchas multiple Cns in functional domains (eg F71L L451VH543Q and L575M) may be useful to elucidate the role ofNrf2 in differential susceptibility to oxidative diseases
4 Oncogenic Somatic Mutations in HumanNF-E2-Related Factor 2
Somatic mutation is a change in the DNA of somatic cellsthat affects derived cells but is not inherited by offspringEfforts to discover somatic mutations have provided insightinto mutagenesis and cancer development Lung cancerparticularly non-small cell lung cancer (NSCLC) is theleading cause of cancer death worldwide Somatic mutationsof NRF2 and KEAP1 discovered in lung cancer patients havedetermined the oncogenic potential ofNRF2 [54 55] KEAP1somatic mutations were associated with its reduced proteinlevels in lung cancer tissues and cells [56 57] Investigationsof NSCLC in various ethnic populations as well as cancersin gastrointestine breast and prostate have coordinatelydemonstrated that multiple Cns somatic mutations in KEAP1cause dysfunction of the translated protein and in turnconstitutive activation of NRF2 increasing risk of neoplasiaand chemoresistance [12 55 58 59] Somatic mutations ofNRF2 have been detected in various cancer tissues (largelysquamous cell carcinomas) in Asian populations (Table 6)NRF2 mutations were significantly associated with NSCLCcases (squamous cell lung carcinoma adenocarcinoma) ofthe Japanese (107 [54]) the Chinese (23 [60]) andthe Koreans (8 [61]) as well as with lung cancer celllines Smoking history was also correlated with mutationoccurrence in all of the studies [54 60 61] In addition tolung cancers laryngeal squamous carcinoma (13 in [61])esophageal squamous cancer (ESC 22 in [60] 114 in[61]) head and neck cancers (25 in [54]) skin (117 casein [61]) and oral cancer cell lines had somatic changesin NRF2 In contrast to wide-spread KEAP1 mutationsmutations in NRF2 were clustered in DLGETGE motifs ofthe Neh2 domain which are critical in the ldquohinge and latchrdquomodel of KEAP1 binding [12] Similar to KEAP1 somaticmutations it has been postulated that NRF2 mutations incancer cells lead to NRF2 accumulation by suppressing itsubiquitination or KEAP1 binding which eventually confersmalignant potential and resistance to chemotherapy
Most variable sites in NRF2 included aa residues 29 (AspD) 31 (Gly G) 77 (Asp D) and 79 (Glu E) (Table 6)Residue 33 (Ser S) in the Neh2 domain is mutated by eithergenetic or somatic processes (Figure 4) Cns in the EDGFmotif of NRF2 was experimentally determined to impairrecognition of KEAP1 [54] NRF2 mutations were signif-icantly correlated with increased (25-fold) copy number(31 of mutants versus 3 wild types) in Japanese NSCLCcases [63] Aberrant mutation of NRF2 also led to increasedexpression of downstream effectors includingRagD known to
16 Oxidative Medicine and Cellular Longevity
pL575M
pF71L
pH543QpL451V
pA151P pA151V pN159D pQ198delpV105D
pA204T pS224I225S226del pT253S pS278ApD305G
pM409V pR410HpP388L pA389G pT395I
Genomic DNA (reversed contig GRCm38 p1 C57BL6JNM 0109023)
Protein (NP 035032)1
51101151201251301351401451501551
minus400
minus500
minus50
minus100
minus200
minus300
minus450
minus350
minus250
minus150
minus950
minus900
minus850
minus800
minus750
minus700
151
101151201
2001
g minus470gg minusg minus
c minus41c minus
c
c
c c
gg minus 6C
g minus125insTTC g minus118insC
g minus281delC
g minus63delCg minus1 (75704642 c minus234)
Translation start site (c +1)Termination codon (c +2025 +2027)
c minus136 minus134in-frame stop codonc minus8585
g minus41
246
440
minus381
minus11
minus91 minus90
minus332
minus153
minus98
c minus197c minus209 minus202
gminus942 minus899 (cminus1175 minus1132) CpG islands [71]
g 460CgtT
g minus103CCgtT [51]
CgtTCgtG
g minus320CgtT
CgtT
CgtT
CgtTg minus426GgtA
GgtACgtA
CgtA
GgtA
g minus238GgtTTgtC
TgtC
TgtC
TgtCTgtG
g minus229AgtG
CgtG
CgtG
GgtC
gminus826 minus654 (cminus1059 ) CpG islands [71]minus887
Figure 3 DNA (partial promoter and exons 1 and 5) and protein sequence ofmouseNrf2 SNPs and amino acid residues for non-synonymousSNPs are marked Promoter regions bearing 5CpG islands are underlined
151
201251301351401451501551601
Genetic mutation (Table 3 and Figure 2)Somatic mutation (Table 6)
Neh2
Neh3
Neh4Neh5
Neh6
Neh1
Protein isoform 1 (NP 006155)
101151
Figure 4 Genetic and somatic mutation loci in human NRF2 protein
Oxidative Medicine and Cellular Longevity 17
Table5Mou
seup
stream
andexon
varia
tions
ofNfe2
l2locusin17
inbred
strainsR
eference
sequ
ence
isC5
7BL6J
(B6strain
1)geno
me(GI14933824975547698ndash75513576)SNPalleleand
geno
typesareshow
nas
chromosom
econ
tigsequ
enceStrains
2129S1S
vlmJ3AJ
4AKR
J5BA
LBcJ6C3
HH
eJ7C
57BL
6NJ8CA
STEiJ9CB
AJ
10D
BA2J11FVBNJ12LPJ
13N
ODShiLtJ14N
ZOH
ILtJ
15P
WK
PhJ16SPR
ETEiJ
17W
SBEiJ
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs2566
08517
75705565
GA
(CT)
51015840Flankingminus924
NC
GG
GG
GG
GG
GG
GG
GG
AA
Grs47274959
75705562
AT
(TA
)51015840Flankingminus921
NC
AT
TA
TT
AA
TT
AT
AT
TT
Ars216940398
75705554ndash75705555
Tadd(A
add)
51015840Flankingminus913minus914
NC
CGrs239114134
75705540
CT(G
A)
51015840Flankingminus899
NC
CT
TC
TT
CC
TT
CT
CT
CT
Crs46
461765
75705528
TG(A
C)oA
C
51015840Flankingminus887
NC
TG
GT
GG
TT
GG
TG
TG
GG
Trs5144
9853
75705525
CA(G
T)
51015840Flankingminus884
NC
CA
AC
AA
CC
AA
CA
CA
CC
Crs249093111
75705512
CT(G
A)
51015840Flankingminus871
NC
CT
TC
TT
CC
TT
CT
CT
TT
Crs263745200
75705510
CG(G
C)
51015840Flankingminus869
NC
GG
GG
GG
GG
GG
GG
GG
CG
Grs45651867
75705498
TG(A
C)
51015840Flankingminus857
NC
TG
GT
GG
TT
GG
TG
TG
GG
Trs219997531
75705495
GA
(CT)
51015840Flankingminus854
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs251918379
75705451
TA(A
T)
51015840Flankingminus810
NC
TT
TT
TT
TT
TT
TT
TT
TA
Trs27978306
75705449
TA(A
T)
51015840Flankingminus808
NC
TA
AT
AA
TT
AA
TA
TA
TT
Trs216087412
75705429
TG(A
C)lowast
51015840Flankingminus788
NC
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
GTlowast
Tlowast
rs261229914
75705423ndash75705424
AGdel(CT
del)
51015840Flankingminus783minus784
NC
AGrs27978307
75705400
CT(G
A)
51015840Flankingminus759
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs243167395
75705359
GA
(CT)
51015840Flankingminus718
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs27978308
75705307
GA
(CT)
51015840Flankingminus66
6NC
GG
GG
GG
GG
GG
GG
GG
AG
Grs254744
09875705294
AT
(TA
)51015840Flankingminus653
NC
AA
AA
AA
AA
AA
AA
AA
AT
Ars228133419
75705179
GT
(CA
)51015840Flankingminus538
NC
GG
GG
GG
GG
GG
GG
GG
GT
Grs214719520
75705111
GA
(CT)
51015840Flankingminus470
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs2440
87730
75705101
GA
(CT)
51015840Flankingminus46
0NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs227781699
75705081
CG(G
C)
51015840Flankingminus44
0NC
CC
CC
CC
CC
CC
CC
CC
GC
Crs257870353
75705067
CT(G
A)
51015840Flankingminus426
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs24487744
075705022
AG
(TC)
51015840Flankingminus381
NC
AA
AA
AA
AA
AA
AA
AA
AG
Ars234628138
75704973
AC
(TG
)51015840Flankingminus332
NC
AA
AA
AA
AA
AA
AA
AA
AC
A
18 Oxidative Medicine and Cellular Longevity
Table5Con
tinued
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs247275247
75704961
GA
(CT)
51015840Flankingminus320
NC
GG
GG
GG
GG
GG
GG
AG
GG
Grs247519047
75704922
Gdel(Cdel)
51015840Flankingminus281
NC
Grs27978309
(rs51915758)
75704887
GA
C(C
TG
)51015840Flankingminus246
NC
GA
AG
AA
GG
AA
GA
GA
CC
G
rs218139102
75704879
CA(G
T)
51015840Flankingminus238
NC
CC
CC
CC
CC
CC
CC
CC
AC
Crs251990355
75704870
TC(A
G)
51015840Flankingminus229
NC
TT
TT
TT
TT
TT
TT
TT
TC
Trs238746955
75704794
AG
(TC)
51015840Flankingminus153
NC
AA
AA
AA
AA
AA
AA
AA
GA
Ars221664
405
75704786
AG
(TC)
51015840Flankingminus145
NC
AA
AA
AA
AA
AA
AA
AA
AG
Ars217054035
75704766
ndash75704767
GAAadd(TTC
add)
51015840Flankingminus125minus126
NC
GA
rs248182931
75704759ndash75704760
Gadd(C
add)
51015840Flankingminus118
minus119
NC
AGrs217197904
75704744
GA
(CT)
51015840Flankingminus103
(+)S
p1[23]
GA
AG
AA
GG
AA
GA
AA
GG
Ars2644
9364975704704
Gdel(Cdel)
51015840Flankingminus63
NC
Grs2404
81112
757046
82CT(G
A)
51015840Flankingminus41
NC
CC
CC
CC
CC
CC
CC
CC
CT
Crs27978310
75704617
AG
(TC)
UTR
-525
NC
AA
AA
AA
AA
AA
AA
AA
GG
Ars27978311
75704610
CG(G
C)
UTR
-532
NC
GG
GG
GG
GG
GG
GG
GG
GG
Crs27978312
757046
05GA
(CT)
UTR
-537
NC
GA
AG
AA
GG
AA
GA
GA
GG
Grs214784220
757044
99AG
(TC)
UTR
-5143
NC
AA
AA
AA
AA
AA
AA
AA
GG
Ars244146318
757044
98GT
(CA
)UTR
-5144
NC
GG
GG
GG
GG
GG
GG
GG
GT
Grs27978313
757044
97GC
(CG
)UTR
-5145
NC
GC
CG
CC
GC
CC
GC
CC
CC
Crs215431944
757044
93CT(G
A)
UTR
-5149
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs257015697
757044
49GA
(CT)
UTR
-5193
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs252234782
757044
19GT
(CA
)UTR
-5223
NC
Grs1346
0861
75679262
AG
(TC)
Exon
244
6Cn
sF71L
AG
GA
GG
AG
GG
AG
AG
GG
Grs1346
0859
75679202
GA
(CT)lowast
Exon
2506
CsH91=
GA
AG
AA
GG
AA
GA
GA
GG
Grs27978436
75679187
GA
(CT)lowast
Exon
2521
CsT9
6=G
GG
GG
GG
AG
GG
GG
GA
GG
rs226131070
75679178
GA
(CT)
Exon
2530
CsS99=
GG
GG
GG
GG
GG
GG
GG
GA
Grs227683834
75678576
AT
(TA
)Ex
on3
547
CnsV
105D
AA
AA
AA
AA
AA
AA
AA
AT
Ars257321187
75678506
TC(A
G)
Exon
3617
CsP128=
TT
TT
TT
TC
TT
TT
TT
TT
Trs27978444
75678500
TA(A
T)
Exon
3623
CsV130=
TT
TT
TT
TA
TT
TT
TT
AT
Trs227743136
75677686
GA
(CT)
Exon
4662
CsH143=
GG
GG
GG
GG
GG
GG
GG
GA
Grs215327202
75677664
CG(G
C)
Exon
4684
CnsA
151P
CC
CC
CC
CG
CC
CC
CC
CC
Crs247539755
75677663
GA
(CT)
Exon
4685
CnsA
151V
GG
GG
GG
GA
GG
GG
GG
GG
Grs233164
668
75677640
TC(A
G)
Exon
4708
CnsN
159D
TT
TT
TT
TT
TT
TT
TT
TC
T
rs2340
95816
75677162ndash75677160
GCT
del
(AGCdel)
Exon
5826minus
828
Cds-in
delQ
198
GCT
Oxidative Medicine and Cellular Longevity 19
Table5Con
tinued
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs27978452
75677145
CT(G
A)
Exon
5843
CnsA
204T
CC
CC
CC
CC
CC
CC
CC
CC
T
rs225047937
75677086ndash75677078
GAG
ATCG
ATdel
(ATG
CATC
TCdel)
Exon
5902minus
910
Cds-in
del
(S224I225S226)
GAG
ATC
GAT
rs241469868
75676998
TA(G
T)
Exon
5990
CnT2
53S
TT
TT
TT
TT
TT
TT
TT
TA
Trs221602571
75676923
AC
(TG
)Ex
on5
1065
CnsS
278A
AA
AA
AA
AA
AA
AA
AA
AC
Ars252576472
75676876
GA
(CT)
Exon
51112
CsS293=
GG
GG
GG
GG
GG
GG
GG
GA
Grs250802933
75676841
TC(A
G)
Exon
5114
7Cn
sD305G
TT
TT
TT
TT
TT
TT
TT
TC
Trs225425698
75676792
CT(G
A)
Exon
5119
6Cs
P321=
CC
CC
CC
CC
CC
CC
CC
CT
Crs13459064
75676732
CT(G
A)
Exon
51256
CsT3
41=
CT
TC
TT
CT
TT
CT
CT
TC
Crs27978453
75676717
CT(G
A)
Exon
51271
CsA346=
CC
CC
CC
CC
CC
CC
CC
TC
Crs214335034
75676636
AG
(TC)
Exon
51352
CsD373=
AA
AA
AA
AA
AA
AA
AA
AG
Ars24958364
4756766
03AC
(TG
)Ex
on5
1385
CsP3
84=
AA
AA
AA
AA
AA
AA
AA
AC
Ars27978454
75676592
GA
(CT)
Exon
51396
CnsP
388L
GG
GG
GG
GG
GG
GG
GG
AG
Grs215384328
75676589
GC
(CG
)Ex
on5
1399
CnsA
389G
GG
GG
GG
GG
GG
GG
GG
GC
Grs251728286
75676571
GA
(CT)
Exon
51417
CnsT
395I
Grs247602334
75676567
TC(A
G)
Exon
51421
CsV396=
TT
TT
TT
TT
TT
TT
TT
TC
Trs234216231
75676530
TC(A
G)
Exon
51458
CnsM
409V
TT
TT
TT
TT
TT
TT
TT
TC
Trs212904337
75676526
CT(G
A)
Exon
51462
CnsR
410H
CC
CC
CC
CC
CC
CC
CC
CT
Crs252650779
75676522
TC(A
G)
Exon
51466
CsE4
11=
TT
TT
TT
TT
TT
TT
TT
TC
Trs231273560
75676516
TC(A
G)
Exon
51472
CsQ413=
TT
TT
TT
TT
TT
TT
TT
TC
Trs257886949
756764
13GT
(CA
)Ex
on5
1575
CsR4
48=
GG
GG
GG
GG
GG
GG
GG
GT
Grs241537608
756764
04GC
(CG
)Ex
on5
1584
CnsL
451V
GG
GG
GG
GG
GG
GG
GG
GC
Grs227619071
75676312
TC(A
G)
Exon
51676
CsQ481=
TT
TT
TT
TT
TT
TT
TT
TC
Trs258913831
75676290
AG
(TC)
Exon
51698
CsL4
89=
AA
AA
AA
AA
AA
AA
AA
AG
Ars225593319
75676126
AT
(TA
)Ex
on5
1862
CnsH
543Q
Ars4223233
75676105
GA
(CT)
Exon
51883
CsS550=
GG
GG
GG
GA
GG
GG
GG
GG
Grs4223232
75676032
GT
(CA
)Ex
on5
1956
CnsL
575M
Grs4223231
75675816
CG
UTR
-32172
NC
GG
GG
GG
GG
GG
GG
GG
GC
Grs1346
860
75675682
AT
UTR
-32306
NC
ASequ
ence
varia
tions
inmou
seNr
f2wereob
tained
from
Mou
sePh
enom
eDatabase(http
ph
enom
ejaxorgSNP)
andNCB
ISNPdatabase
(http
wwwncbinlm
nihgovSNP)N
CBIreference
sequ
ence
isMus
Musculusstra
inC5
7BL6J
chromosom
e2GRC
m38p1(NC
0000
687
GI37209910875704641ndash756755192
9123
bp)To
tal968
genetic
mutations
arerepo
rted
inNr
f2gene
and5k
bup
stream
(ge75704
642minus
1)and2k
bdo
wnstre
am(le75675512)sequences
asof
Janu
ary2003A
llSN
Psandallelesa
representedas
geno
miccontig
(reversedsequ
ence
indicated)E
xon175704
641ndash75704364(278
bpT
TS=75704
408)
exon
275679431ndash756791
65(267
bp)exon
375678579ndash75678490(90b
p)exon475677713ndash756775
46(168
bp)andexon
57567718
4ndash75678849
(1666
bp)Proteinam
inoacid
(aa)
resid
uesinNP00
61552
(597
aa)Cs
cod
ing-syno
nymou
sCn
scoding
-non
syno
nymou
sAminoacid
A-alanineD-aspartic
acidE
-glutamicacidF-phenylalanineG
-glycineH
-histidineI-iso
leucineL-leu
cineand
M-m
ethion
ineN-
asparagineP-prolin
eQ-glutamineR-arginineS-serineT-threon
ineandV-valin
elowastErrorsin
databasesfi
xedNC
notcon
firmed
20 Oxidative Medicine and Cellular Longevity
Table 6 NRF2 somatic mutations revealed in various human cancers
Domain Amino acid residues DNA mutation Cancer types (cases) ReferencesLocus Wild type Mutant
DLG motif
24 W (Trp) C (Cys) c72GgtCGgtT NSCLC neck ESC [54 62]K (Lys) c72TgtC ESC [62]
26 Q (Gln) E (Glu) c76CgtG NSCLC ESC [54 62]
27 D (Asp) G (Gly)lowast c80GgtA NSCLC [60]Y (Tyr) c79GgtT ESC [61]
28 I (Ile) T (Thr) c83CgtT NSCLC [54]
29 D (Asp) G (Gly) c86AgtG Head and neck ESC [54 62]H (His) c85GgtC NSCLC laynx [60 61]
30 L (Leu) F (Phe) c88CgtT NSCLC ESC [54 62]
31 G (Gly) A (Ala) c92GgtC NSCLC ESC skin [54 60ndash62]
32 V (Val) T (Thr) c95TgtG NSCLC [54]del c93 95delAGT ESC [61]
33ndash36 S-R-E-V S-R-E-V-S-R-E-Vlowast c97 108dupAGTCGAGCCGTA ESC [61]
34 R (Arg) Q (Gln) c101GgtA NCSCL [54 60 61]P (Pro) c101GgtC NSCLC [63]
ETGF motif
75 Q (Gln) H (His) c225AgtC Head and neck ESC [54 62]
77 D (Asp)
V (Val) c230AgtT NSCLC ESC [54 60 62]G (Gly) c230AgtG ESC [62]A (Ala) c230AgtC NSCLC [61]N (Asn) c229GgtA Larynx [61]
78 E (Glu) K (Lys) c232GgtA NSCLC ESC [54 62]
79 E (Glu)
K (Lys) c235GgtA NSCLC ESC [54 61 62]Q (Gln) c235GgtC NSCLC ESC [54 60ndash62]G (Gly) c236AgtG Larynx [61]E-E c234 236dupAGA ESC [61]
80 T (Thr) K (Lys) c239CgtACgtG NSCLC ESC [54 61 62]
80 T (Thr)P (Phe) c238AgtC ESC [62]I (Ile) c239CgtT Head and neck [54]A (Ala) c238AgtG NSCLC [63]
81 G (Gly) V (Val) c242GgtT ESC [61]D (Asp) c242GgtA NSCLC ESC [60ndash62]
82 E (Glu)
D (Asp) c246AgtT ESC oral cancer cell line [54 62]G (Gly) c245AgtG NSCLC [54]Q (Gln)lowast c244GgtC NSCLC ESC [60 61]V (Val)lowast c245AgtT ESC [61]
83 F (Phe) L (Leu)lowast c247TgtC NSCLC [60]NSCLC non-small cell lung cancer ESC esophageal squamous cancer and lowasterrors in reference fixed Number of cases 82 NSCLC and 10 ESC in [62] 125NSCLC 70 ESC 23 larynx and 17 skin in [61] 103 NSCLC and 12 head and neck in [54] 90 NSCLC in [63] 103 NSCLC in [60]
be involved in squamous lung cancer cell proliferation [64]suggesting that the mutation is functional and overcomesKEAP1 inhibition Singh et al [65] determined in vitrothat RNAi-mediated depletion of NRF2 in lung cancer cellsenhanced ROS production and susceptibility to cell deathby ionizing radiation These studies support the concept thatelevated NRF2 and ARE responsiveness provides cancer cellswith proliferative advantage for malignant transformation
and undue protection from anti-cancer therapy Onco-genic epidermal growth factor receptor (EGFR) signaling isrecently found to be critical in NRF2-mediated proliferationof NSCLC cells [66]
Collectively ldquogain of functionrdquo mutations in NRF2 thatreduce KEAP1 recognition are suggested to be predictivemarkers for poor responsiveness to chemotherapy and radi-ation therapy Although NRF2-mediated cellular defense
Oxidative Medicine and Cellular Longevity 21
processes are essential in the initiation stage enhancedNRF2-ARE activity in advanced stages of cancer developmentmay create a favorable intracellular environment for tumorcell growth and survival [67 68] In this context NRF2may be a potential molecular target for the treatment ofradio-resistance cancers especially those that have ldquoloss offunctionrdquo mutations in EGFR KRAS or KEAP1 as well asldquogain of functionrdquo mutations in NRF2
5 Epigenetic Alterations of NF-E2-RelatedFactor 2
Epigenetic modifications are alterations of molecules inter-acting with genes without changes to the primary DNAsequence They include post-translational modification ofhistones DNA methylation events chromatin conforma-tional changes and alterations to noncoding regulatoryRNAs Epigenetic alterations are stable and often inheritablebut are reversible and may affect expression of the geneDysregulation or defects in epigenetic processes particularlyhypermethylation of tumor suppressor gene promoters (egCpG islands) or histone modifications are thought to beassociated with carcinogenesis Investigators have reportedhypermethylation in CpG islands of KEAP1 which wereassociated with reduced KEAP1 expression in human cancersfrom lung prostate colon and so forth [69ndash72] Similar tosomatic mutations epigenetic changes on KEAP1 impairedthe function of its encoded protein leading to constitutiveNRF2 activation
Supporting the role of ldquopathogenic mutationsrdquo in NRF2expression of Nrf2 and downstream Nqo1 was suppressedin prostate tumors of mice (transgenic adenocarcinomaof mouse prostate TRAMP) Among 15 promoter CpGislands located between minus942 and minus654 (cminus1175 cminus1132and cminus1059 cminus887 gap in cminus1131 cminus1060 see Figure 3)hypermethylation of the first 5 CpG islands (minus942 minus899 andcminus1175 cminus1132) was significantly associated with tumorige-nesis [73] Moreover treatment with inhibitors for DNAmethyltransferase and histone deacetylase restored Nrf2expression in these tumor cells [73] A dietary phytochemicalcurcumin known as a DNA hypomethylation agent restoredepigenetically silent Nrf2 expression through CpG demethy-lation in carcinogen-induced mouse tumor cells [74]
The whole genome epigenetic datasets for 5 species arepublicly accessible at NCBI Epigenomics [75 76]The humanNRF2 epigenome of primary cells (breast penis) and H1stem cell line as well as mouse Nrf2 CpG island methylationdata for sperm blood and cerebellum are currently avail-able (httpwwwncbinlmnihgovepigenomics) Althoughno direct evidence of disease-associated epigenetic modu-lation has been identified in human NRF2 various phyto-chemical NRF2 agonists such as sulforaphane and curcuminhave shown their roles in DNA methylation and histonemodification (see reviews by Lee and colleagues eg [77])Taken together epigenetic modifications of theNRF2KEAP1axle are predicted to cause dysregulation of ARE-mediatedcellular defense leading to deleterious health effects and
phytochemical antioxidants as epigenetic modulators forNRF2 are suggested to be useful in cancer prevention
6 Conclusions
NRF2 is evolutionally conserved with high-sequence homol-ogy in many species However it is a highly mutable geneand numerous genetic variants have been discovered inhuman ethnic groups Importantly certain SNPs or haplo-types have been identified in various diseases as ldquoat-riskrdquoalleles and are related to functional alterations In additionto genetic variations multiple somatic mutations identifiedin the KEAP1 recognition domain of NRF2 in cancer cellshave been found to be oncogenic due to dysregulation ofNRF2 homeostasis by its excess ldquogain of functionrdquo Epigeneticalteration of theNRF2 is under investigation and is predictedto have pathogenic influences as learned from mouse andphytochemical agonist studies Continuous updates of Nrf2allelic variants in inbred mouse strains will provide a usefultool for effective experimental designs formodels of oxidativedisorders to provide insight into the disease mechanisms andintervention strategies
Abbreviations
AD Alzheimerrsquos diseaseALI acute lung injuryARE antioxidant response elementbZIP basic leucine zippercds coding DNA sequencesCI confidence intervalCOPD chronic obstructive pulmonary diseaseESC esophageal squamous cancerFEV1 forced expiratory volume in one second
Gbp giga base pairsGST glutathione S-transferaseGWAS genome-wide association studyHGVS Human Genome Variation SocietyHO-1 heme oxygenase-1119867 119901119910119897119900119903119894 119867119890119897119894119888119900119887119886119888119905119890119903 119901119910119897119900119903119894KEAP1 Kelch-like ECH activating protein 1MPD Mouse Phenome DatabaseMRP multidrug resistance proteinNCBI National Center for Biotechnology InformationNeh NRF2-ECH homologyNfe2l2 nuclear factor (erythroid-derived 2)-like 2Nrf2 NF-E2-related factor 2NSCLC nonsmall cell lung cancerNQO1 NAD(P)Hquinone oxidoreductase 1OR odds ratioPD Parkinsonrsquos diseasePM particulate matterROS reactive oxygen speciesSLE systemic lupus erythematosusSNP single nucleotide polymorphismSOD superoxide dismutaseURT untranslated region
22 Oxidative Medicine and Cellular Longevity
Disclosure
Authorrsquos contribution to the Work was done as part of theAuthorrsquos official duties as a NIH employee and is a Work ofthe United States Government Therefore copyright may notbe established in the United States
Conflict of Interests
The author declares that there is no conflict of interests
Acknowledgments
The research related to this paper was supported by theIntramural Research Program of the National Institutes ofHealth of the National Institute of Environmental HealthSciences (NIEHS) Drs Steven Kleeberger and StephanieLondon at the NIEHS provided excellent critical review ofthis paper The author thanks Mrs Jacqui Marzec for herhelpful comments and English editing
References
[1] J Y Chan M C Cheung P Moi K Chan and Y W KanldquoChromosomal localization of the humanNF-E2 family of bZIPtranscription factors by fluorescence in situ hybridizationrdquoHuman Genetics vol 95 no 3 pp 265ndash269 1995
[2] K Itoh K Igarashi N Hayashi M Nishizawa and MYamamoto ldquoCloning and characterization of a novel erythroidcell-derived CNC family transcription factor heterodimerizingwith the small Maf family proteinsrdquo Molecular and CellularBiology vol 15 no 8 pp 4184ndash4193 1995
[3] W O Osburn and T W Kensler ldquoNRF2 signaling an adaptiveresponse pathway for protection against environmental toxicinsultsrdquoMutation Research vol 659 no 1-2 pp 31ndash39 2008
[4] D Papp K Lenti D Modos D Fazekas Z Dul D Tureiet al ldquoThe NRF2-related interactome and regulome containmultifunctional proteins and fine-tuned autoregulatory loopsrdquoFEBS Letters vol 586 no 13 pp 1795ndash1802 2012
[5] X Wang D J Tomso B N Chorley et al ldquoIdentificationof polymorphic antioxidant response elements in the humangenomerdquo Human Molecular Genetics vol 16 no 10 pp 1188ndash1200 2007
[6] D Malhotra E Portales-Casamar A Singh et al ldquoGlobalmapping of binding sites for NRF2 identifies novel targets incell survival response through ChIP-Seq profiling and networkanalysisrdquo Nucleic Acids Research vol 38 no 17 pp 5718ndash57342010
[7] K Chan R Lu J C Chang and Y W Kan ldquoNRF2 a memberof the NFE2 family of transcription factors is not essential formurine erythropoiesis growth and developmentrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 93 no 24 pp 13943ndash13948 1996
[8] K Itoh T Chiba S Takahashi et al ldquoAn NRF2small Mafheterodimer mediates the induction of phase II detoxifyingenzyme genes through antioxidant response elementsrdquo Bio-chemical and Biophysical Research Communications vol 236no 2 pp 313ndash322 1997
[9] FMartin J M vanDeursen R A Shivdasani CW Jackson AG Troutman and P A Ney ldquoErythroid maturation and globin
gene expression inmicewith combined deficiency ofNF-E2 andNrf-2rdquo Blood vol 91 no 9 pp 3459ndash3466 1998
[10] K Itoh N Wakabayashi Y Katoh et al ldquoKEAP1 repressesnuclear activation of antioxidant responsive elements by NRF2through binding to the amino-terminal Neh2 domainrdquo Genesand Development vol 13 no 1 pp 76ndash86 1999
[11] A Kobayashi M I Kang H Okawa et al ldquoOxidative stresssensor KEAP1 functions as an adaptor for Cul3-based E3 ligaseto regulate proteasomal degradation of NRF2rdquo Molecular andCellular Biology vol 24 no 16 pp 7130ndash7139 2004
[12] K Taguchi H Motohashi and M Yamamoto ldquoMolecularmechanisms of the KEAP1-NRF2 pathway in stress responseand cancer evolutionrdquo Genes to Cells vol 16 no 2 pp 123ndash1402011
[13] K Mukaigasa L T Nguyen L Li H Nakajima M Yamamotoand M Kobayashi ldquoGenetic evidence of an evolutionarilyconserved role for NRF2 in the protection against oxidativestressrdquoMolecular and Cellular Biology vol 32 no 21 pp 4455ndash4461 2012
[14] JMaher andMYamamoto ldquoThe rise of antioxidant signalingmdashthe evolution and hormetic actions of NRF2rdquo Toxicology andApplied Pharmacology vol 244 no 1 pp 4ndash15 2010
[15] S C Lo X Li M T Henzl L J Beamer and M HanninkldquoStructure of the KEAP1NRF2 interface provides mechanisticinsight intoNRF2 signalingrdquoThe EMBO Journal vol 25 no 15pp 3605ndash3617 2006
[16] V A McKusick ldquoMendelian inheritance in man and its onlineversion OMIMrdquo American Journal of Human Genetics vol 80no 4 pp 588ndash604 2007
[17] B Yalcin D J Adams J Flint and T M Keane ldquoNext-generation sequencing of experimental mouse strainsrdquo Mam-malian Genome vol 23 no 9-10 pp 490ndash498 2012
[18] D R Bentley S Balasubramanian H P Swerdlow et alldquoAccurate whole human genome sequencing using reversibleterminator chemistryrdquo Nature vol 456 no 7218 pp 53ndash592008
[19] P S G Chain D V Grafham R S Fulton et al ldquoGenomeproject standards in a new era of sequencingrdquo Science vol 326no 5950 pp 236ndash237 2009
[20] RHWaterstonK Lindblad-Toh E Birney J Rogers J F AbrilP Agarwal et al ldquoInitial sequencing and comparative analysis ofthe mouse genomerdquo Nature vol 420 pp 520ndash562 2002
[21] T M Keane L Goodstadt P Danecek M A White K WongB Yalcin et al ldquoMouse genomic variation and its effect onphenotypes and gene regulationrdquoNature vol 477 pp 289ndash2942011
[22] T Yamamoto K Yoh A Kobayashi et al ldquoIdentification ofpolymorphisms in the promoter region of the human NRF2generdquo Biochemical and Biophysical Research Communicationsvol 321 no 1 pp 72ndash79 2004
[23] J M Marzec J D Christie S P Reddy et al ldquoFunctionalpolymorphisms in the transcription factor NRF2 in humansincrease the risk of acute lung injuryrdquo The FASEB Journal vol21 no 9 pp 2237ndash2246 2007
[24] H Fukushima-Uesaka Y Saito K Maekawa et al ldquoGeneticvariations and haplotype structures of transcriptional factorNRF2 and its cytosolic reservoir protein KEAP1 in JapaneserdquoDrug Metabolism and Pharmacokinetics vol 22 no 3 pp 212ndash219 2007
[25] E J Cordova R Velazquez-Cruz F Centeno V Baca and LOrozco ldquoThe NRF2 gene variant -653GA is associated with
Oxidative Medicine and Cellular Longevity 23
nephritis in childhood-onset systemic lupus erythematosusrdquoLupus vol 19 no 10 pp 1237ndash1242 2010
[26] M von Otter S Landgren S Nilsson et al ldquoAssociation ofNRF2-encoding NFE2L2 haplotypes with Parkinsonrsquos diseaserdquoBMCMedical Genetics vol 11 no 1 article 36 2010
[27] JMHartikainenM TengstromVMKosmaV L Kinnula AMannermaa andY Soini ldquoGenetic polymorphisms and proteinexpression of NRF2 and sulfiredoxin predict survival outcomesin breast cancerrdquo Cancer Research vol 72 pp 5537ndash5546 2012
[28] T Arisawa T Tahara T Shibata et al ldquoThe relationshipbetween Helicobacter pylori infection and promoter polymor-phism of the NRF2 gene in chronic gastritisrdquo InternationalJournal of Molecular Medicine vol 19 no 1 pp 143ndash148 2007
[29] T Arisawa T Tahara T Shibata et al ldquoThe influence ofpromoter polymorphism of nuclear factor-erythroid 2-relatedfactor 2 gene on the aberrant DNA methylation in gastricepitheliumrdquo Oncology Reports vol 19 no 1 pp 211ndash216 2008
[30] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene pro-moter polymorphism and gastric carcinogenesisrdquo Hepato-Gastroenterology vol 55 no 82-83 pp 750ndash754 2008
[31] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene promoterpolymorphism is associated with ulcerative colitis in a Japanesepopulationrdquo Hepato-Gastroenterology vol 55 no 82-83 pp394ndash397 2008
[32] C C Hua L C Chang J C Tseng C M Chu Y C Liu andW B Shieh ldquoFunctional haplotypes in the promoter region oftranscription factor NRF2 in chronic obstructive pulmonarydiseaserdquo Disease Markers vol 28 no 3 pp 185ndash193 2010
[33] S O Shaheen R B Newson S M Ring M J Rose-ZerilliJ W Holloway and A J Henderson ldquoPrenatal and infantacetaminophen exposure antioxidant gene polymorphismsand childhood asthmardquo The Journal of Allergy and ClinicalImmunology vol 126 no 6 pp 1141e7ndash1148e7 2010
[34] H Masuko T Sakamoto Y Kaneko et al ldquoLower FEV1 innon-COPD nonasthmatic subjects association with smokingannual decline in FEV1 total IgE levels and TSLP genotypesrdquoInternational Journal of Chronic Obstructive Pulmonary Diseasevol 6 pp 181ndash189 2011
[35] C P Guan M N Zhou A E Xu et al ldquoThe susceptibilityto vitiligo is associated with NF-E2-related factor 2 (NRF2)gene polymorphisms a study on Chinese Han populationrdquoExperimental Dermatology vol 17 no 12 pp 1059ndash1062 2008
[36] J Bouligand O Cabaret M Canonico et al ldquoEffect of NFE2L2genetic polymorphism on the association between oral estrogentherapy and the risk of venous thromboembolism in post-menopausal womenrdquo Clinical Pharmacology amp Therapeuticsvol 89 no 1 pp 60ndash64 2011
[37] D S OrsquoMahony B J Glavan T D Holden C Fong R A BlackG Rona et al ldquoInflammation and immune-related candidategene associations with acute lung injury susceptibility andseverity a validation studyrdquo PLoS ONE vol 7 no 12 Article IDe51104 2012
[38] I Ungvari E Hadadi V Virag et al ldquoRelationship between airpollutionNFE2L2 gene polymorphisms and childhood asthmain aHungarian populationrdquo Journal of CommunityGenetics vol3 no 1 pp 25ndash33 2012
[39] M SiedlinskiD S Postma JMA Boer et al ldquoLevel and courseof FEV1 in relation to polymorphisms inNFE2L2 andKEAP1 inthe general populationrdquo Respiratory Research vol 10 article 732009
[40] C C Hong C B Ambrosone J Ahn et al ldquoGenetic variabilityin iron-related oxidative stress pathways (NRF2 NQ01 NOS3
and HO-1) iron intake and risk of postmenopausal breastcancerrdquo Cancer Epidemiology Biomarkers and Prevention vol16 no 9 pp 1784ndash1794 2007
[41] C Canova C Dunster F J Kelly C Minelli P L Shah CCaneja et al ldquoPM10-induced hospital admissions for asthmaand chronic obstructive pulmonary disease the modifyingeffect of individual characteristicsrdquo Epidemiology vol 23 no 4pp 607ndash615 2012
[42] A J Henderson R B Newson M Rose-Zerilli S M RingJ W Holloway and S O Shaheen ldquoMaternal NRF2 andgluthathione-S-transferase polymorphisms donotmodify asso-ciations of prenatal tobacco smoke exposure with asthma andlung function in school-aged childrenrdquo Thorax vol 65 no 10pp 897ndash902 2010
[43] C Xing A L Sestak J A Kelly et al ldquoLocalization andreplication of the systemic lupus erythematosus linkage signalat 4p16 interaction with 2p11 12q24 and 19q13 in EuropeanAmericansrdquo Human Genetics vol 120 no 5 pp 623ndash631 2007
[44] T F Wong K Yoshinaga Y Monma K Ito H NiikuraS Nagase et al ldquoAssociation of KEAP1 and NRF2 geneticmutations and polymorphisms with endometrioid endometrialadenocarcinoma survivalrdquo International Journal of Gynecologi-cal Cancer vol 21 no 8 pp 1428ndash1435 2011
[45] M von Otter S Landgren S Nilsson et al ldquoNRF2-encodingNFE2L2 haplotypes influence disease progression but not riskin Alzheimerrsquos disease and age-related cataractrdquoMechanisms ofAgeing and Development vol 131 no 2 pp 105ndash110 2010
[46] S Tsang Z Sun B Luke et al ldquoA comprehensive SNP-basedgenetic analysis of inbred mouse strainsrdquoMammalian Genomevol 16 no 7 pp 476ndash480 2005
[47] C M Wade E J Kulbokas III A W Kirby et al ldquoThe mosaicstructure of variation in the laboratory mouse genomerdquoNaturevol 420 no 6915 pp 574ndash578 2002
[48] T Wiltshire M T Pletcher S Batalov et al ldquoGenome-widesingle-nucleotide polymorphism analysis defines haplotypepatterns in mouserdquo Proceedings of the National Academy ofSciences of the United States of America vol 100 no 6 pp 3380ndash3385 2003
[49] K A Frazer E Eskin H M Kang et al ldquoA sequence-basedvariation map of 827 million SNPs in inbred mouse strainsrdquoNature vol 448 no 7157 pp 1050ndash1053 2007
[50] T P Maddatu S C Grubb C J Bult andM A Bogue ldquoMousephenome database (MPD)rdquo Nucleic Acids Research vol 40 ppD887ndashD894 2007
[51] A Kirby H M Kang C M Wade et al ldquoFine mapping in 94inbred mouse strains using a high-density haplotype resourcerdquoGenetics vol 185 no 3 pp 1081ndash1095 2010
[52] H Y Cho A E Jedlicka S P M Reddy L Y Zhang T WKensler and S R Kleeberger ldquoLinkage analysis of susceptibilityto hyperoxia NRF2 is a candidate generdquo American Journal ofRespiratory Cell and Molecular Biology vol 26 no 1 pp 42ndash512002
[53] H Y Cho A E Jedlicka S P M Reddy et al ldquoRole of NRF2in protection against hyperoxic lung injury in micerdquo AmericanJournal of Respiratory Cell and Molecular Biology vol 26 no 2pp 175ndash182 2002
[54] T Shibata T Ohta K I Tong et al ldquoCancer related mutationsin NRF2 impair its recognition by KEAP1-Cul3 E3 ligase andpromote malignancyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 36 pp13568ndash13573 2008
24 Oxidative Medicine and Cellular Longevity
[55] A Singh V Misra R K Thimmulappa et al ldquoDysfunctionalKEAP1-NRF2 interaction in non-small-cell lung cancerrdquo PLoSMedicine vol 3 no 10 article e420 2006
[56] T Ohta K Iijima M Miyamoto et al ldquoLoss of KEAP1 functionactivates NRF2 and provides advantages for lung cancer cellgrowthrdquo Cancer Research vol 68 no 5 pp 1303ndash1309 2008
[57] B Padmanabhan K I Tong T Ohta et al ldquoStructural basisfor defects of KEAP1 activity provoked by its point mutationsin lung cancerrdquoMolecular Cell vol 21 no 5 pp 689ndash700 2006
[58] T Shibata A Kokubu M Gotoh et al ldquoGenetic alteration ofKEAP1 confers constitutive NRF2 activation and resistance tochemotherapy in gallbladder cancerrdquoGastroenterology vol 135no 4 pp 1358e4ndash1368e4 2008
[59] N J Yoo H R Kim Y R Kim C H An and S H LeeldquoSomatic mutations of the KEAP1 gene in common solidcancersrdquo Histopathology vol 60 no 6 pp 943ndash952 2012
[60] Y Hu Y Ju D Lin Z Wang Y Huang S Zhang et alldquoMutation of the NRF2 gene in non-small cell lung cancerrdquoMolecular Biology Reports vol 39 no 4 pp 4743ndash4747 2012
[61] Y R Kim J EOhM S Kim et al ldquoOncogenicNRF2mutationsin squamous cell carcinomas of oesophagus and skinrdquo TheJournal of Pathology vol 220 no 4 pp 446ndash451 2010
[62] T Shibata A Kokubu S Saito et al ldquoNRF2 mutation confersmalignant potential and resistance to chemoradiation therapyin advanced esophageal squamous cancerrdquo Neoplasia vol 13pp 864ndash873 2011
[63] H Sasaki M Shitara K Yokota Y Hikosaka S MoriyamaM Yano et al ldquoIncreased NRF2 gene (NFE2L2) copy numbercorrelates with mutations in lung squamous cell carcinomasrdquoMolecular Medicine Reports vol 6 no 2 pp 391ndash394 2012
[64] H Sasaki M Shitara K Yokota Y Hikosaka S Moriyama MYano et al ldquoRagD gene expression andNRF2mutations in lungsquamous cell carcinomasrdquo Oncology Letters vol 4 pp 1167ndash1170 2012
[65] A Singh M Bodas N Wakabayashi F Bunz and S BiswalldquoGain of NRF2 function in non-small-cell lung cancer cellsconfers radioresistancerdquo Antioxidants and Redox Signaling vol13 no 11 pp 1627ndash1637 2010
[66] T Yamadori Y Ishii SHommaYMorishimaKKurishimaKItoh et al ldquoMolecular mechanisms for the regulation of NRF2-mediated cell proliferation in non-small-cell lung cancersrdquoOncogene vol 31 pp 4768ndash4777 2012
[67] A K Bauer H Y Cho L Miller-Degraff C Walker K HelmsJ Fostel et al ldquoTargeted deletion of NRF2 reduces urethane-induced lung tumor development in micerdquo PLoS ONE vol 6no 10 Article ID e26590 2011
[68] H Y Cho and S R Kleeberger ldquoNRF2 protects against airwaydisordersrdquo Toxicology and Applied Pharmacology vol 244 no1 pp 43ndash56 2010
[69] R Wang J An F Ji H Jiao H Sun and D Zhou ldquoHyperme-thylation of the KEAP1 gene in human lung cancer cell linesand lung cancer tissuesrdquo Biochemical and Biophysical ResearchCommunications vol 373 no 1 pp 151ndash154 2008
[70] P Zhang A Singh S Yegnasubramanian et al ldquoLoss ofkelch-like ECH-associated protein 1 function in prostate cancercells causes chemoresistance and radioresistance and promotestumor growthrdquoMolecular Cancer Therapeutics vol 9 no 2 pp336ndash346 2010
[71] N Hanada T Takahata Q Zhou et al ldquoMethylation of theKEAP1 gene promoter region in human colorectal cancerrdquoBMCCancer vol 12 article 66 2012
[72] L A Muscarella R Barbano V DrsquoAngelo et al ldquoRegulationof KEAP1 expression by promoter methylation in malignantgliomas and association with patientrsquos outcomerdquo Epigeneticsvol 6 no 3 pp 317ndash325 2011
[73] S Yu T O Khor K L Cheung et al ldquoNRF2 expressionis regulated by epigenetic mechanisms in prostate cancer ofTRAMP micerdquo PLoS ONE vol 5 no 1 Article ID e8579 2010
[74] T O Khor Y Huang T Y Wu L Shu J Lee and A N KongldquoPharmacodynamics of curcumin as DNA hypomethylationagent in restoring the expression of NRF2 via promoter CpGsdemethylationrdquo Biochemical Pharmacology vol 82 no 9 pp1073ndash1078 2011
[75] I M Fingerman L McDaniel X Zhang et al ldquoNCBI epige-nomics a new public resource for exploring epigenomic datasetsrdquo Nucleic Acids Research vol 39 supplemen 1 pp D908ndashD912 2011
[76] I M Fingerman X Zhang W Ratzat N Husain R F Cohenand G D Schuler ldquoNCBI epigenomics whatrsquos new for 2013rdquoNucleic Acids Research vol 41 no 1 pp D221ndashD225 2013
[77] J H Lee T O Khor L Shu Z Y Su F Fuentes andA N Kong ldquoDietary phytochemicals and cancer preventionNRF2 signaling epigenetics and cell death mechanisms inblocking cancer initiation and progressionrdquo Pharmacology ampTherapeutics vol 137 no 2 pp 153ndash171 2012
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
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Diabetes ResearchJournal of
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Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
12 Oxidative Medicine and Cellular Longevity
Table4Con
tinued
IDMap
onchromosom
e2 (H
GVS
name)
Region
class
Dise
asea
ssociatio
nandreferences
Risk
allelesa
ndstatistics
Ethn
icgrou
p(num
bero
fcase)
COPD
(2010[32])
G=098Daggerin
haplotype
hazard
ratio
=095C
I=091ndash0
99
German
(69)
Parkinsonrsquos
disease(2010
[26])
G=0972343Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
03ndash06
Swedish
Polish
Caucasian
(357)
Acutelun
ginjury
follo
wingtrauma
(2007[23])
CA=0119Dagger
OR=644
CI=
134ndash
3080
Caucasia
nAfrican-
American
(164
)
Ann
ualF
EV1decline
(2011[34])
A=0082in
haplotype
Japanese
(915)
Vitiligo(2008[35])
CAA
AOR=17
24C
I=13
5ndash221
Chinese(300)
COPD
(2010[32])
G=073Daggerin
haplotype
hazard
ratio
=095C
I=091ndash0
99
German
(69)
rs6721961
(rs117801448)
g178130037TgtC
TgtG
minus178Agt
GAgt
C(previou
slyminus617[23]
orminus650[22])
Parkinsonrsquos
disease(2010
[26])
C=0980346Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
Postm
enop
ausalvenou
sthrombo
embo
lism
(2011[36])
A=033311
OR=25CI
=370ndash8570
Caucasia
n(161)
Breastcancer
survivalandNRF
2proteinexpressio
n(2012[27])
AA
OR=4656CI
=13
5ndash1606
FinlandKB
CP(452)
Acutelun
ginjury-related
mortality
followingsyste
micinflammatory
respon
sesynd
rome(2012
[37])
GG
OR=97
3CI
=12
7ndash7480
Caucasia
n(750)
Infection-indu
cedasthma(
2012
[38])
AC
OR=0437CI
=028ndash0
80
Hun
garia
nCaucasia
nGypsy
(307)
rs143406266
g178129391178
129393delGGC
Exon
1(467ndash4
69G
CC)
COPD
(2010[32])
GCC
4=053Dagger
hazard
ratio
=095C
I=091ndash0
99
Taiwanese(69)
rs2886161
g178127839Tgt
ClowastIntro
n1
Parkinsonrsquos
disease(2010
[26])
T=0878309Dagger
inhaplotype
OR=09or
04CI
=06ndash
14or
03ndash06
Swedish
Polish
Caucasian
(357)
rs2364723
g178126546Ggt
CIntro
n1
Basaland
smoker
FEV
1(200
9[39])
CI=minus6360simminus1780C
=0525Dagger
Also
ashaplotype
Netherla
nd(2542)
rs2364722
g178124787Agt
GIntro
n1
Ann
ualF
EV1decline
(2011[34])
A=0082in
haplotype
Japanese
(915)
rs13001694
g178118990Agt
GIntro
n1
Basaland
smoker
FEV
1(200
9[39])
G=040
11578Dagger
inhaplotype
Netherla
nd(2542)
Oxidative Medicine and Cellular Longevity 13
Table4Con
tinued
IDMap
onchromosom
e2 (H
GVS
name)
Region
class
Dise
asea
ssociatio
nandreferences
Risk
allelesa
ndstatistics
Ethn
icgrou
p(num
bero
fcase)
Breastcancer
[40]
Twith
NQO1N
OS3H
O1risk
allelesOR=15
6CI
=097ndash251
Caucasia
nandothers(505)
rs1806
649
(rs58745895)
g178118152Cgt
TIntro
n1
Basaland
smoker
FEV
1(200
9[39])
T=02631119Daggerin
haplotype
CI=minus8730ndash
(minus17
0)Netherla
nd(2542)
Parkinsonrsquos
disease(2010
[26])
T=0422148Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
Particulatem
attera
ndasthmaCO
PDadmiss
ion(2012[41])
Cwith
lowvitamin
Clevel(OR=
31CI
=15
0ndash630)
UK(209)
rs4243387
(rs6003846
4)g178117765Cgt
TIntro
n1
Basaland
smoker
FEV
1(200
9[39])
T=00914
25Daggerin
haplotype
Netherla
nd(2542)
rs1962142
(rs58448508)
g178113484Agt
GIntro
n1
Ann
ualF
EV1decline
(2011[34])
A=0082in
haplotype
Japanese
(915)
Breastcancer
NRF
2andARE
expressio
n(2012[27])
A(119875=0036)
FinlandKB
CP(452)
rs6726395
(rs57309289)
g178103229Agt
GIntro
n1
Smok
ing-relatedFE
V1decline
and
annu
alFE
V1decline
(2011[34])
G=0884Dagger
A=0082in
haplotype
Japanese
(915)
Basaland
smoker
FEV
1(200
9[39])
G=046
41764Daggerin
haplotype
Netherla
nd(2542)
rs2001350
(rs17515179
rs60883775)
g178100
425Cgt
TIntro
n1
Ann
ualF
EV1decline
(2011[34])
T=0082in
haplotype
Japanese
(915)
Parkinsonrsquos
disease(2010
[26])
T=0986350Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
rs10183914
(rs58731187
rs61374844
)g17809766
6Cgt
TIntro
n3
Parkinsonrsquos
disease(2010
[26])
T=0536188Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
rs2706110
g178092162Tgt
C31015840Flanking
Breastcancer
(2012[27])
TT
OR=2079CI
=118ndash368
FinlandKB
CP(452)
rs2588882
g178087165Ggt
T31015840Flanking
Infection-indu
cedasthma(
2012
[38])
TG
OR=0290CI
=013ndash0
62
Hun
garia
nCaucasia
nGypsy
(307)
minus686in
reference[22]
=minus653in
reference[23]
=currentlyminus214minus684in
reference[22]
=minus651inreference[23]
=currentlyminus212minus651inreference[22]
=minus617in
reference[23]
=currentlyminus178Ch
romosom
econtig(in
tron
31015840flank
ing)
orreversed
(51015840flank
ingprom
oter)a
llelesinbo
ldhave
been
used
inthetextand
TableOR
odds
ratio
CI95confi
denceinterval
14 Oxidative Medicine and Cellular Longevity
minus212GG (98 rs67006649 previously minus684minus651) minus178Callele (73) and GCC
4(53) was predicted to increase
respiratory failure development (hazard ratio = 095 CI091ndash099) in German COPD patients [32] Significant inter-action was also identified between an intronic SNP Gallele (rs6726395 g178103229AgtG 884 frequency) andsmoking status on FEV
1decline relative to the reference
AA allele in the above Japanese cohort [34] Siedlinskiet al [39] reported that the CC genotype of anotherintronic SNP (rs2364723 g178126546GgtC) was associatedwith a lower FEV
1level compared to the wild-type geno-
type (GG) in two Netherland cohorts (CI minus636minus178frequency = 0525 and pooled cohort size = 2542) ThisSNP alone or as a haplotype with 4 more intronic SNPs(rs13001694Grs1806649Trs4243387Trs6726395G)was alsoassociated with high FEV
1levels in individuals that ever
smoked [39] In a Hungarian population of childhoodasthma SNPs at minus178 (CA) and 31015840 flanking (rs2588882TG)loci were inversely associated with infection-induced asthma(OR 0437 CI 028ndash080 OR 0290 CI 013ndash062 resp) andthese SNPs significantly influenced an asthma-environmentalpollution interaction [38]The intronic SNP rs1806649 (CgtT)was associated but not significantly with an increased risk ofhospitalization during high-level particulate matter (PM
10)
periods in asthma or COPD patients (119899 = 209) of the UnitedKingdom (UK) [41] Asthma andCOPDadmission rateswererelated to the increase in environmental PM
10concentration
Importantly effects of interaction between prenatal stressand NRF2 SNPs on descendant pulmonary health wereinvestigated by the Avalon Longitudinal Study in the UKmaternal smoking during pregnancy was not associated withlung function change determined by maximum mild expi-ratory flow (FEF
25ndash75) or with asthma incidence in school-aged children and this relation was not modified by NRF2SNP genotypes [42]However early gestation acetaminophenexposure significantly influenced the risk of asthma andwheezing at the age of 7 years in gt4000 mothers and gt5000children [33] When maternal copies of the minus212A allele werepresent association with asthma (11374891 OR 173 CI122ndash245) and wheezing (11494949 OR 153 95 CI 106ndash220) was significantly increased [33]
Gastrointestinal Disorders While there was no evidence inlung cancer cases studies in Japanese populations suggesteda potential association of NRF2 variations with gastro-intestinal tumorigenesisHelicobacter pylori (H pylori) causesgastritis which can lead to gastric atrophy and cancer Ingastric epithelium from the Japanese cancer cohorts (39gastric cancers 46 controls) H pylori infection was posi-tively correlated with aberrant CpG island methylation oftumor suppressor genes (eg p14) and minus214Gminus212G orminus214Aminus212G NRF2 haplotype was significantly associatedwith increased (OR 290 95 CI 114ndash736) or decreased(OR 033 95 CI 013ndash088) risk of the CpG methyla-tion respectively in the H pylori-infected patients [29]Further study from the same investigators determined thatminus214Aminus212G allele carriers had significantly (119875 = 0022)reduced risk of gastric cancer inH pylori-negative cases [30]The minus214AGminus212AG genotypes were negatively associated
(OR 045 CI 022ndash093) and the minus214Gminus212G genotypeswere positively associated (chronic continuous phenotypeOR 257 CI 101ndash660) with ulcerative colitis (89 patients 141controls) in a Japanese population [31]
Autoimmune Disorders Systemic lupus erythematosus (SLE)is a long-term autoimmune disease more frequently found infemales than in males It affects organs including skin jointskidneys and brain and nephritis is an aggressive character-istic in some patients Genome-wide association studies inhumans identified a suggestive quantitative trait locus nearNRF2 [43] A study of a Mexican Mestizo population (362patients with childhood-onset SLE 379 controls and 212nephritis diagnosed) determined that lupus with nephritiswas significantly (OR 181 CI 104ndash312) associated with theminus214GA SNP in females [25] The same SNPs were notclosely associated with SLE risk in a Japanese cohort [22]Vitiligo is a skin condition in which there is a loss of browncolor (pigment) from areas of skin resulting in irregularwhite patches It is thought to be an autoimmune diseasecaused by loss of cells (melanocytes) that produce brownpigment A study indicated that theminus178A allele increased therisk of vitiligo dose-dependently (OR 1724 95 CI 135ndash221for CA OR 2902 CI 162ndash519 for AA) [35]
Female Disorders It is well known that estrogen metabolites(eg catechols) cause ROS formation suggesting correla-tion of NRF2 and downstream effectors in postmenopausalmammary cancer In a study of a Finish population (KuopioBreast Cancer Project 119899 = 452 patients 370 controls)the minus178AA homozygous genotype (OR 4656 CI = 135ndash1606) and 31015840 flanking rs2706110 (TT OR 2079 CI 118ndash368) genotype were associated with increased risk of breastcancer while the 51015840 flanking minus3306TT homozygous allelewas significantly associated with lower survival (frequency =71219 OR 1687 CI 1105ndash275) [27] suggesting that NRF2genetic polymorphisms affect susceptibility and outcome ofthe patients The minus178A allele carriers together with intronicrs1962142A allele carriers were associated with lowered tissuelevels of NRF2 proteins [27] In postmenopausal women theminus178A allele (OR 179 95CI 370ndash8570) appeared tomodifythe risk of venous thromboembolism caused by oral estrogentherapy (AA or AC frequency = 333) as demonstratedby the French ESTHER study (161 cases 474 controls) [36]An intronic rs1806649CgtT SNP did not associate with breastcancer risk in postmenopausal women [40] However whenthis SNP and other at-risk alleles of ARE-responsive genes(NQO1 HO-1 NOS3) were combined there was a significantgene-dose effect on the breast cancer risk [40] Althoughcoding region SNPs in NRF2 and KEAP1 were identifiedin the Japanese endometrial adenocarcinoma patients noassociation of NRF2 SNPs with the disease was found [44]
Neurodegenerative Diseases Oxidative stress is known to beinvolved in Parkinsonrsquos disease (PD) presumably due toproduction of ROS fromhigh-dopaminemetabolism and lowlevels of antioxidants in the substantia nigra of the brainInvestigators found a protective NRF2 haplotype consistingof four 51015840 flanking SNPs (minus5238Gminus214Aminus212Gminus178C)
Oxidative Medicine and Cellular Longevity 15
and 4 intronic SNPs (rs2886161Ars1806649Ars2001350Ars10183914A) from Swedish (OR 09 CI 060ndash140) and Polish(OR 04 CI 030ndash060) populations (total 165+192 PD cases190+192 controls) [26]The investigators also suggested thatNRF2 haplotype alleles were associated with 2 years earlierage of Alzheimerrsquos disease (AD) onset 4 years earlier age ofposterior subcapsular cataract surgery and 4 years later ageof cortical cataract surgery while they were not significantlyrelated to AD or age-related cataract risk [45]
34 Genetic Mutations in Mouse Nrf2 Tsang et al [46]compiled 673 SNPs in 55 mouse strains and constructedtheir phylogenetic tree to correlate and clarify the originsof strains based on the assembled mouse genome sequenceand SNP data [20 47 48] Recently using the completegenome sequence of C57BL6J (B6) mouse as a referencehigh-density SNP screening in other laboratory strains orin panels of strains has been published (see [17]) Althoughmillions of mouse SNPs (gt10089892 as of December 2012)and haplotype mappings from more than 120 strains havebeen published as valuable references for dissecting thegenetic basis of complex traits [49ndash51] little attention hasbeen paid to polymorphisms of Nrf2 and their correlationwith disease phenotypes
Figure 3 demonstrates the proximal promoter region(minus1 to minus950)51015840-UTR (exon 1 up to TSS) and proteinsequence of mouse Nrf2 based on GRCm38p1 PrimaryAssembly (75704641ndash75675513 bp) mRNA variant1 and protein (NP 035032 encoded by NM 010902234ndash2027 bp) sequences Genetic variations in the Nrf2genome of inbred strains collected from public databasesare listed in Tables S2 and 5 (See Supplementary TableS2) Overall 968 genetic mutations are compiled forNrf2 gene and 5 kb upstream2 kb downstream regions785 SNPs between B6 and another 16 strains wereacquired from the Mouse Phenome Database (MPDhttpphenomejaxorg dbqrtn=snpret1) and additionalSNPs and other mutations were acquired from NCBI dbSNP(httpwwwncbinlmnihgovsnpterm=mus+musculus20nfe2l2) In total 132 mutations are in the promoter (37in proximal 1 kb) 49 in exons (38 in coding region 19 Cns)727 in introns and 60 in the 31015840 flanking region Excludingmutations in the 51015840 and 31015840 flanking sequences murine Nrf2sequences appear to be more highly variable (1 variationper 375 bp) than much of the mouse genome which has anapproximate frequency of one SNP per every 245 bp (httpwwwinformaticsjaxorgmgihomehomepagesstatsallstatsshtmlallstats snp)
Nrf2 was found to be a susceptibility gene from genome-wide linkage analysis in a murine model of hyperoxia-induced ALI [52] A promoter SNP minus103TgtC (previouslypublished as minus336TgtC) in Nrf2 was found and predicted toadd an additional Sp1 binding site in hyperoxia-susceptibleB6 mice but not in resistant C3HHeJ mice [52] Genotypesfrom the SNP and from simple-sequence length polymor-phism markers of the Nrf2 locus (D2Mit248 and D2Mit94)cosegregated in the B6C3F
2mouse cohort [52] and Nrf2
deficient mice were significantly more susceptible to ALI
sub-phenotypes caused by hyperoxia than similarly exposedwild-type mice supporting Nrf2 as a contributor to thephenotypic traits [53] Although no other functional analyseson Nrf2 SNPs or haplotype association studies have beenconducted in inbred mice strains bearing haplotypes suchas multiple Cns in functional domains (eg F71L L451VH543Q and L575M) may be useful to elucidate the role ofNrf2 in differential susceptibility to oxidative diseases
4 Oncogenic Somatic Mutations in HumanNF-E2-Related Factor 2
Somatic mutation is a change in the DNA of somatic cellsthat affects derived cells but is not inherited by offspringEfforts to discover somatic mutations have provided insightinto mutagenesis and cancer development Lung cancerparticularly non-small cell lung cancer (NSCLC) is theleading cause of cancer death worldwide Somatic mutationsof NRF2 and KEAP1 discovered in lung cancer patients havedetermined the oncogenic potential ofNRF2 [54 55] KEAP1somatic mutations were associated with its reduced proteinlevels in lung cancer tissues and cells [56 57] Investigationsof NSCLC in various ethnic populations as well as cancersin gastrointestine breast and prostate have coordinatelydemonstrated that multiple Cns somatic mutations in KEAP1cause dysfunction of the translated protein and in turnconstitutive activation of NRF2 increasing risk of neoplasiaand chemoresistance [12 55 58 59] Somatic mutations ofNRF2 have been detected in various cancer tissues (largelysquamous cell carcinomas) in Asian populations (Table 6)NRF2 mutations were significantly associated with NSCLCcases (squamous cell lung carcinoma adenocarcinoma) ofthe Japanese (107 [54]) the Chinese (23 [60]) andthe Koreans (8 [61]) as well as with lung cancer celllines Smoking history was also correlated with mutationoccurrence in all of the studies [54 60 61] In addition tolung cancers laryngeal squamous carcinoma (13 in [61])esophageal squamous cancer (ESC 22 in [60] 114 in[61]) head and neck cancers (25 in [54]) skin (117 casein [61]) and oral cancer cell lines had somatic changesin NRF2 In contrast to wide-spread KEAP1 mutationsmutations in NRF2 were clustered in DLGETGE motifs ofthe Neh2 domain which are critical in the ldquohinge and latchrdquomodel of KEAP1 binding [12] Similar to KEAP1 somaticmutations it has been postulated that NRF2 mutations incancer cells lead to NRF2 accumulation by suppressing itsubiquitination or KEAP1 binding which eventually confersmalignant potential and resistance to chemotherapy
Most variable sites in NRF2 included aa residues 29 (AspD) 31 (Gly G) 77 (Asp D) and 79 (Glu E) (Table 6)Residue 33 (Ser S) in the Neh2 domain is mutated by eithergenetic or somatic processes (Figure 4) Cns in the EDGFmotif of NRF2 was experimentally determined to impairrecognition of KEAP1 [54] NRF2 mutations were signif-icantly correlated with increased (25-fold) copy number(31 of mutants versus 3 wild types) in Japanese NSCLCcases [63] Aberrant mutation of NRF2 also led to increasedexpression of downstream effectors includingRagD known to
16 Oxidative Medicine and Cellular Longevity
pL575M
pF71L
pH543QpL451V
pA151P pA151V pN159D pQ198delpV105D
pA204T pS224I225S226del pT253S pS278ApD305G
pM409V pR410HpP388L pA389G pT395I
Genomic DNA (reversed contig GRCm38 p1 C57BL6JNM 0109023)
Protein (NP 035032)1
51101151201251301351401451501551
minus400
minus500
minus50
minus100
minus200
minus300
minus450
minus350
minus250
minus150
minus950
minus900
minus850
minus800
minus750
minus700
151
101151201
2001
g minus470gg minusg minus
c minus41c minus
c
c
c c
gg minus 6C
g minus125insTTC g minus118insC
g minus281delC
g minus63delCg minus1 (75704642 c minus234)
Translation start site (c +1)Termination codon (c +2025 +2027)
c minus136 minus134in-frame stop codonc minus8585
g minus41
246
440
minus381
minus11
minus91 minus90
minus332
minus153
minus98
c minus197c minus209 minus202
gminus942 minus899 (cminus1175 minus1132) CpG islands [71]
g 460CgtT
g minus103CCgtT [51]
CgtTCgtG
g minus320CgtT
CgtT
CgtT
CgtTg minus426GgtA
GgtACgtA
CgtA
GgtA
g minus238GgtTTgtC
TgtC
TgtC
TgtCTgtG
g minus229AgtG
CgtG
CgtG
GgtC
gminus826 minus654 (cminus1059 ) CpG islands [71]minus887
Figure 3 DNA (partial promoter and exons 1 and 5) and protein sequence ofmouseNrf2 SNPs and amino acid residues for non-synonymousSNPs are marked Promoter regions bearing 5CpG islands are underlined
151
201251301351401451501551601
Genetic mutation (Table 3 and Figure 2)Somatic mutation (Table 6)
Neh2
Neh3
Neh4Neh5
Neh6
Neh1
Protein isoform 1 (NP 006155)
101151
Figure 4 Genetic and somatic mutation loci in human NRF2 protein
Oxidative Medicine and Cellular Longevity 17
Table5Mou
seup
stream
andexon
varia
tions
ofNfe2
l2locusin17
inbred
strainsR
eference
sequ
ence
isC5
7BL6J
(B6strain
1)geno
me(GI14933824975547698ndash75513576)SNPalleleand
geno
typesareshow
nas
chromosom
econ
tigsequ
enceStrains
2129S1S
vlmJ3AJ
4AKR
J5BA
LBcJ6C3
HH
eJ7C
57BL
6NJ8CA
STEiJ9CB
AJ
10D
BA2J11FVBNJ12LPJ
13N
ODShiLtJ14N
ZOH
ILtJ
15P
WK
PhJ16SPR
ETEiJ
17W
SBEiJ
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs2566
08517
75705565
GA
(CT)
51015840Flankingminus924
NC
GG
GG
GG
GG
GG
GG
GG
AA
Grs47274959
75705562
AT
(TA
)51015840Flankingminus921
NC
AT
TA
TT
AA
TT
AT
AT
TT
Ars216940398
75705554ndash75705555
Tadd(A
add)
51015840Flankingminus913minus914
NC
CGrs239114134
75705540
CT(G
A)
51015840Flankingminus899
NC
CT
TC
TT
CC
TT
CT
CT
CT
Crs46
461765
75705528
TG(A
C)oA
C
51015840Flankingminus887
NC
TG
GT
GG
TT
GG
TG
TG
GG
Trs5144
9853
75705525
CA(G
T)
51015840Flankingminus884
NC
CA
AC
AA
CC
AA
CA
CA
CC
Crs249093111
75705512
CT(G
A)
51015840Flankingminus871
NC
CT
TC
TT
CC
TT
CT
CT
TT
Crs263745200
75705510
CG(G
C)
51015840Flankingminus869
NC
GG
GG
GG
GG
GG
GG
GG
CG
Grs45651867
75705498
TG(A
C)
51015840Flankingminus857
NC
TG
GT
GG
TT
GG
TG
TG
GG
Trs219997531
75705495
GA
(CT)
51015840Flankingminus854
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs251918379
75705451
TA(A
T)
51015840Flankingminus810
NC
TT
TT
TT
TT
TT
TT
TT
TA
Trs27978306
75705449
TA(A
T)
51015840Flankingminus808
NC
TA
AT
AA
TT
AA
TA
TA
TT
Trs216087412
75705429
TG(A
C)lowast
51015840Flankingminus788
NC
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
GTlowast
Tlowast
rs261229914
75705423ndash75705424
AGdel(CT
del)
51015840Flankingminus783minus784
NC
AGrs27978307
75705400
CT(G
A)
51015840Flankingminus759
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs243167395
75705359
GA
(CT)
51015840Flankingminus718
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs27978308
75705307
GA
(CT)
51015840Flankingminus66
6NC
GG
GG
GG
GG
GG
GG
GG
AG
Grs254744
09875705294
AT
(TA
)51015840Flankingminus653
NC
AA
AA
AA
AA
AA
AA
AA
AT
Ars228133419
75705179
GT
(CA
)51015840Flankingminus538
NC
GG
GG
GG
GG
GG
GG
GG
GT
Grs214719520
75705111
GA
(CT)
51015840Flankingminus470
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs2440
87730
75705101
GA
(CT)
51015840Flankingminus46
0NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs227781699
75705081
CG(G
C)
51015840Flankingminus44
0NC
CC
CC
CC
CC
CC
CC
CC
GC
Crs257870353
75705067
CT(G
A)
51015840Flankingminus426
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs24487744
075705022
AG
(TC)
51015840Flankingminus381
NC
AA
AA
AA
AA
AA
AA
AA
AG
Ars234628138
75704973
AC
(TG
)51015840Flankingminus332
NC
AA
AA
AA
AA
AA
AA
AA
AC
A
18 Oxidative Medicine and Cellular Longevity
Table5Con
tinued
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs247275247
75704961
GA
(CT)
51015840Flankingminus320
NC
GG
GG
GG
GG
GG
GG
AG
GG
Grs247519047
75704922
Gdel(Cdel)
51015840Flankingminus281
NC
Grs27978309
(rs51915758)
75704887
GA
C(C
TG
)51015840Flankingminus246
NC
GA
AG
AA
GG
AA
GA
GA
CC
G
rs218139102
75704879
CA(G
T)
51015840Flankingminus238
NC
CC
CC
CC
CC
CC
CC
CC
AC
Crs251990355
75704870
TC(A
G)
51015840Flankingminus229
NC
TT
TT
TT
TT
TT
TT
TT
TC
Trs238746955
75704794
AG
(TC)
51015840Flankingminus153
NC
AA
AA
AA
AA
AA
AA
AA
GA
Ars221664
405
75704786
AG
(TC)
51015840Flankingminus145
NC
AA
AA
AA
AA
AA
AA
AA
AG
Ars217054035
75704766
ndash75704767
GAAadd(TTC
add)
51015840Flankingminus125minus126
NC
GA
rs248182931
75704759ndash75704760
Gadd(C
add)
51015840Flankingminus118
minus119
NC
AGrs217197904
75704744
GA
(CT)
51015840Flankingminus103
(+)S
p1[23]
GA
AG
AA
GG
AA
GA
AA
GG
Ars2644
9364975704704
Gdel(Cdel)
51015840Flankingminus63
NC
Grs2404
81112
757046
82CT(G
A)
51015840Flankingminus41
NC
CC
CC
CC
CC
CC
CC
CC
CT
Crs27978310
75704617
AG
(TC)
UTR
-525
NC
AA
AA
AA
AA
AA
AA
AA
GG
Ars27978311
75704610
CG(G
C)
UTR
-532
NC
GG
GG
GG
GG
GG
GG
GG
GG
Crs27978312
757046
05GA
(CT)
UTR
-537
NC
GA
AG
AA
GG
AA
GA
GA
GG
Grs214784220
757044
99AG
(TC)
UTR
-5143
NC
AA
AA
AA
AA
AA
AA
AA
GG
Ars244146318
757044
98GT
(CA
)UTR
-5144
NC
GG
GG
GG
GG
GG
GG
GG
GT
Grs27978313
757044
97GC
(CG
)UTR
-5145
NC
GC
CG
CC
GC
CC
GC
CC
CC
Crs215431944
757044
93CT(G
A)
UTR
-5149
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs257015697
757044
49GA
(CT)
UTR
-5193
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs252234782
757044
19GT
(CA
)UTR
-5223
NC
Grs1346
0861
75679262
AG
(TC)
Exon
244
6Cn
sF71L
AG
GA
GG
AG
GG
AG
AG
GG
Grs1346
0859
75679202
GA
(CT)lowast
Exon
2506
CsH91=
GA
AG
AA
GG
AA
GA
GA
GG
Grs27978436
75679187
GA
(CT)lowast
Exon
2521
CsT9
6=G
GG
GG
GG
AG
GG
GG
GA
GG
rs226131070
75679178
GA
(CT)
Exon
2530
CsS99=
GG
GG
GG
GG
GG
GG
GG
GA
Grs227683834
75678576
AT
(TA
)Ex
on3
547
CnsV
105D
AA
AA
AA
AA
AA
AA
AA
AT
Ars257321187
75678506
TC(A
G)
Exon
3617
CsP128=
TT
TT
TT
TC
TT
TT
TT
TT
Trs27978444
75678500
TA(A
T)
Exon
3623
CsV130=
TT
TT
TT
TA
TT
TT
TT
AT
Trs227743136
75677686
GA
(CT)
Exon
4662
CsH143=
GG
GG
GG
GG
GG
GG
GG
GA
Grs215327202
75677664
CG(G
C)
Exon
4684
CnsA
151P
CC
CC
CC
CG
CC
CC
CC
CC
Crs247539755
75677663
GA
(CT)
Exon
4685
CnsA
151V
GG
GG
GG
GA
GG
GG
GG
GG
Grs233164
668
75677640
TC(A
G)
Exon
4708
CnsN
159D
TT
TT
TT
TT
TT
TT
TT
TC
T
rs2340
95816
75677162ndash75677160
GCT
del
(AGCdel)
Exon
5826minus
828
Cds-in
delQ
198
GCT
Oxidative Medicine and Cellular Longevity 19
Table5Con
tinued
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs27978452
75677145
CT(G
A)
Exon
5843
CnsA
204T
CC
CC
CC
CC
CC
CC
CC
CC
T
rs225047937
75677086ndash75677078
GAG
ATCG
ATdel
(ATG
CATC
TCdel)
Exon
5902minus
910
Cds-in
del
(S224I225S226)
GAG
ATC
GAT
rs241469868
75676998
TA(G
T)
Exon
5990
CnT2
53S
TT
TT
TT
TT
TT
TT
TT
TA
Trs221602571
75676923
AC
(TG
)Ex
on5
1065
CnsS
278A
AA
AA
AA
AA
AA
AA
AA
AC
Ars252576472
75676876
GA
(CT)
Exon
51112
CsS293=
GG
GG
GG
GG
GG
GG
GG
GA
Grs250802933
75676841
TC(A
G)
Exon
5114
7Cn
sD305G
TT
TT
TT
TT
TT
TT
TT
TC
Trs225425698
75676792
CT(G
A)
Exon
5119
6Cs
P321=
CC
CC
CC
CC
CC
CC
CC
CT
Crs13459064
75676732
CT(G
A)
Exon
51256
CsT3
41=
CT
TC
TT
CT
TT
CT
CT
TC
Crs27978453
75676717
CT(G
A)
Exon
51271
CsA346=
CC
CC
CC
CC
CC
CC
CC
TC
Crs214335034
75676636
AG
(TC)
Exon
51352
CsD373=
AA
AA
AA
AA
AA
AA
AA
AG
Ars24958364
4756766
03AC
(TG
)Ex
on5
1385
CsP3
84=
AA
AA
AA
AA
AA
AA
AA
AC
Ars27978454
75676592
GA
(CT)
Exon
51396
CnsP
388L
GG
GG
GG
GG
GG
GG
GG
AG
Grs215384328
75676589
GC
(CG
)Ex
on5
1399
CnsA
389G
GG
GG
GG
GG
GG
GG
GG
GC
Grs251728286
75676571
GA
(CT)
Exon
51417
CnsT
395I
Grs247602334
75676567
TC(A
G)
Exon
51421
CsV396=
TT
TT
TT
TT
TT
TT
TT
TC
Trs234216231
75676530
TC(A
G)
Exon
51458
CnsM
409V
TT
TT
TT
TT
TT
TT
TT
TC
Trs212904337
75676526
CT(G
A)
Exon
51462
CnsR
410H
CC
CC
CC
CC
CC
CC
CC
CT
Crs252650779
75676522
TC(A
G)
Exon
51466
CsE4
11=
TT
TT
TT
TT
TT
TT
TT
TC
Trs231273560
75676516
TC(A
G)
Exon
51472
CsQ413=
TT
TT
TT
TT
TT
TT
TT
TC
Trs257886949
756764
13GT
(CA
)Ex
on5
1575
CsR4
48=
GG
GG
GG
GG
GG
GG
GG
GT
Grs241537608
756764
04GC
(CG
)Ex
on5
1584
CnsL
451V
GG
GG
GG
GG
GG
GG
GG
GC
Grs227619071
75676312
TC(A
G)
Exon
51676
CsQ481=
TT
TT
TT
TT
TT
TT
TT
TC
Trs258913831
75676290
AG
(TC)
Exon
51698
CsL4
89=
AA
AA
AA
AA
AA
AA
AA
AG
Ars225593319
75676126
AT
(TA
)Ex
on5
1862
CnsH
543Q
Ars4223233
75676105
GA
(CT)
Exon
51883
CsS550=
GG
GG
GG
GA
GG
GG
GG
GG
Grs4223232
75676032
GT
(CA
)Ex
on5
1956
CnsL
575M
Grs4223231
75675816
CG
UTR
-32172
NC
GG
GG
GG
GG
GG
GG
GG
GC
Grs1346
860
75675682
AT
UTR
-32306
NC
ASequ
ence
varia
tions
inmou
seNr
f2wereob
tained
from
Mou
sePh
enom
eDatabase(http
ph
enom
ejaxorgSNP)
andNCB
ISNPdatabase
(http
wwwncbinlm
nihgovSNP)N
CBIreference
sequ
ence
isMus
Musculusstra
inC5
7BL6J
chromosom
e2GRC
m38p1(NC
0000
687
GI37209910875704641ndash756755192
9123
bp)To
tal968
genetic
mutations
arerepo
rted
inNr
f2gene
and5k
bup
stream
(ge75704
642minus
1)and2k
bdo
wnstre
am(le75675512)sequences
asof
Janu
ary2003A
llSN
Psandallelesa
representedas
geno
miccontig
(reversedsequ
ence
indicated)E
xon175704
641ndash75704364(278
bpT
TS=75704
408)
exon
275679431ndash756791
65(267
bp)exon
375678579ndash75678490(90b
p)exon475677713ndash756775
46(168
bp)andexon
57567718
4ndash75678849
(1666
bp)Proteinam
inoacid
(aa)
resid
uesinNP00
61552
(597
aa)Cs
cod
ing-syno
nymou
sCn
scoding
-non
syno
nymou
sAminoacid
A-alanineD-aspartic
acidE
-glutamicacidF-phenylalanineG
-glycineH
-histidineI-iso
leucineL-leu
cineand
M-m
ethion
ineN-
asparagineP-prolin
eQ-glutamineR-arginineS-serineT-threon
ineandV-valin
elowastErrorsin
databasesfi
xedNC
notcon
firmed
20 Oxidative Medicine and Cellular Longevity
Table 6 NRF2 somatic mutations revealed in various human cancers
Domain Amino acid residues DNA mutation Cancer types (cases) ReferencesLocus Wild type Mutant
DLG motif
24 W (Trp) C (Cys) c72GgtCGgtT NSCLC neck ESC [54 62]K (Lys) c72TgtC ESC [62]
26 Q (Gln) E (Glu) c76CgtG NSCLC ESC [54 62]
27 D (Asp) G (Gly)lowast c80GgtA NSCLC [60]Y (Tyr) c79GgtT ESC [61]
28 I (Ile) T (Thr) c83CgtT NSCLC [54]
29 D (Asp) G (Gly) c86AgtG Head and neck ESC [54 62]H (His) c85GgtC NSCLC laynx [60 61]
30 L (Leu) F (Phe) c88CgtT NSCLC ESC [54 62]
31 G (Gly) A (Ala) c92GgtC NSCLC ESC skin [54 60ndash62]
32 V (Val) T (Thr) c95TgtG NSCLC [54]del c93 95delAGT ESC [61]
33ndash36 S-R-E-V S-R-E-V-S-R-E-Vlowast c97 108dupAGTCGAGCCGTA ESC [61]
34 R (Arg) Q (Gln) c101GgtA NCSCL [54 60 61]P (Pro) c101GgtC NSCLC [63]
ETGF motif
75 Q (Gln) H (His) c225AgtC Head and neck ESC [54 62]
77 D (Asp)
V (Val) c230AgtT NSCLC ESC [54 60 62]G (Gly) c230AgtG ESC [62]A (Ala) c230AgtC NSCLC [61]N (Asn) c229GgtA Larynx [61]
78 E (Glu) K (Lys) c232GgtA NSCLC ESC [54 62]
79 E (Glu)
K (Lys) c235GgtA NSCLC ESC [54 61 62]Q (Gln) c235GgtC NSCLC ESC [54 60ndash62]G (Gly) c236AgtG Larynx [61]E-E c234 236dupAGA ESC [61]
80 T (Thr) K (Lys) c239CgtACgtG NSCLC ESC [54 61 62]
80 T (Thr)P (Phe) c238AgtC ESC [62]I (Ile) c239CgtT Head and neck [54]A (Ala) c238AgtG NSCLC [63]
81 G (Gly) V (Val) c242GgtT ESC [61]D (Asp) c242GgtA NSCLC ESC [60ndash62]
82 E (Glu)
D (Asp) c246AgtT ESC oral cancer cell line [54 62]G (Gly) c245AgtG NSCLC [54]Q (Gln)lowast c244GgtC NSCLC ESC [60 61]V (Val)lowast c245AgtT ESC [61]
83 F (Phe) L (Leu)lowast c247TgtC NSCLC [60]NSCLC non-small cell lung cancer ESC esophageal squamous cancer and lowasterrors in reference fixed Number of cases 82 NSCLC and 10 ESC in [62] 125NSCLC 70 ESC 23 larynx and 17 skin in [61] 103 NSCLC and 12 head and neck in [54] 90 NSCLC in [63] 103 NSCLC in [60]
be involved in squamous lung cancer cell proliferation [64]suggesting that the mutation is functional and overcomesKEAP1 inhibition Singh et al [65] determined in vitrothat RNAi-mediated depletion of NRF2 in lung cancer cellsenhanced ROS production and susceptibility to cell deathby ionizing radiation These studies support the concept thatelevated NRF2 and ARE responsiveness provides cancer cellswith proliferative advantage for malignant transformation
and undue protection from anti-cancer therapy Onco-genic epidermal growth factor receptor (EGFR) signaling isrecently found to be critical in NRF2-mediated proliferationof NSCLC cells [66]
Collectively ldquogain of functionrdquo mutations in NRF2 thatreduce KEAP1 recognition are suggested to be predictivemarkers for poor responsiveness to chemotherapy and radi-ation therapy Although NRF2-mediated cellular defense
Oxidative Medicine and Cellular Longevity 21
processes are essential in the initiation stage enhancedNRF2-ARE activity in advanced stages of cancer developmentmay create a favorable intracellular environment for tumorcell growth and survival [67 68] In this context NRF2may be a potential molecular target for the treatment ofradio-resistance cancers especially those that have ldquoloss offunctionrdquo mutations in EGFR KRAS or KEAP1 as well asldquogain of functionrdquo mutations in NRF2
5 Epigenetic Alterations of NF-E2-RelatedFactor 2
Epigenetic modifications are alterations of molecules inter-acting with genes without changes to the primary DNAsequence They include post-translational modification ofhistones DNA methylation events chromatin conforma-tional changes and alterations to noncoding regulatoryRNAs Epigenetic alterations are stable and often inheritablebut are reversible and may affect expression of the geneDysregulation or defects in epigenetic processes particularlyhypermethylation of tumor suppressor gene promoters (egCpG islands) or histone modifications are thought to beassociated with carcinogenesis Investigators have reportedhypermethylation in CpG islands of KEAP1 which wereassociated with reduced KEAP1 expression in human cancersfrom lung prostate colon and so forth [69ndash72] Similar tosomatic mutations epigenetic changes on KEAP1 impairedthe function of its encoded protein leading to constitutiveNRF2 activation
Supporting the role of ldquopathogenic mutationsrdquo in NRF2expression of Nrf2 and downstream Nqo1 was suppressedin prostate tumors of mice (transgenic adenocarcinomaof mouse prostate TRAMP) Among 15 promoter CpGislands located between minus942 and minus654 (cminus1175 cminus1132and cminus1059 cminus887 gap in cminus1131 cminus1060 see Figure 3)hypermethylation of the first 5 CpG islands (minus942 minus899 andcminus1175 cminus1132) was significantly associated with tumorige-nesis [73] Moreover treatment with inhibitors for DNAmethyltransferase and histone deacetylase restored Nrf2expression in these tumor cells [73] A dietary phytochemicalcurcumin known as a DNA hypomethylation agent restoredepigenetically silent Nrf2 expression through CpG demethy-lation in carcinogen-induced mouse tumor cells [74]
The whole genome epigenetic datasets for 5 species arepublicly accessible at NCBI Epigenomics [75 76]The humanNRF2 epigenome of primary cells (breast penis) and H1stem cell line as well as mouse Nrf2 CpG island methylationdata for sperm blood and cerebellum are currently avail-able (httpwwwncbinlmnihgovepigenomics) Althoughno direct evidence of disease-associated epigenetic modu-lation has been identified in human NRF2 various phyto-chemical NRF2 agonists such as sulforaphane and curcuminhave shown their roles in DNA methylation and histonemodification (see reviews by Lee and colleagues eg [77])Taken together epigenetic modifications of theNRF2KEAP1axle are predicted to cause dysregulation of ARE-mediatedcellular defense leading to deleterious health effects and
phytochemical antioxidants as epigenetic modulators forNRF2 are suggested to be useful in cancer prevention
6 Conclusions
NRF2 is evolutionally conserved with high-sequence homol-ogy in many species However it is a highly mutable geneand numerous genetic variants have been discovered inhuman ethnic groups Importantly certain SNPs or haplo-types have been identified in various diseases as ldquoat-riskrdquoalleles and are related to functional alterations In additionto genetic variations multiple somatic mutations identifiedin the KEAP1 recognition domain of NRF2 in cancer cellshave been found to be oncogenic due to dysregulation ofNRF2 homeostasis by its excess ldquogain of functionrdquo Epigeneticalteration of theNRF2 is under investigation and is predictedto have pathogenic influences as learned from mouse andphytochemical agonist studies Continuous updates of Nrf2allelic variants in inbred mouse strains will provide a usefultool for effective experimental designs formodels of oxidativedisorders to provide insight into the disease mechanisms andintervention strategies
Abbreviations
AD Alzheimerrsquos diseaseALI acute lung injuryARE antioxidant response elementbZIP basic leucine zippercds coding DNA sequencesCI confidence intervalCOPD chronic obstructive pulmonary diseaseESC esophageal squamous cancerFEV1 forced expiratory volume in one second
Gbp giga base pairsGST glutathione S-transferaseGWAS genome-wide association studyHGVS Human Genome Variation SocietyHO-1 heme oxygenase-1119867 119901119910119897119900119903119894 119867119890119897119894119888119900119887119886119888119905119890119903 119901119910119897119900119903119894KEAP1 Kelch-like ECH activating protein 1MPD Mouse Phenome DatabaseMRP multidrug resistance proteinNCBI National Center for Biotechnology InformationNeh NRF2-ECH homologyNfe2l2 nuclear factor (erythroid-derived 2)-like 2Nrf2 NF-E2-related factor 2NSCLC nonsmall cell lung cancerNQO1 NAD(P)Hquinone oxidoreductase 1OR odds ratioPD Parkinsonrsquos diseasePM particulate matterROS reactive oxygen speciesSLE systemic lupus erythematosusSNP single nucleotide polymorphismSOD superoxide dismutaseURT untranslated region
22 Oxidative Medicine and Cellular Longevity
Disclosure
Authorrsquos contribution to the Work was done as part of theAuthorrsquos official duties as a NIH employee and is a Work ofthe United States Government Therefore copyright may notbe established in the United States
Conflict of Interests
The author declares that there is no conflict of interests
Acknowledgments
The research related to this paper was supported by theIntramural Research Program of the National Institutes ofHealth of the National Institute of Environmental HealthSciences (NIEHS) Drs Steven Kleeberger and StephanieLondon at the NIEHS provided excellent critical review ofthis paper The author thanks Mrs Jacqui Marzec for herhelpful comments and English editing
References
[1] J Y Chan M C Cheung P Moi K Chan and Y W KanldquoChromosomal localization of the humanNF-E2 family of bZIPtranscription factors by fluorescence in situ hybridizationrdquoHuman Genetics vol 95 no 3 pp 265ndash269 1995
[2] K Itoh K Igarashi N Hayashi M Nishizawa and MYamamoto ldquoCloning and characterization of a novel erythroidcell-derived CNC family transcription factor heterodimerizingwith the small Maf family proteinsrdquo Molecular and CellularBiology vol 15 no 8 pp 4184ndash4193 1995
[3] W O Osburn and T W Kensler ldquoNRF2 signaling an adaptiveresponse pathway for protection against environmental toxicinsultsrdquoMutation Research vol 659 no 1-2 pp 31ndash39 2008
[4] D Papp K Lenti D Modos D Fazekas Z Dul D Tureiet al ldquoThe NRF2-related interactome and regulome containmultifunctional proteins and fine-tuned autoregulatory loopsrdquoFEBS Letters vol 586 no 13 pp 1795ndash1802 2012
[5] X Wang D J Tomso B N Chorley et al ldquoIdentificationof polymorphic antioxidant response elements in the humangenomerdquo Human Molecular Genetics vol 16 no 10 pp 1188ndash1200 2007
[6] D Malhotra E Portales-Casamar A Singh et al ldquoGlobalmapping of binding sites for NRF2 identifies novel targets incell survival response through ChIP-Seq profiling and networkanalysisrdquo Nucleic Acids Research vol 38 no 17 pp 5718ndash57342010
[7] K Chan R Lu J C Chang and Y W Kan ldquoNRF2 a memberof the NFE2 family of transcription factors is not essential formurine erythropoiesis growth and developmentrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 93 no 24 pp 13943ndash13948 1996
[8] K Itoh T Chiba S Takahashi et al ldquoAn NRF2small Mafheterodimer mediates the induction of phase II detoxifyingenzyme genes through antioxidant response elementsrdquo Bio-chemical and Biophysical Research Communications vol 236no 2 pp 313ndash322 1997
[9] FMartin J M vanDeursen R A Shivdasani CW Jackson AG Troutman and P A Ney ldquoErythroid maturation and globin
gene expression inmicewith combined deficiency ofNF-E2 andNrf-2rdquo Blood vol 91 no 9 pp 3459ndash3466 1998
[10] K Itoh N Wakabayashi Y Katoh et al ldquoKEAP1 repressesnuclear activation of antioxidant responsive elements by NRF2through binding to the amino-terminal Neh2 domainrdquo Genesand Development vol 13 no 1 pp 76ndash86 1999
[11] A Kobayashi M I Kang H Okawa et al ldquoOxidative stresssensor KEAP1 functions as an adaptor for Cul3-based E3 ligaseto regulate proteasomal degradation of NRF2rdquo Molecular andCellular Biology vol 24 no 16 pp 7130ndash7139 2004
[12] K Taguchi H Motohashi and M Yamamoto ldquoMolecularmechanisms of the KEAP1-NRF2 pathway in stress responseand cancer evolutionrdquo Genes to Cells vol 16 no 2 pp 123ndash1402011
[13] K Mukaigasa L T Nguyen L Li H Nakajima M Yamamotoand M Kobayashi ldquoGenetic evidence of an evolutionarilyconserved role for NRF2 in the protection against oxidativestressrdquoMolecular and Cellular Biology vol 32 no 21 pp 4455ndash4461 2012
[14] JMaher andMYamamoto ldquoThe rise of antioxidant signalingmdashthe evolution and hormetic actions of NRF2rdquo Toxicology andApplied Pharmacology vol 244 no 1 pp 4ndash15 2010
[15] S C Lo X Li M T Henzl L J Beamer and M HanninkldquoStructure of the KEAP1NRF2 interface provides mechanisticinsight intoNRF2 signalingrdquoThe EMBO Journal vol 25 no 15pp 3605ndash3617 2006
[16] V A McKusick ldquoMendelian inheritance in man and its onlineversion OMIMrdquo American Journal of Human Genetics vol 80no 4 pp 588ndash604 2007
[17] B Yalcin D J Adams J Flint and T M Keane ldquoNext-generation sequencing of experimental mouse strainsrdquo Mam-malian Genome vol 23 no 9-10 pp 490ndash498 2012
[18] D R Bentley S Balasubramanian H P Swerdlow et alldquoAccurate whole human genome sequencing using reversibleterminator chemistryrdquo Nature vol 456 no 7218 pp 53ndash592008
[19] P S G Chain D V Grafham R S Fulton et al ldquoGenomeproject standards in a new era of sequencingrdquo Science vol 326no 5950 pp 236ndash237 2009
[20] RHWaterstonK Lindblad-Toh E Birney J Rogers J F AbrilP Agarwal et al ldquoInitial sequencing and comparative analysis ofthe mouse genomerdquo Nature vol 420 pp 520ndash562 2002
[21] T M Keane L Goodstadt P Danecek M A White K WongB Yalcin et al ldquoMouse genomic variation and its effect onphenotypes and gene regulationrdquoNature vol 477 pp 289ndash2942011
[22] T Yamamoto K Yoh A Kobayashi et al ldquoIdentification ofpolymorphisms in the promoter region of the human NRF2generdquo Biochemical and Biophysical Research Communicationsvol 321 no 1 pp 72ndash79 2004
[23] J M Marzec J D Christie S P Reddy et al ldquoFunctionalpolymorphisms in the transcription factor NRF2 in humansincrease the risk of acute lung injuryrdquo The FASEB Journal vol21 no 9 pp 2237ndash2246 2007
[24] H Fukushima-Uesaka Y Saito K Maekawa et al ldquoGeneticvariations and haplotype structures of transcriptional factorNRF2 and its cytosolic reservoir protein KEAP1 in JapaneserdquoDrug Metabolism and Pharmacokinetics vol 22 no 3 pp 212ndash219 2007
[25] E J Cordova R Velazquez-Cruz F Centeno V Baca and LOrozco ldquoThe NRF2 gene variant -653GA is associated with
Oxidative Medicine and Cellular Longevity 23
nephritis in childhood-onset systemic lupus erythematosusrdquoLupus vol 19 no 10 pp 1237ndash1242 2010
[26] M von Otter S Landgren S Nilsson et al ldquoAssociation ofNRF2-encoding NFE2L2 haplotypes with Parkinsonrsquos diseaserdquoBMCMedical Genetics vol 11 no 1 article 36 2010
[27] JMHartikainenM TengstromVMKosmaV L Kinnula AMannermaa andY Soini ldquoGenetic polymorphisms and proteinexpression of NRF2 and sulfiredoxin predict survival outcomesin breast cancerrdquo Cancer Research vol 72 pp 5537ndash5546 2012
[28] T Arisawa T Tahara T Shibata et al ldquoThe relationshipbetween Helicobacter pylori infection and promoter polymor-phism of the NRF2 gene in chronic gastritisrdquo InternationalJournal of Molecular Medicine vol 19 no 1 pp 143ndash148 2007
[29] T Arisawa T Tahara T Shibata et al ldquoThe influence ofpromoter polymorphism of nuclear factor-erythroid 2-relatedfactor 2 gene on the aberrant DNA methylation in gastricepitheliumrdquo Oncology Reports vol 19 no 1 pp 211ndash216 2008
[30] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene pro-moter polymorphism and gastric carcinogenesisrdquo Hepato-Gastroenterology vol 55 no 82-83 pp 750ndash754 2008
[31] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene promoterpolymorphism is associated with ulcerative colitis in a Japanesepopulationrdquo Hepato-Gastroenterology vol 55 no 82-83 pp394ndash397 2008
[32] C C Hua L C Chang J C Tseng C M Chu Y C Liu andW B Shieh ldquoFunctional haplotypes in the promoter region oftranscription factor NRF2 in chronic obstructive pulmonarydiseaserdquo Disease Markers vol 28 no 3 pp 185ndash193 2010
[33] S O Shaheen R B Newson S M Ring M J Rose-ZerilliJ W Holloway and A J Henderson ldquoPrenatal and infantacetaminophen exposure antioxidant gene polymorphismsand childhood asthmardquo The Journal of Allergy and ClinicalImmunology vol 126 no 6 pp 1141e7ndash1148e7 2010
[34] H Masuko T Sakamoto Y Kaneko et al ldquoLower FEV1 innon-COPD nonasthmatic subjects association with smokingannual decline in FEV1 total IgE levels and TSLP genotypesrdquoInternational Journal of Chronic Obstructive Pulmonary Diseasevol 6 pp 181ndash189 2011
[35] C P Guan M N Zhou A E Xu et al ldquoThe susceptibilityto vitiligo is associated with NF-E2-related factor 2 (NRF2)gene polymorphisms a study on Chinese Han populationrdquoExperimental Dermatology vol 17 no 12 pp 1059ndash1062 2008
[36] J Bouligand O Cabaret M Canonico et al ldquoEffect of NFE2L2genetic polymorphism on the association between oral estrogentherapy and the risk of venous thromboembolism in post-menopausal womenrdquo Clinical Pharmacology amp Therapeuticsvol 89 no 1 pp 60ndash64 2011
[37] D S OrsquoMahony B J Glavan T D Holden C Fong R A BlackG Rona et al ldquoInflammation and immune-related candidategene associations with acute lung injury susceptibility andseverity a validation studyrdquo PLoS ONE vol 7 no 12 Article IDe51104 2012
[38] I Ungvari E Hadadi V Virag et al ldquoRelationship between airpollutionNFE2L2 gene polymorphisms and childhood asthmain aHungarian populationrdquo Journal of CommunityGenetics vol3 no 1 pp 25ndash33 2012
[39] M SiedlinskiD S Postma JMA Boer et al ldquoLevel and courseof FEV1 in relation to polymorphisms inNFE2L2 andKEAP1 inthe general populationrdquo Respiratory Research vol 10 article 732009
[40] C C Hong C B Ambrosone J Ahn et al ldquoGenetic variabilityin iron-related oxidative stress pathways (NRF2 NQ01 NOS3
and HO-1) iron intake and risk of postmenopausal breastcancerrdquo Cancer Epidemiology Biomarkers and Prevention vol16 no 9 pp 1784ndash1794 2007
[41] C Canova C Dunster F J Kelly C Minelli P L Shah CCaneja et al ldquoPM10-induced hospital admissions for asthmaand chronic obstructive pulmonary disease the modifyingeffect of individual characteristicsrdquo Epidemiology vol 23 no 4pp 607ndash615 2012
[42] A J Henderson R B Newson M Rose-Zerilli S M RingJ W Holloway and S O Shaheen ldquoMaternal NRF2 andgluthathione-S-transferase polymorphisms donotmodify asso-ciations of prenatal tobacco smoke exposure with asthma andlung function in school-aged childrenrdquo Thorax vol 65 no 10pp 897ndash902 2010
[43] C Xing A L Sestak J A Kelly et al ldquoLocalization andreplication of the systemic lupus erythematosus linkage signalat 4p16 interaction with 2p11 12q24 and 19q13 in EuropeanAmericansrdquo Human Genetics vol 120 no 5 pp 623ndash631 2007
[44] T F Wong K Yoshinaga Y Monma K Ito H NiikuraS Nagase et al ldquoAssociation of KEAP1 and NRF2 geneticmutations and polymorphisms with endometrioid endometrialadenocarcinoma survivalrdquo International Journal of Gynecologi-cal Cancer vol 21 no 8 pp 1428ndash1435 2011
[45] M von Otter S Landgren S Nilsson et al ldquoNRF2-encodingNFE2L2 haplotypes influence disease progression but not riskin Alzheimerrsquos disease and age-related cataractrdquoMechanisms ofAgeing and Development vol 131 no 2 pp 105ndash110 2010
[46] S Tsang Z Sun B Luke et al ldquoA comprehensive SNP-basedgenetic analysis of inbred mouse strainsrdquoMammalian Genomevol 16 no 7 pp 476ndash480 2005
[47] C M Wade E J Kulbokas III A W Kirby et al ldquoThe mosaicstructure of variation in the laboratory mouse genomerdquoNaturevol 420 no 6915 pp 574ndash578 2002
[48] T Wiltshire M T Pletcher S Batalov et al ldquoGenome-widesingle-nucleotide polymorphism analysis defines haplotypepatterns in mouserdquo Proceedings of the National Academy ofSciences of the United States of America vol 100 no 6 pp 3380ndash3385 2003
[49] K A Frazer E Eskin H M Kang et al ldquoA sequence-basedvariation map of 827 million SNPs in inbred mouse strainsrdquoNature vol 448 no 7157 pp 1050ndash1053 2007
[50] T P Maddatu S C Grubb C J Bult andM A Bogue ldquoMousephenome database (MPD)rdquo Nucleic Acids Research vol 40 ppD887ndashD894 2007
[51] A Kirby H M Kang C M Wade et al ldquoFine mapping in 94inbred mouse strains using a high-density haplotype resourcerdquoGenetics vol 185 no 3 pp 1081ndash1095 2010
[52] H Y Cho A E Jedlicka S P M Reddy L Y Zhang T WKensler and S R Kleeberger ldquoLinkage analysis of susceptibilityto hyperoxia NRF2 is a candidate generdquo American Journal ofRespiratory Cell and Molecular Biology vol 26 no 1 pp 42ndash512002
[53] H Y Cho A E Jedlicka S P M Reddy et al ldquoRole of NRF2in protection against hyperoxic lung injury in micerdquo AmericanJournal of Respiratory Cell and Molecular Biology vol 26 no 2pp 175ndash182 2002
[54] T Shibata T Ohta K I Tong et al ldquoCancer related mutationsin NRF2 impair its recognition by KEAP1-Cul3 E3 ligase andpromote malignancyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 36 pp13568ndash13573 2008
24 Oxidative Medicine and Cellular Longevity
[55] A Singh V Misra R K Thimmulappa et al ldquoDysfunctionalKEAP1-NRF2 interaction in non-small-cell lung cancerrdquo PLoSMedicine vol 3 no 10 article e420 2006
[56] T Ohta K Iijima M Miyamoto et al ldquoLoss of KEAP1 functionactivates NRF2 and provides advantages for lung cancer cellgrowthrdquo Cancer Research vol 68 no 5 pp 1303ndash1309 2008
[57] B Padmanabhan K I Tong T Ohta et al ldquoStructural basisfor defects of KEAP1 activity provoked by its point mutationsin lung cancerrdquoMolecular Cell vol 21 no 5 pp 689ndash700 2006
[58] T Shibata A Kokubu M Gotoh et al ldquoGenetic alteration ofKEAP1 confers constitutive NRF2 activation and resistance tochemotherapy in gallbladder cancerrdquoGastroenterology vol 135no 4 pp 1358e4ndash1368e4 2008
[59] N J Yoo H R Kim Y R Kim C H An and S H LeeldquoSomatic mutations of the KEAP1 gene in common solidcancersrdquo Histopathology vol 60 no 6 pp 943ndash952 2012
[60] Y Hu Y Ju D Lin Z Wang Y Huang S Zhang et alldquoMutation of the NRF2 gene in non-small cell lung cancerrdquoMolecular Biology Reports vol 39 no 4 pp 4743ndash4747 2012
[61] Y R Kim J EOhM S Kim et al ldquoOncogenicNRF2mutationsin squamous cell carcinomas of oesophagus and skinrdquo TheJournal of Pathology vol 220 no 4 pp 446ndash451 2010
[62] T Shibata A Kokubu S Saito et al ldquoNRF2 mutation confersmalignant potential and resistance to chemoradiation therapyin advanced esophageal squamous cancerrdquo Neoplasia vol 13pp 864ndash873 2011
[63] H Sasaki M Shitara K Yokota Y Hikosaka S MoriyamaM Yano et al ldquoIncreased NRF2 gene (NFE2L2) copy numbercorrelates with mutations in lung squamous cell carcinomasrdquoMolecular Medicine Reports vol 6 no 2 pp 391ndash394 2012
[64] H Sasaki M Shitara K Yokota Y Hikosaka S Moriyama MYano et al ldquoRagD gene expression andNRF2mutations in lungsquamous cell carcinomasrdquo Oncology Letters vol 4 pp 1167ndash1170 2012
[65] A Singh M Bodas N Wakabayashi F Bunz and S BiswalldquoGain of NRF2 function in non-small-cell lung cancer cellsconfers radioresistancerdquo Antioxidants and Redox Signaling vol13 no 11 pp 1627ndash1637 2010
[66] T Yamadori Y Ishii SHommaYMorishimaKKurishimaKItoh et al ldquoMolecular mechanisms for the regulation of NRF2-mediated cell proliferation in non-small-cell lung cancersrdquoOncogene vol 31 pp 4768ndash4777 2012
[67] A K Bauer H Y Cho L Miller-Degraff C Walker K HelmsJ Fostel et al ldquoTargeted deletion of NRF2 reduces urethane-induced lung tumor development in micerdquo PLoS ONE vol 6no 10 Article ID e26590 2011
[68] H Y Cho and S R Kleeberger ldquoNRF2 protects against airwaydisordersrdquo Toxicology and Applied Pharmacology vol 244 no1 pp 43ndash56 2010
[69] R Wang J An F Ji H Jiao H Sun and D Zhou ldquoHyperme-thylation of the KEAP1 gene in human lung cancer cell linesand lung cancer tissuesrdquo Biochemical and Biophysical ResearchCommunications vol 373 no 1 pp 151ndash154 2008
[70] P Zhang A Singh S Yegnasubramanian et al ldquoLoss ofkelch-like ECH-associated protein 1 function in prostate cancercells causes chemoresistance and radioresistance and promotestumor growthrdquoMolecular Cancer Therapeutics vol 9 no 2 pp336ndash346 2010
[71] N Hanada T Takahata Q Zhou et al ldquoMethylation of theKEAP1 gene promoter region in human colorectal cancerrdquoBMCCancer vol 12 article 66 2012
[72] L A Muscarella R Barbano V DrsquoAngelo et al ldquoRegulationof KEAP1 expression by promoter methylation in malignantgliomas and association with patientrsquos outcomerdquo Epigeneticsvol 6 no 3 pp 317ndash325 2011
[73] S Yu T O Khor K L Cheung et al ldquoNRF2 expressionis regulated by epigenetic mechanisms in prostate cancer ofTRAMP micerdquo PLoS ONE vol 5 no 1 Article ID e8579 2010
[74] T O Khor Y Huang T Y Wu L Shu J Lee and A N KongldquoPharmacodynamics of curcumin as DNA hypomethylationagent in restoring the expression of NRF2 via promoter CpGsdemethylationrdquo Biochemical Pharmacology vol 82 no 9 pp1073ndash1078 2011
[75] I M Fingerman L McDaniel X Zhang et al ldquoNCBI epige-nomics a new public resource for exploring epigenomic datasetsrdquo Nucleic Acids Research vol 39 supplemen 1 pp D908ndashD912 2011
[76] I M Fingerman X Zhang W Ratzat N Husain R F Cohenand G D Schuler ldquoNCBI epigenomics whatrsquos new for 2013rdquoNucleic Acids Research vol 41 no 1 pp D221ndashD225 2013
[77] J H Lee T O Khor L Shu Z Y Su F Fuentes andA N Kong ldquoDietary phytochemicals and cancer preventionNRF2 signaling epigenetics and cell death mechanisms inblocking cancer initiation and progressionrdquo Pharmacology ampTherapeutics vol 137 no 2 pp 153ndash171 2012
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Disease Markers
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BioMed Research International
OncologyJournal of
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Oxidative Medicine and Cellular Longevity
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PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
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Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Oxidative Medicine and Cellular Longevity 13
Table4Con
tinued
IDMap
onchromosom
e2 (H
GVS
name)
Region
class
Dise
asea
ssociatio
nandreferences
Risk
allelesa
ndstatistics
Ethn
icgrou
p(num
bero
fcase)
Breastcancer
[40]
Twith
NQO1N
OS3H
O1risk
allelesOR=15
6CI
=097ndash251
Caucasia
nandothers(505)
rs1806
649
(rs58745895)
g178118152Cgt
TIntro
n1
Basaland
smoker
FEV
1(200
9[39])
T=02631119Daggerin
haplotype
CI=minus8730ndash
(minus17
0)Netherla
nd(2542)
Parkinsonrsquos
disease(2010
[26])
T=0422148Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
Particulatem
attera
ndasthmaCO
PDadmiss
ion(2012[41])
Cwith
lowvitamin
Clevel(OR=
31CI
=15
0ndash630)
UK(209)
rs4243387
(rs6003846
4)g178117765Cgt
TIntro
n1
Basaland
smoker
FEV
1(200
9[39])
T=00914
25Daggerin
haplotype
Netherla
nd(2542)
rs1962142
(rs58448508)
g178113484Agt
GIntro
n1
Ann
ualF
EV1decline
(2011[34])
A=0082in
haplotype
Japanese
(915)
Breastcancer
NRF
2andARE
expressio
n(2012[27])
A(119875=0036)
FinlandKB
CP(452)
rs6726395
(rs57309289)
g178103229Agt
GIntro
n1
Smok
ing-relatedFE
V1decline
and
annu
alFE
V1decline
(2011[34])
G=0884Dagger
A=0082in
haplotype
Japanese
(915)
Basaland
smoker
FEV
1(200
9[39])
G=046
41764Daggerin
haplotype
Netherla
nd(2542)
rs2001350
(rs17515179
rs60883775)
g178100
425Cgt
TIntro
n1
Ann
ualF
EV1decline
(2011[34])
T=0082in
haplotype
Japanese
(915)
Parkinsonrsquos
disease(2010
[26])
T=0986350Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
rs10183914
(rs58731187
rs61374844
)g17809766
6Cgt
TIntro
n3
Parkinsonrsquos
disease(2010
[26])
T=0536188Dagger
inhaplotype
OR=09or
04CI
=060ndash140or
030ndash0
60
Swedish
Polish
Caucasian
(357)
rs2706110
g178092162Tgt
C31015840Flanking
Breastcancer
(2012[27])
TT
OR=2079CI
=118ndash368
FinlandKB
CP(452)
rs2588882
g178087165Ggt
T31015840Flanking
Infection-indu
cedasthma(
2012
[38])
TG
OR=0290CI
=013ndash0
62
Hun
garia
nCaucasia
nGypsy
(307)
minus686in
reference[22]
=minus653in
reference[23]
=currentlyminus214minus684in
reference[22]
=minus651inreference[23]
=currentlyminus212minus651inreference[22]
=minus617in
reference[23]
=currentlyminus178Ch
romosom
econtig(in
tron
31015840flank
ing)
orreversed
(51015840flank
ingprom
oter)a
llelesinbo
ldhave
been
used
inthetextand
TableOR
odds
ratio
CI95confi
denceinterval
14 Oxidative Medicine and Cellular Longevity
minus212GG (98 rs67006649 previously minus684minus651) minus178Callele (73) and GCC
4(53) was predicted to increase
respiratory failure development (hazard ratio = 095 CI091ndash099) in German COPD patients [32] Significant inter-action was also identified between an intronic SNP Gallele (rs6726395 g178103229AgtG 884 frequency) andsmoking status on FEV
1decline relative to the reference
AA allele in the above Japanese cohort [34] Siedlinskiet al [39] reported that the CC genotype of anotherintronic SNP (rs2364723 g178126546GgtC) was associatedwith a lower FEV
1level compared to the wild-type geno-
type (GG) in two Netherland cohorts (CI minus636minus178frequency = 0525 and pooled cohort size = 2542) ThisSNP alone or as a haplotype with 4 more intronic SNPs(rs13001694Grs1806649Trs4243387Trs6726395G)was alsoassociated with high FEV
1levels in individuals that ever
smoked [39] In a Hungarian population of childhoodasthma SNPs at minus178 (CA) and 31015840 flanking (rs2588882TG)loci were inversely associated with infection-induced asthma(OR 0437 CI 028ndash080 OR 0290 CI 013ndash062 resp) andthese SNPs significantly influenced an asthma-environmentalpollution interaction [38]The intronic SNP rs1806649 (CgtT)was associated but not significantly with an increased risk ofhospitalization during high-level particulate matter (PM
10)
periods in asthma or COPD patients (119899 = 209) of the UnitedKingdom (UK) [41] Asthma andCOPDadmission rateswererelated to the increase in environmental PM
10concentration
Importantly effects of interaction between prenatal stressand NRF2 SNPs on descendant pulmonary health wereinvestigated by the Avalon Longitudinal Study in the UKmaternal smoking during pregnancy was not associated withlung function change determined by maximum mild expi-ratory flow (FEF
25ndash75) or with asthma incidence in school-aged children and this relation was not modified by NRF2SNP genotypes [42]However early gestation acetaminophenexposure significantly influenced the risk of asthma andwheezing at the age of 7 years in gt4000 mothers and gt5000children [33] When maternal copies of the minus212A allele werepresent association with asthma (11374891 OR 173 CI122ndash245) and wheezing (11494949 OR 153 95 CI 106ndash220) was significantly increased [33]
Gastrointestinal Disorders While there was no evidence inlung cancer cases studies in Japanese populations suggesteda potential association of NRF2 variations with gastro-intestinal tumorigenesisHelicobacter pylori (H pylori) causesgastritis which can lead to gastric atrophy and cancer Ingastric epithelium from the Japanese cancer cohorts (39gastric cancers 46 controls) H pylori infection was posi-tively correlated with aberrant CpG island methylation oftumor suppressor genes (eg p14) and minus214Gminus212G orminus214Aminus212G NRF2 haplotype was significantly associatedwith increased (OR 290 95 CI 114ndash736) or decreased(OR 033 95 CI 013ndash088) risk of the CpG methyla-tion respectively in the H pylori-infected patients [29]Further study from the same investigators determined thatminus214Aminus212G allele carriers had significantly (119875 = 0022)reduced risk of gastric cancer inH pylori-negative cases [30]The minus214AGminus212AG genotypes were negatively associated
(OR 045 CI 022ndash093) and the minus214Gminus212G genotypeswere positively associated (chronic continuous phenotypeOR 257 CI 101ndash660) with ulcerative colitis (89 patients 141controls) in a Japanese population [31]
Autoimmune Disorders Systemic lupus erythematosus (SLE)is a long-term autoimmune disease more frequently found infemales than in males It affects organs including skin jointskidneys and brain and nephritis is an aggressive character-istic in some patients Genome-wide association studies inhumans identified a suggestive quantitative trait locus nearNRF2 [43] A study of a Mexican Mestizo population (362patients with childhood-onset SLE 379 controls and 212nephritis diagnosed) determined that lupus with nephritiswas significantly (OR 181 CI 104ndash312) associated with theminus214GA SNP in females [25] The same SNPs were notclosely associated with SLE risk in a Japanese cohort [22]Vitiligo is a skin condition in which there is a loss of browncolor (pigment) from areas of skin resulting in irregularwhite patches It is thought to be an autoimmune diseasecaused by loss of cells (melanocytes) that produce brownpigment A study indicated that theminus178A allele increased therisk of vitiligo dose-dependently (OR 1724 95 CI 135ndash221for CA OR 2902 CI 162ndash519 for AA) [35]
Female Disorders It is well known that estrogen metabolites(eg catechols) cause ROS formation suggesting correla-tion of NRF2 and downstream effectors in postmenopausalmammary cancer In a study of a Finish population (KuopioBreast Cancer Project 119899 = 452 patients 370 controls)the minus178AA homozygous genotype (OR 4656 CI = 135ndash1606) and 31015840 flanking rs2706110 (TT OR 2079 CI 118ndash368) genotype were associated with increased risk of breastcancer while the 51015840 flanking minus3306TT homozygous allelewas significantly associated with lower survival (frequency =71219 OR 1687 CI 1105ndash275) [27] suggesting that NRF2genetic polymorphisms affect susceptibility and outcome ofthe patients The minus178A allele carriers together with intronicrs1962142A allele carriers were associated with lowered tissuelevels of NRF2 proteins [27] In postmenopausal women theminus178A allele (OR 179 95CI 370ndash8570) appeared tomodifythe risk of venous thromboembolism caused by oral estrogentherapy (AA or AC frequency = 333) as demonstratedby the French ESTHER study (161 cases 474 controls) [36]An intronic rs1806649CgtT SNP did not associate with breastcancer risk in postmenopausal women [40] However whenthis SNP and other at-risk alleles of ARE-responsive genes(NQO1 HO-1 NOS3) were combined there was a significantgene-dose effect on the breast cancer risk [40] Althoughcoding region SNPs in NRF2 and KEAP1 were identifiedin the Japanese endometrial adenocarcinoma patients noassociation of NRF2 SNPs with the disease was found [44]
Neurodegenerative Diseases Oxidative stress is known to beinvolved in Parkinsonrsquos disease (PD) presumably due toproduction of ROS fromhigh-dopaminemetabolism and lowlevels of antioxidants in the substantia nigra of the brainInvestigators found a protective NRF2 haplotype consistingof four 51015840 flanking SNPs (minus5238Gminus214Aminus212Gminus178C)
Oxidative Medicine and Cellular Longevity 15
and 4 intronic SNPs (rs2886161Ars1806649Ars2001350Ars10183914A) from Swedish (OR 09 CI 060ndash140) and Polish(OR 04 CI 030ndash060) populations (total 165+192 PD cases190+192 controls) [26]The investigators also suggested thatNRF2 haplotype alleles were associated with 2 years earlierage of Alzheimerrsquos disease (AD) onset 4 years earlier age ofposterior subcapsular cataract surgery and 4 years later ageof cortical cataract surgery while they were not significantlyrelated to AD or age-related cataract risk [45]
34 Genetic Mutations in Mouse Nrf2 Tsang et al [46]compiled 673 SNPs in 55 mouse strains and constructedtheir phylogenetic tree to correlate and clarify the originsof strains based on the assembled mouse genome sequenceand SNP data [20 47 48] Recently using the completegenome sequence of C57BL6J (B6) mouse as a referencehigh-density SNP screening in other laboratory strains orin panels of strains has been published (see [17]) Althoughmillions of mouse SNPs (gt10089892 as of December 2012)and haplotype mappings from more than 120 strains havebeen published as valuable references for dissecting thegenetic basis of complex traits [49ndash51] little attention hasbeen paid to polymorphisms of Nrf2 and their correlationwith disease phenotypes
Figure 3 demonstrates the proximal promoter region(minus1 to minus950)51015840-UTR (exon 1 up to TSS) and proteinsequence of mouse Nrf2 based on GRCm38p1 PrimaryAssembly (75704641ndash75675513 bp) mRNA variant1 and protein (NP 035032 encoded by NM 010902234ndash2027 bp) sequences Genetic variations in the Nrf2genome of inbred strains collected from public databasesare listed in Tables S2 and 5 (See Supplementary TableS2) Overall 968 genetic mutations are compiled forNrf2 gene and 5 kb upstream2 kb downstream regions785 SNPs between B6 and another 16 strains wereacquired from the Mouse Phenome Database (MPDhttpphenomejaxorg dbqrtn=snpret1) and additionalSNPs and other mutations were acquired from NCBI dbSNP(httpwwwncbinlmnihgovsnpterm=mus+musculus20nfe2l2) In total 132 mutations are in the promoter (37in proximal 1 kb) 49 in exons (38 in coding region 19 Cns)727 in introns and 60 in the 31015840 flanking region Excludingmutations in the 51015840 and 31015840 flanking sequences murine Nrf2sequences appear to be more highly variable (1 variationper 375 bp) than much of the mouse genome which has anapproximate frequency of one SNP per every 245 bp (httpwwwinformaticsjaxorgmgihomehomepagesstatsallstatsshtmlallstats snp)
Nrf2 was found to be a susceptibility gene from genome-wide linkage analysis in a murine model of hyperoxia-induced ALI [52] A promoter SNP minus103TgtC (previouslypublished as minus336TgtC) in Nrf2 was found and predicted toadd an additional Sp1 binding site in hyperoxia-susceptibleB6 mice but not in resistant C3HHeJ mice [52] Genotypesfrom the SNP and from simple-sequence length polymor-phism markers of the Nrf2 locus (D2Mit248 and D2Mit94)cosegregated in the B6C3F
2mouse cohort [52] and Nrf2
deficient mice were significantly more susceptible to ALI
sub-phenotypes caused by hyperoxia than similarly exposedwild-type mice supporting Nrf2 as a contributor to thephenotypic traits [53] Although no other functional analyseson Nrf2 SNPs or haplotype association studies have beenconducted in inbred mice strains bearing haplotypes suchas multiple Cns in functional domains (eg F71L L451VH543Q and L575M) may be useful to elucidate the role ofNrf2 in differential susceptibility to oxidative diseases
4 Oncogenic Somatic Mutations in HumanNF-E2-Related Factor 2
Somatic mutation is a change in the DNA of somatic cellsthat affects derived cells but is not inherited by offspringEfforts to discover somatic mutations have provided insightinto mutagenesis and cancer development Lung cancerparticularly non-small cell lung cancer (NSCLC) is theleading cause of cancer death worldwide Somatic mutationsof NRF2 and KEAP1 discovered in lung cancer patients havedetermined the oncogenic potential ofNRF2 [54 55] KEAP1somatic mutations were associated with its reduced proteinlevels in lung cancer tissues and cells [56 57] Investigationsof NSCLC in various ethnic populations as well as cancersin gastrointestine breast and prostate have coordinatelydemonstrated that multiple Cns somatic mutations in KEAP1cause dysfunction of the translated protein and in turnconstitutive activation of NRF2 increasing risk of neoplasiaand chemoresistance [12 55 58 59] Somatic mutations ofNRF2 have been detected in various cancer tissues (largelysquamous cell carcinomas) in Asian populations (Table 6)NRF2 mutations were significantly associated with NSCLCcases (squamous cell lung carcinoma adenocarcinoma) ofthe Japanese (107 [54]) the Chinese (23 [60]) andthe Koreans (8 [61]) as well as with lung cancer celllines Smoking history was also correlated with mutationoccurrence in all of the studies [54 60 61] In addition tolung cancers laryngeal squamous carcinoma (13 in [61])esophageal squamous cancer (ESC 22 in [60] 114 in[61]) head and neck cancers (25 in [54]) skin (117 casein [61]) and oral cancer cell lines had somatic changesin NRF2 In contrast to wide-spread KEAP1 mutationsmutations in NRF2 were clustered in DLGETGE motifs ofthe Neh2 domain which are critical in the ldquohinge and latchrdquomodel of KEAP1 binding [12] Similar to KEAP1 somaticmutations it has been postulated that NRF2 mutations incancer cells lead to NRF2 accumulation by suppressing itsubiquitination or KEAP1 binding which eventually confersmalignant potential and resistance to chemotherapy
Most variable sites in NRF2 included aa residues 29 (AspD) 31 (Gly G) 77 (Asp D) and 79 (Glu E) (Table 6)Residue 33 (Ser S) in the Neh2 domain is mutated by eithergenetic or somatic processes (Figure 4) Cns in the EDGFmotif of NRF2 was experimentally determined to impairrecognition of KEAP1 [54] NRF2 mutations were signif-icantly correlated with increased (25-fold) copy number(31 of mutants versus 3 wild types) in Japanese NSCLCcases [63] Aberrant mutation of NRF2 also led to increasedexpression of downstream effectors includingRagD known to
16 Oxidative Medicine and Cellular Longevity
pL575M
pF71L
pH543QpL451V
pA151P pA151V pN159D pQ198delpV105D
pA204T pS224I225S226del pT253S pS278ApD305G
pM409V pR410HpP388L pA389G pT395I
Genomic DNA (reversed contig GRCm38 p1 C57BL6JNM 0109023)
Protein (NP 035032)1
51101151201251301351401451501551
minus400
minus500
minus50
minus100
minus200
minus300
minus450
minus350
minus250
minus150
minus950
minus900
minus850
minus800
minus750
minus700
151
101151201
2001
g minus470gg minusg minus
c minus41c minus
c
c
c c
gg minus 6C
g minus125insTTC g minus118insC
g minus281delC
g minus63delCg minus1 (75704642 c minus234)
Translation start site (c +1)Termination codon (c +2025 +2027)
c minus136 minus134in-frame stop codonc minus8585
g minus41
246
440
minus381
minus11
minus91 minus90
minus332
minus153
minus98
c minus197c minus209 minus202
gminus942 minus899 (cminus1175 minus1132) CpG islands [71]
g 460CgtT
g minus103CCgtT [51]
CgtTCgtG
g minus320CgtT
CgtT
CgtT
CgtTg minus426GgtA
GgtACgtA
CgtA
GgtA
g minus238GgtTTgtC
TgtC
TgtC
TgtCTgtG
g minus229AgtG
CgtG
CgtG
GgtC
gminus826 minus654 (cminus1059 ) CpG islands [71]minus887
Figure 3 DNA (partial promoter and exons 1 and 5) and protein sequence ofmouseNrf2 SNPs and amino acid residues for non-synonymousSNPs are marked Promoter regions bearing 5CpG islands are underlined
151
201251301351401451501551601
Genetic mutation (Table 3 and Figure 2)Somatic mutation (Table 6)
Neh2
Neh3
Neh4Neh5
Neh6
Neh1
Protein isoform 1 (NP 006155)
101151
Figure 4 Genetic and somatic mutation loci in human NRF2 protein
Oxidative Medicine and Cellular Longevity 17
Table5Mou
seup
stream
andexon
varia
tions
ofNfe2
l2locusin17
inbred
strainsR
eference
sequ
ence
isC5
7BL6J
(B6strain
1)geno
me(GI14933824975547698ndash75513576)SNPalleleand
geno
typesareshow
nas
chromosom
econ
tigsequ
enceStrains
2129S1S
vlmJ3AJ
4AKR
J5BA
LBcJ6C3
HH
eJ7C
57BL
6NJ8CA
STEiJ9CB
AJ
10D
BA2J11FVBNJ12LPJ
13N
ODShiLtJ14N
ZOH
ILtJ
15P
WK
PhJ16SPR
ETEiJ
17W
SBEiJ
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs2566
08517
75705565
GA
(CT)
51015840Flankingminus924
NC
GG
GG
GG
GG
GG
GG
GG
AA
Grs47274959
75705562
AT
(TA
)51015840Flankingminus921
NC
AT
TA
TT
AA
TT
AT
AT
TT
Ars216940398
75705554ndash75705555
Tadd(A
add)
51015840Flankingminus913minus914
NC
CGrs239114134
75705540
CT(G
A)
51015840Flankingminus899
NC
CT
TC
TT
CC
TT
CT
CT
CT
Crs46
461765
75705528
TG(A
C)oA
C
51015840Flankingminus887
NC
TG
GT
GG
TT
GG
TG
TG
GG
Trs5144
9853
75705525
CA(G
T)
51015840Flankingminus884
NC
CA
AC
AA
CC
AA
CA
CA
CC
Crs249093111
75705512
CT(G
A)
51015840Flankingminus871
NC
CT
TC
TT
CC
TT
CT
CT
TT
Crs263745200
75705510
CG(G
C)
51015840Flankingminus869
NC
GG
GG
GG
GG
GG
GG
GG
CG
Grs45651867
75705498
TG(A
C)
51015840Flankingminus857
NC
TG
GT
GG
TT
GG
TG
TG
GG
Trs219997531
75705495
GA
(CT)
51015840Flankingminus854
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs251918379
75705451
TA(A
T)
51015840Flankingminus810
NC
TT
TT
TT
TT
TT
TT
TT
TA
Trs27978306
75705449
TA(A
T)
51015840Flankingminus808
NC
TA
AT
AA
TT
AA
TA
TA
TT
Trs216087412
75705429
TG(A
C)lowast
51015840Flankingminus788
NC
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
GTlowast
Tlowast
rs261229914
75705423ndash75705424
AGdel(CT
del)
51015840Flankingminus783minus784
NC
AGrs27978307
75705400
CT(G
A)
51015840Flankingminus759
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs243167395
75705359
GA
(CT)
51015840Flankingminus718
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs27978308
75705307
GA
(CT)
51015840Flankingminus66
6NC
GG
GG
GG
GG
GG
GG
GG
AG
Grs254744
09875705294
AT
(TA
)51015840Flankingminus653
NC
AA
AA
AA
AA
AA
AA
AA
AT
Ars228133419
75705179
GT
(CA
)51015840Flankingminus538
NC
GG
GG
GG
GG
GG
GG
GG
GT
Grs214719520
75705111
GA
(CT)
51015840Flankingminus470
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs2440
87730
75705101
GA
(CT)
51015840Flankingminus46
0NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs227781699
75705081
CG(G
C)
51015840Flankingminus44
0NC
CC
CC
CC
CC
CC
CC
CC
GC
Crs257870353
75705067
CT(G
A)
51015840Flankingminus426
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs24487744
075705022
AG
(TC)
51015840Flankingminus381
NC
AA
AA
AA
AA
AA
AA
AA
AG
Ars234628138
75704973
AC
(TG
)51015840Flankingminus332
NC
AA
AA
AA
AA
AA
AA
AA
AC
A
18 Oxidative Medicine and Cellular Longevity
Table5Con
tinued
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs247275247
75704961
GA
(CT)
51015840Flankingminus320
NC
GG
GG
GG
GG
GG
GG
AG
GG
Grs247519047
75704922
Gdel(Cdel)
51015840Flankingminus281
NC
Grs27978309
(rs51915758)
75704887
GA
C(C
TG
)51015840Flankingminus246
NC
GA
AG
AA
GG
AA
GA
GA
CC
G
rs218139102
75704879
CA(G
T)
51015840Flankingminus238
NC
CC
CC
CC
CC
CC
CC
CC
AC
Crs251990355
75704870
TC(A
G)
51015840Flankingminus229
NC
TT
TT
TT
TT
TT
TT
TT
TC
Trs238746955
75704794
AG
(TC)
51015840Flankingminus153
NC
AA
AA
AA
AA
AA
AA
AA
GA
Ars221664
405
75704786
AG
(TC)
51015840Flankingminus145
NC
AA
AA
AA
AA
AA
AA
AA
AG
Ars217054035
75704766
ndash75704767
GAAadd(TTC
add)
51015840Flankingminus125minus126
NC
GA
rs248182931
75704759ndash75704760
Gadd(C
add)
51015840Flankingminus118
minus119
NC
AGrs217197904
75704744
GA
(CT)
51015840Flankingminus103
(+)S
p1[23]
GA
AG
AA
GG
AA
GA
AA
GG
Ars2644
9364975704704
Gdel(Cdel)
51015840Flankingminus63
NC
Grs2404
81112
757046
82CT(G
A)
51015840Flankingminus41
NC
CC
CC
CC
CC
CC
CC
CC
CT
Crs27978310
75704617
AG
(TC)
UTR
-525
NC
AA
AA
AA
AA
AA
AA
AA
GG
Ars27978311
75704610
CG(G
C)
UTR
-532
NC
GG
GG
GG
GG
GG
GG
GG
GG
Crs27978312
757046
05GA
(CT)
UTR
-537
NC
GA
AG
AA
GG
AA
GA
GA
GG
Grs214784220
757044
99AG
(TC)
UTR
-5143
NC
AA
AA
AA
AA
AA
AA
AA
GG
Ars244146318
757044
98GT
(CA
)UTR
-5144
NC
GG
GG
GG
GG
GG
GG
GG
GT
Grs27978313
757044
97GC
(CG
)UTR
-5145
NC
GC
CG
CC
GC
CC
GC
CC
CC
Crs215431944
757044
93CT(G
A)
UTR
-5149
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs257015697
757044
49GA
(CT)
UTR
-5193
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs252234782
757044
19GT
(CA
)UTR
-5223
NC
Grs1346
0861
75679262
AG
(TC)
Exon
244
6Cn
sF71L
AG
GA
GG
AG
GG
AG
AG
GG
Grs1346
0859
75679202
GA
(CT)lowast
Exon
2506
CsH91=
GA
AG
AA
GG
AA
GA
GA
GG
Grs27978436
75679187
GA
(CT)lowast
Exon
2521
CsT9
6=G
GG
GG
GG
AG
GG
GG
GA
GG
rs226131070
75679178
GA
(CT)
Exon
2530
CsS99=
GG
GG
GG
GG
GG
GG
GG
GA
Grs227683834
75678576
AT
(TA
)Ex
on3
547
CnsV
105D
AA
AA
AA
AA
AA
AA
AA
AT
Ars257321187
75678506
TC(A
G)
Exon
3617
CsP128=
TT
TT
TT
TC
TT
TT
TT
TT
Trs27978444
75678500
TA(A
T)
Exon
3623
CsV130=
TT
TT
TT
TA
TT
TT
TT
AT
Trs227743136
75677686
GA
(CT)
Exon
4662
CsH143=
GG
GG
GG
GG
GG
GG
GG
GA
Grs215327202
75677664
CG(G
C)
Exon
4684
CnsA
151P
CC
CC
CC
CG
CC
CC
CC
CC
Crs247539755
75677663
GA
(CT)
Exon
4685
CnsA
151V
GG
GG
GG
GA
GG
GG
GG
GG
Grs233164
668
75677640
TC(A
G)
Exon
4708
CnsN
159D
TT
TT
TT
TT
TT
TT
TT
TC
T
rs2340
95816
75677162ndash75677160
GCT
del
(AGCdel)
Exon
5826minus
828
Cds-in
delQ
198
GCT
Oxidative Medicine and Cellular Longevity 19
Table5Con
tinued
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs27978452
75677145
CT(G
A)
Exon
5843
CnsA
204T
CC
CC
CC
CC
CC
CC
CC
CC
T
rs225047937
75677086ndash75677078
GAG
ATCG
ATdel
(ATG
CATC
TCdel)
Exon
5902minus
910
Cds-in
del
(S224I225S226)
GAG
ATC
GAT
rs241469868
75676998
TA(G
T)
Exon
5990
CnT2
53S
TT
TT
TT
TT
TT
TT
TT
TA
Trs221602571
75676923
AC
(TG
)Ex
on5
1065
CnsS
278A
AA
AA
AA
AA
AA
AA
AA
AC
Ars252576472
75676876
GA
(CT)
Exon
51112
CsS293=
GG
GG
GG
GG
GG
GG
GG
GA
Grs250802933
75676841
TC(A
G)
Exon
5114
7Cn
sD305G
TT
TT
TT
TT
TT
TT
TT
TC
Trs225425698
75676792
CT(G
A)
Exon
5119
6Cs
P321=
CC
CC
CC
CC
CC
CC
CC
CT
Crs13459064
75676732
CT(G
A)
Exon
51256
CsT3
41=
CT
TC
TT
CT
TT
CT
CT
TC
Crs27978453
75676717
CT(G
A)
Exon
51271
CsA346=
CC
CC
CC
CC
CC
CC
CC
TC
Crs214335034
75676636
AG
(TC)
Exon
51352
CsD373=
AA
AA
AA
AA
AA
AA
AA
AG
Ars24958364
4756766
03AC
(TG
)Ex
on5
1385
CsP3
84=
AA
AA
AA
AA
AA
AA
AA
AC
Ars27978454
75676592
GA
(CT)
Exon
51396
CnsP
388L
GG
GG
GG
GG
GG
GG
GG
AG
Grs215384328
75676589
GC
(CG
)Ex
on5
1399
CnsA
389G
GG
GG
GG
GG
GG
GG
GG
GC
Grs251728286
75676571
GA
(CT)
Exon
51417
CnsT
395I
Grs247602334
75676567
TC(A
G)
Exon
51421
CsV396=
TT
TT
TT
TT
TT
TT
TT
TC
Trs234216231
75676530
TC(A
G)
Exon
51458
CnsM
409V
TT
TT
TT
TT
TT
TT
TT
TC
Trs212904337
75676526
CT(G
A)
Exon
51462
CnsR
410H
CC
CC
CC
CC
CC
CC
CC
CT
Crs252650779
75676522
TC(A
G)
Exon
51466
CsE4
11=
TT
TT
TT
TT
TT
TT
TT
TC
Trs231273560
75676516
TC(A
G)
Exon
51472
CsQ413=
TT
TT
TT
TT
TT
TT
TT
TC
Trs257886949
756764
13GT
(CA
)Ex
on5
1575
CsR4
48=
GG
GG
GG
GG
GG
GG
GG
GT
Grs241537608
756764
04GC
(CG
)Ex
on5
1584
CnsL
451V
GG
GG
GG
GG
GG
GG
GG
GC
Grs227619071
75676312
TC(A
G)
Exon
51676
CsQ481=
TT
TT
TT
TT
TT
TT
TT
TC
Trs258913831
75676290
AG
(TC)
Exon
51698
CsL4
89=
AA
AA
AA
AA
AA
AA
AA
AG
Ars225593319
75676126
AT
(TA
)Ex
on5
1862
CnsH
543Q
Ars4223233
75676105
GA
(CT)
Exon
51883
CsS550=
GG
GG
GG
GA
GG
GG
GG
GG
Grs4223232
75676032
GT
(CA
)Ex
on5
1956
CnsL
575M
Grs4223231
75675816
CG
UTR
-32172
NC
GG
GG
GG
GG
GG
GG
GG
GC
Grs1346
860
75675682
AT
UTR
-32306
NC
ASequ
ence
varia
tions
inmou
seNr
f2wereob
tained
from
Mou
sePh
enom
eDatabase(http
ph
enom
ejaxorgSNP)
andNCB
ISNPdatabase
(http
wwwncbinlm
nihgovSNP)N
CBIreference
sequ
ence
isMus
Musculusstra
inC5
7BL6J
chromosom
e2GRC
m38p1(NC
0000
687
GI37209910875704641ndash756755192
9123
bp)To
tal968
genetic
mutations
arerepo
rted
inNr
f2gene
and5k
bup
stream
(ge75704
642minus
1)and2k
bdo
wnstre
am(le75675512)sequences
asof
Janu
ary2003A
llSN
Psandallelesa
representedas
geno
miccontig
(reversedsequ
ence
indicated)E
xon175704
641ndash75704364(278
bpT
TS=75704
408)
exon
275679431ndash756791
65(267
bp)exon
375678579ndash75678490(90b
p)exon475677713ndash756775
46(168
bp)andexon
57567718
4ndash75678849
(1666
bp)Proteinam
inoacid
(aa)
resid
uesinNP00
61552
(597
aa)Cs
cod
ing-syno
nymou
sCn
scoding
-non
syno
nymou
sAminoacid
A-alanineD-aspartic
acidE
-glutamicacidF-phenylalanineG
-glycineH
-histidineI-iso
leucineL-leu
cineand
M-m
ethion
ineN-
asparagineP-prolin
eQ-glutamineR-arginineS-serineT-threon
ineandV-valin
elowastErrorsin
databasesfi
xedNC
notcon
firmed
20 Oxidative Medicine and Cellular Longevity
Table 6 NRF2 somatic mutations revealed in various human cancers
Domain Amino acid residues DNA mutation Cancer types (cases) ReferencesLocus Wild type Mutant
DLG motif
24 W (Trp) C (Cys) c72GgtCGgtT NSCLC neck ESC [54 62]K (Lys) c72TgtC ESC [62]
26 Q (Gln) E (Glu) c76CgtG NSCLC ESC [54 62]
27 D (Asp) G (Gly)lowast c80GgtA NSCLC [60]Y (Tyr) c79GgtT ESC [61]
28 I (Ile) T (Thr) c83CgtT NSCLC [54]
29 D (Asp) G (Gly) c86AgtG Head and neck ESC [54 62]H (His) c85GgtC NSCLC laynx [60 61]
30 L (Leu) F (Phe) c88CgtT NSCLC ESC [54 62]
31 G (Gly) A (Ala) c92GgtC NSCLC ESC skin [54 60ndash62]
32 V (Val) T (Thr) c95TgtG NSCLC [54]del c93 95delAGT ESC [61]
33ndash36 S-R-E-V S-R-E-V-S-R-E-Vlowast c97 108dupAGTCGAGCCGTA ESC [61]
34 R (Arg) Q (Gln) c101GgtA NCSCL [54 60 61]P (Pro) c101GgtC NSCLC [63]
ETGF motif
75 Q (Gln) H (His) c225AgtC Head and neck ESC [54 62]
77 D (Asp)
V (Val) c230AgtT NSCLC ESC [54 60 62]G (Gly) c230AgtG ESC [62]A (Ala) c230AgtC NSCLC [61]N (Asn) c229GgtA Larynx [61]
78 E (Glu) K (Lys) c232GgtA NSCLC ESC [54 62]
79 E (Glu)
K (Lys) c235GgtA NSCLC ESC [54 61 62]Q (Gln) c235GgtC NSCLC ESC [54 60ndash62]G (Gly) c236AgtG Larynx [61]E-E c234 236dupAGA ESC [61]
80 T (Thr) K (Lys) c239CgtACgtG NSCLC ESC [54 61 62]
80 T (Thr)P (Phe) c238AgtC ESC [62]I (Ile) c239CgtT Head and neck [54]A (Ala) c238AgtG NSCLC [63]
81 G (Gly) V (Val) c242GgtT ESC [61]D (Asp) c242GgtA NSCLC ESC [60ndash62]
82 E (Glu)
D (Asp) c246AgtT ESC oral cancer cell line [54 62]G (Gly) c245AgtG NSCLC [54]Q (Gln)lowast c244GgtC NSCLC ESC [60 61]V (Val)lowast c245AgtT ESC [61]
83 F (Phe) L (Leu)lowast c247TgtC NSCLC [60]NSCLC non-small cell lung cancer ESC esophageal squamous cancer and lowasterrors in reference fixed Number of cases 82 NSCLC and 10 ESC in [62] 125NSCLC 70 ESC 23 larynx and 17 skin in [61] 103 NSCLC and 12 head and neck in [54] 90 NSCLC in [63] 103 NSCLC in [60]
be involved in squamous lung cancer cell proliferation [64]suggesting that the mutation is functional and overcomesKEAP1 inhibition Singh et al [65] determined in vitrothat RNAi-mediated depletion of NRF2 in lung cancer cellsenhanced ROS production and susceptibility to cell deathby ionizing radiation These studies support the concept thatelevated NRF2 and ARE responsiveness provides cancer cellswith proliferative advantage for malignant transformation
and undue protection from anti-cancer therapy Onco-genic epidermal growth factor receptor (EGFR) signaling isrecently found to be critical in NRF2-mediated proliferationof NSCLC cells [66]
Collectively ldquogain of functionrdquo mutations in NRF2 thatreduce KEAP1 recognition are suggested to be predictivemarkers for poor responsiveness to chemotherapy and radi-ation therapy Although NRF2-mediated cellular defense
Oxidative Medicine and Cellular Longevity 21
processes are essential in the initiation stage enhancedNRF2-ARE activity in advanced stages of cancer developmentmay create a favorable intracellular environment for tumorcell growth and survival [67 68] In this context NRF2may be a potential molecular target for the treatment ofradio-resistance cancers especially those that have ldquoloss offunctionrdquo mutations in EGFR KRAS or KEAP1 as well asldquogain of functionrdquo mutations in NRF2
5 Epigenetic Alterations of NF-E2-RelatedFactor 2
Epigenetic modifications are alterations of molecules inter-acting with genes without changes to the primary DNAsequence They include post-translational modification ofhistones DNA methylation events chromatin conforma-tional changes and alterations to noncoding regulatoryRNAs Epigenetic alterations are stable and often inheritablebut are reversible and may affect expression of the geneDysregulation or defects in epigenetic processes particularlyhypermethylation of tumor suppressor gene promoters (egCpG islands) or histone modifications are thought to beassociated with carcinogenesis Investigators have reportedhypermethylation in CpG islands of KEAP1 which wereassociated with reduced KEAP1 expression in human cancersfrom lung prostate colon and so forth [69ndash72] Similar tosomatic mutations epigenetic changes on KEAP1 impairedthe function of its encoded protein leading to constitutiveNRF2 activation
Supporting the role of ldquopathogenic mutationsrdquo in NRF2expression of Nrf2 and downstream Nqo1 was suppressedin prostate tumors of mice (transgenic adenocarcinomaof mouse prostate TRAMP) Among 15 promoter CpGislands located between minus942 and minus654 (cminus1175 cminus1132and cminus1059 cminus887 gap in cminus1131 cminus1060 see Figure 3)hypermethylation of the first 5 CpG islands (minus942 minus899 andcminus1175 cminus1132) was significantly associated with tumorige-nesis [73] Moreover treatment with inhibitors for DNAmethyltransferase and histone deacetylase restored Nrf2expression in these tumor cells [73] A dietary phytochemicalcurcumin known as a DNA hypomethylation agent restoredepigenetically silent Nrf2 expression through CpG demethy-lation in carcinogen-induced mouse tumor cells [74]
The whole genome epigenetic datasets for 5 species arepublicly accessible at NCBI Epigenomics [75 76]The humanNRF2 epigenome of primary cells (breast penis) and H1stem cell line as well as mouse Nrf2 CpG island methylationdata for sperm blood and cerebellum are currently avail-able (httpwwwncbinlmnihgovepigenomics) Althoughno direct evidence of disease-associated epigenetic modu-lation has been identified in human NRF2 various phyto-chemical NRF2 agonists such as sulforaphane and curcuminhave shown their roles in DNA methylation and histonemodification (see reviews by Lee and colleagues eg [77])Taken together epigenetic modifications of theNRF2KEAP1axle are predicted to cause dysregulation of ARE-mediatedcellular defense leading to deleterious health effects and
phytochemical antioxidants as epigenetic modulators forNRF2 are suggested to be useful in cancer prevention
6 Conclusions
NRF2 is evolutionally conserved with high-sequence homol-ogy in many species However it is a highly mutable geneand numerous genetic variants have been discovered inhuman ethnic groups Importantly certain SNPs or haplo-types have been identified in various diseases as ldquoat-riskrdquoalleles and are related to functional alterations In additionto genetic variations multiple somatic mutations identifiedin the KEAP1 recognition domain of NRF2 in cancer cellshave been found to be oncogenic due to dysregulation ofNRF2 homeostasis by its excess ldquogain of functionrdquo Epigeneticalteration of theNRF2 is under investigation and is predictedto have pathogenic influences as learned from mouse andphytochemical agonist studies Continuous updates of Nrf2allelic variants in inbred mouse strains will provide a usefultool for effective experimental designs formodels of oxidativedisorders to provide insight into the disease mechanisms andintervention strategies
Abbreviations
AD Alzheimerrsquos diseaseALI acute lung injuryARE antioxidant response elementbZIP basic leucine zippercds coding DNA sequencesCI confidence intervalCOPD chronic obstructive pulmonary diseaseESC esophageal squamous cancerFEV1 forced expiratory volume in one second
Gbp giga base pairsGST glutathione S-transferaseGWAS genome-wide association studyHGVS Human Genome Variation SocietyHO-1 heme oxygenase-1119867 119901119910119897119900119903119894 119867119890119897119894119888119900119887119886119888119905119890119903 119901119910119897119900119903119894KEAP1 Kelch-like ECH activating protein 1MPD Mouse Phenome DatabaseMRP multidrug resistance proteinNCBI National Center for Biotechnology InformationNeh NRF2-ECH homologyNfe2l2 nuclear factor (erythroid-derived 2)-like 2Nrf2 NF-E2-related factor 2NSCLC nonsmall cell lung cancerNQO1 NAD(P)Hquinone oxidoreductase 1OR odds ratioPD Parkinsonrsquos diseasePM particulate matterROS reactive oxygen speciesSLE systemic lupus erythematosusSNP single nucleotide polymorphismSOD superoxide dismutaseURT untranslated region
22 Oxidative Medicine and Cellular Longevity
Disclosure
Authorrsquos contribution to the Work was done as part of theAuthorrsquos official duties as a NIH employee and is a Work ofthe United States Government Therefore copyright may notbe established in the United States
Conflict of Interests
The author declares that there is no conflict of interests
Acknowledgments
The research related to this paper was supported by theIntramural Research Program of the National Institutes ofHealth of the National Institute of Environmental HealthSciences (NIEHS) Drs Steven Kleeberger and StephanieLondon at the NIEHS provided excellent critical review ofthis paper The author thanks Mrs Jacqui Marzec for herhelpful comments and English editing
References
[1] J Y Chan M C Cheung P Moi K Chan and Y W KanldquoChromosomal localization of the humanNF-E2 family of bZIPtranscription factors by fluorescence in situ hybridizationrdquoHuman Genetics vol 95 no 3 pp 265ndash269 1995
[2] K Itoh K Igarashi N Hayashi M Nishizawa and MYamamoto ldquoCloning and characterization of a novel erythroidcell-derived CNC family transcription factor heterodimerizingwith the small Maf family proteinsrdquo Molecular and CellularBiology vol 15 no 8 pp 4184ndash4193 1995
[3] W O Osburn and T W Kensler ldquoNRF2 signaling an adaptiveresponse pathway for protection against environmental toxicinsultsrdquoMutation Research vol 659 no 1-2 pp 31ndash39 2008
[4] D Papp K Lenti D Modos D Fazekas Z Dul D Tureiet al ldquoThe NRF2-related interactome and regulome containmultifunctional proteins and fine-tuned autoregulatory loopsrdquoFEBS Letters vol 586 no 13 pp 1795ndash1802 2012
[5] X Wang D J Tomso B N Chorley et al ldquoIdentificationof polymorphic antioxidant response elements in the humangenomerdquo Human Molecular Genetics vol 16 no 10 pp 1188ndash1200 2007
[6] D Malhotra E Portales-Casamar A Singh et al ldquoGlobalmapping of binding sites for NRF2 identifies novel targets incell survival response through ChIP-Seq profiling and networkanalysisrdquo Nucleic Acids Research vol 38 no 17 pp 5718ndash57342010
[7] K Chan R Lu J C Chang and Y W Kan ldquoNRF2 a memberof the NFE2 family of transcription factors is not essential formurine erythropoiesis growth and developmentrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 93 no 24 pp 13943ndash13948 1996
[8] K Itoh T Chiba S Takahashi et al ldquoAn NRF2small Mafheterodimer mediates the induction of phase II detoxifyingenzyme genes through antioxidant response elementsrdquo Bio-chemical and Biophysical Research Communications vol 236no 2 pp 313ndash322 1997
[9] FMartin J M vanDeursen R A Shivdasani CW Jackson AG Troutman and P A Ney ldquoErythroid maturation and globin
gene expression inmicewith combined deficiency ofNF-E2 andNrf-2rdquo Blood vol 91 no 9 pp 3459ndash3466 1998
[10] K Itoh N Wakabayashi Y Katoh et al ldquoKEAP1 repressesnuclear activation of antioxidant responsive elements by NRF2through binding to the amino-terminal Neh2 domainrdquo Genesand Development vol 13 no 1 pp 76ndash86 1999
[11] A Kobayashi M I Kang H Okawa et al ldquoOxidative stresssensor KEAP1 functions as an adaptor for Cul3-based E3 ligaseto regulate proteasomal degradation of NRF2rdquo Molecular andCellular Biology vol 24 no 16 pp 7130ndash7139 2004
[12] K Taguchi H Motohashi and M Yamamoto ldquoMolecularmechanisms of the KEAP1-NRF2 pathway in stress responseand cancer evolutionrdquo Genes to Cells vol 16 no 2 pp 123ndash1402011
[13] K Mukaigasa L T Nguyen L Li H Nakajima M Yamamotoand M Kobayashi ldquoGenetic evidence of an evolutionarilyconserved role for NRF2 in the protection against oxidativestressrdquoMolecular and Cellular Biology vol 32 no 21 pp 4455ndash4461 2012
[14] JMaher andMYamamoto ldquoThe rise of antioxidant signalingmdashthe evolution and hormetic actions of NRF2rdquo Toxicology andApplied Pharmacology vol 244 no 1 pp 4ndash15 2010
[15] S C Lo X Li M T Henzl L J Beamer and M HanninkldquoStructure of the KEAP1NRF2 interface provides mechanisticinsight intoNRF2 signalingrdquoThe EMBO Journal vol 25 no 15pp 3605ndash3617 2006
[16] V A McKusick ldquoMendelian inheritance in man and its onlineversion OMIMrdquo American Journal of Human Genetics vol 80no 4 pp 588ndash604 2007
[17] B Yalcin D J Adams J Flint and T M Keane ldquoNext-generation sequencing of experimental mouse strainsrdquo Mam-malian Genome vol 23 no 9-10 pp 490ndash498 2012
[18] D R Bentley S Balasubramanian H P Swerdlow et alldquoAccurate whole human genome sequencing using reversibleterminator chemistryrdquo Nature vol 456 no 7218 pp 53ndash592008
[19] P S G Chain D V Grafham R S Fulton et al ldquoGenomeproject standards in a new era of sequencingrdquo Science vol 326no 5950 pp 236ndash237 2009
[20] RHWaterstonK Lindblad-Toh E Birney J Rogers J F AbrilP Agarwal et al ldquoInitial sequencing and comparative analysis ofthe mouse genomerdquo Nature vol 420 pp 520ndash562 2002
[21] T M Keane L Goodstadt P Danecek M A White K WongB Yalcin et al ldquoMouse genomic variation and its effect onphenotypes and gene regulationrdquoNature vol 477 pp 289ndash2942011
[22] T Yamamoto K Yoh A Kobayashi et al ldquoIdentification ofpolymorphisms in the promoter region of the human NRF2generdquo Biochemical and Biophysical Research Communicationsvol 321 no 1 pp 72ndash79 2004
[23] J M Marzec J D Christie S P Reddy et al ldquoFunctionalpolymorphisms in the transcription factor NRF2 in humansincrease the risk of acute lung injuryrdquo The FASEB Journal vol21 no 9 pp 2237ndash2246 2007
[24] H Fukushima-Uesaka Y Saito K Maekawa et al ldquoGeneticvariations and haplotype structures of transcriptional factorNRF2 and its cytosolic reservoir protein KEAP1 in JapaneserdquoDrug Metabolism and Pharmacokinetics vol 22 no 3 pp 212ndash219 2007
[25] E J Cordova R Velazquez-Cruz F Centeno V Baca and LOrozco ldquoThe NRF2 gene variant -653GA is associated with
Oxidative Medicine and Cellular Longevity 23
nephritis in childhood-onset systemic lupus erythematosusrdquoLupus vol 19 no 10 pp 1237ndash1242 2010
[26] M von Otter S Landgren S Nilsson et al ldquoAssociation ofNRF2-encoding NFE2L2 haplotypes with Parkinsonrsquos diseaserdquoBMCMedical Genetics vol 11 no 1 article 36 2010
[27] JMHartikainenM TengstromVMKosmaV L Kinnula AMannermaa andY Soini ldquoGenetic polymorphisms and proteinexpression of NRF2 and sulfiredoxin predict survival outcomesin breast cancerrdquo Cancer Research vol 72 pp 5537ndash5546 2012
[28] T Arisawa T Tahara T Shibata et al ldquoThe relationshipbetween Helicobacter pylori infection and promoter polymor-phism of the NRF2 gene in chronic gastritisrdquo InternationalJournal of Molecular Medicine vol 19 no 1 pp 143ndash148 2007
[29] T Arisawa T Tahara T Shibata et al ldquoThe influence ofpromoter polymorphism of nuclear factor-erythroid 2-relatedfactor 2 gene on the aberrant DNA methylation in gastricepitheliumrdquo Oncology Reports vol 19 no 1 pp 211ndash216 2008
[30] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene pro-moter polymorphism and gastric carcinogenesisrdquo Hepato-Gastroenterology vol 55 no 82-83 pp 750ndash754 2008
[31] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene promoterpolymorphism is associated with ulcerative colitis in a Japanesepopulationrdquo Hepato-Gastroenterology vol 55 no 82-83 pp394ndash397 2008
[32] C C Hua L C Chang J C Tseng C M Chu Y C Liu andW B Shieh ldquoFunctional haplotypes in the promoter region oftranscription factor NRF2 in chronic obstructive pulmonarydiseaserdquo Disease Markers vol 28 no 3 pp 185ndash193 2010
[33] S O Shaheen R B Newson S M Ring M J Rose-ZerilliJ W Holloway and A J Henderson ldquoPrenatal and infantacetaminophen exposure antioxidant gene polymorphismsand childhood asthmardquo The Journal of Allergy and ClinicalImmunology vol 126 no 6 pp 1141e7ndash1148e7 2010
[34] H Masuko T Sakamoto Y Kaneko et al ldquoLower FEV1 innon-COPD nonasthmatic subjects association with smokingannual decline in FEV1 total IgE levels and TSLP genotypesrdquoInternational Journal of Chronic Obstructive Pulmonary Diseasevol 6 pp 181ndash189 2011
[35] C P Guan M N Zhou A E Xu et al ldquoThe susceptibilityto vitiligo is associated with NF-E2-related factor 2 (NRF2)gene polymorphisms a study on Chinese Han populationrdquoExperimental Dermatology vol 17 no 12 pp 1059ndash1062 2008
[36] J Bouligand O Cabaret M Canonico et al ldquoEffect of NFE2L2genetic polymorphism on the association between oral estrogentherapy and the risk of venous thromboembolism in post-menopausal womenrdquo Clinical Pharmacology amp Therapeuticsvol 89 no 1 pp 60ndash64 2011
[37] D S OrsquoMahony B J Glavan T D Holden C Fong R A BlackG Rona et al ldquoInflammation and immune-related candidategene associations with acute lung injury susceptibility andseverity a validation studyrdquo PLoS ONE vol 7 no 12 Article IDe51104 2012
[38] I Ungvari E Hadadi V Virag et al ldquoRelationship between airpollutionNFE2L2 gene polymorphisms and childhood asthmain aHungarian populationrdquo Journal of CommunityGenetics vol3 no 1 pp 25ndash33 2012
[39] M SiedlinskiD S Postma JMA Boer et al ldquoLevel and courseof FEV1 in relation to polymorphisms inNFE2L2 andKEAP1 inthe general populationrdquo Respiratory Research vol 10 article 732009
[40] C C Hong C B Ambrosone J Ahn et al ldquoGenetic variabilityin iron-related oxidative stress pathways (NRF2 NQ01 NOS3
and HO-1) iron intake and risk of postmenopausal breastcancerrdquo Cancer Epidemiology Biomarkers and Prevention vol16 no 9 pp 1784ndash1794 2007
[41] C Canova C Dunster F J Kelly C Minelli P L Shah CCaneja et al ldquoPM10-induced hospital admissions for asthmaand chronic obstructive pulmonary disease the modifyingeffect of individual characteristicsrdquo Epidemiology vol 23 no 4pp 607ndash615 2012
[42] A J Henderson R B Newson M Rose-Zerilli S M RingJ W Holloway and S O Shaheen ldquoMaternal NRF2 andgluthathione-S-transferase polymorphisms donotmodify asso-ciations of prenatal tobacco smoke exposure with asthma andlung function in school-aged childrenrdquo Thorax vol 65 no 10pp 897ndash902 2010
[43] C Xing A L Sestak J A Kelly et al ldquoLocalization andreplication of the systemic lupus erythematosus linkage signalat 4p16 interaction with 2p11 12q24 and 19q13 in EuropeanAmericansrdquo Human Genetics vol 120 no 5 pp 623ndash631 2007
[44] T F Wong K Yoshinaga Y Monma K Ito H NiikuraS Nagase et al ldquoAssociation of KEAP1 and NRF2 geneticmutations and polymorphisms with endometrioid endometrialadenocarcinoma survivalrdquo International Journal of Gynecologi-cal Cancer vol 21 no 8 pp 1428ndash1435 2011
[45] M von Otter S Landgren S Nilsson et al ldquoNRF2-encodingNFE2L2 haplotypes influence disease progression but not riskin Alzheimerrsquos disease and age-related cataractrdquoMechanisms ofAgeing and Development vol 131 no 2 pp 105ndash110 2010
[46] S Tsang Z Sun B Luke et al ldquoA comprehensive SNP-basedgenetic analysis of inbred mouse strainsrdquoMammalian Genomevol 16 no 7 pp 476ndash480 2005
[47] C M Wade E J Kulbokas III A W Kirby et al ldquoThe mosaicstructure of variation in the laboratory mouse genomerdquoNaturevol 420 no 6915 pp 574ndash578 2002
[48] T Wiltshire M T Pletcher S Batalov et al ldquoGenome-widesingle-nucleotide polymorphism analysis defines haplotypepatterns in mouserdquo Proceedings of the National Academy ofSciences of the United States of America vol 100 no 6 pp 3380ndash3385 2003
[49] K A Frazer E Eskin H M Kang et al ldquoA sequence-basedvariation map of 827 million SNPs in inbred mouse strainsrdquoNature vol 448 no 7157 pp 1050ndash1053 2007
[50] T P Maddatu S C Grubb C J Bult andM A Bogue ldquoMousephenome database (MPD)rdquo Nucleic Acids Research vol 40 ppD887ndashD894 2007
[51] A Kirby H M Kang C M Wade et al ldquoFine mapping in 94inbred mouse strains using a high-density haplotype resourcerdquoGenetics vol 185 no 3 pp 1081ndash1095 2010
[52] H Y Cho A E Jedlicka S P M Reddy L Y Zhang T WKensler and S R Kleeberger ldquoLinkage analysis of susceptibilityto hyperoxia NRF2 is a candidate generdquo American Journal ofRespiratory Cell and Molecular Biology vol 26 no 1 pp 42ndash512002
[53] H Y Cho A E Jedlicka S P M Reddy et al ldquoRole of NRF2in protection against hyperoxic lung injury in micerdquo AmericanJournal of Respiratory Cell and Molecular Biology vol 26 no 2pp 175ndash182 2002
[54] T Shibata T Ohta K I Tong et al ldquoCancer related mutationsin NRF2 impair its recognition by KEAP1-Cul3 E3 ligase andpromote malignancyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 36 pp13568ndash13573 2008
24 Oxidative Medicine and Cellular Longevity
[55] A Singh V Misra R K Thimmulappa et al ldquoDysfunctionalKEAP1-NRF2 interaction in non-small-cell lung cancerrdquo PLoSMedicine vol 3 no 10 article e420 2006
[56] T Ohta K Iijima M Miyamoto et al ldquoLoss of KEAP1 functionactivates NRF2 and provides advantages for lung cancer cellgrowthrdquo Cancer Research vol 68 no 5 pp 1303ndash1309 2008
[57] B Padmanabhan K I Tong T Ohta et al ldquoStructural basisfor defects of KEAP1 activity provoked by its point mutationsin lung cancerrdquoMolecular Cell vol 21 no 5 pp 689ndash700 2006
[58] T Shibata A Kokubu M Gotoh et al ldquoGenetic alteration ofKEAP1 confers constitutive NRF2 activation and resistance tochemotherapy in gallbladder cancerrdquoGastroenterology vol 135no 4 pp 1358e4ndash1368e4 2008
[59] N J Yoo H R Kim Y R Kim C H An and S H LeeldquoSomatic mutations of the KEAP1 gene in common solidcancersrdquo Histopathology vol 60 no 6 pp 943ndash952 2012
[60] Y Hu Y Ju D Lin Z Wang Y Huang S Zhang et alldquoMutation of the NRF2 gene in non-small cell lung cancerrdquoMolecular Biology Reports vol 39 no 4 pp 4743ndash4747 2012
[61] Y R Kim J EOhM S Kim et al ldquoOncogenicNRF2mutationsin squamous cell carcinomas of oesophagus and skinrdquo TheJournal of Pathology vol 220 no 4 pp 446ndash451 2010
[62] T Shibata A Kokubu S Saito et al ldquoNRF2 mutation confersmalignant potential and resistance to chemoradiation therapyin advanced esophageal squamous cancerrdquo Neoplasia vol 13pp 864ndash873 2011
[63] H Sasaki M Shitara K Yokota Y Hikosaka S MoriyamaM Yano et al ldquoIncreased NRF2 gene (NFE2L2) copy numbercorrelates with mutations in lung squamous cell carcinomasrdquoMolecular Medicine Reports vol 6 no 2 pp 391ndash394 2012
[64] H Sasaki M Shitara K Yokota Y Hikosaka S Moriyama MYano et al ldquoRagD gene expression andNRF2mutations in lungsquamous cell carcinomasrdquo Oncology Letters vol 4 pp 1167ndash1170 2012
[65] A Singh M Bodas N Wakabayashi F Bunz and S BiswalldquoGain of NRF2 function in non-small-cell lung cancer cellsconfers radioresistancerdquo Antioxidants and Redox Signaling vol13 no 11 pp 1627ndash1637 2010
[66] T Yamadori Y Ishii SHommaYMorishimaKKurishimaKItoh et al ldquoMolecular mechanisms for the regulation of NRF2-mediated cell proliferation in non-small-cell lung cancersrdquoOncogene vol 31 pp 4768ndash4777 2012
[67] A K Bauer H Y Cho L Miller-Degraff C Walker K HelmsJ Fostel et al ldquoTargeted deletion of NRF2 reduces urethane-induced lung tumor development in micerdquo PLoS ONE vol 6no 10 Article ID e26590 2011
[68] H Y Cho and S R Kleeberger ldquoNRF2 protects against airwaydisordersrdquo Toxicology and Applied Pharmacology vol 244 no1 pp 43ndash56 2010
[69] R Wang J An F Ji H Jiao H Sun and D Zhou ldquoHyperme-thylation of the KEAP1 gene in human lung cancer cell linesand lung cancer tissuesrdquo Biochemical and Biophysical ResearchCommunications vol 373 no 1 pp 151ndash154 2008
[70] P Zhang A Singh S Yegnasubramanian et al ldquoLoss ofkelch-like ECH-associated protein 1 function in prostate cancercells causes chemoresistance and radioresistance and promotestumor growthrdquoMolecular Cancer Therapeutics vol 9 no 2 pp336ndash346 2010
[71] N Hanada T Takahata Q Zhou et al ldquoMethylation of theKEAP1 gene promoter region in human colorectal cancerrdquoBMCCancer vol 12 article 66 2012
[72] L A Muscarella R Barbano V DrsquoAngelo et al ldquoRegulationof KEAP1 expression by promoter methylation in malignantgliomas and association with patientrsquos outcomerdquo Epigeneticsvol 6 no 3 pp 317ndash325 2011
[73] S Yu T O Khor K L Cheung et al ldquoNRF2 expressionis regulated by epigenetic mechanisms in prostate cancer ofTRAMP micerdquo PLoS ONE vol 5 no 1 Article ID e8579 2010
[74] T O Khor Y Huang T Y Wu L Shu J Lee and A N KongldquoPharmacodynamics of curcumin as DNA hypomethylationagent in restoring the expression of NRF2 via promoter CpGsdemethylationrdquo Biochemical Pharmacology vol 82 no 9 pp1073ndash1078 2011
[75] I M Fingerman L McDaniel X Zhang et al ldquoNCBI epige-nomics a new public resource for exploring epigenomic datasetsrdquo Nucleic Acids Research vol 39 supplemen 1 pp D908ndashD912 2011
[76] I M Fingerman X Zhang W Ratzat N Husain R F Cohenand G D Schuler ldquoNCBI epigenomics whatrsquos new for 2013rdquoNucleic Acids Research vol 41 no 1 pp D221ndashD225 2013
[77] J H Lee T O Khor L Shu Z Y Su F Fuentes andA N Kong ldquoDietary phytochemicals and cancer preventionNRF2 signaling epigenetics and cell death mechanisms inblocking cancer initiation and progressionrdquo Pharmacology ampTherapeutics vol 137 no 2 pp 153ndash171 2012
Submit your manuscripts athttpwwwhindawicom
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Disease Markers
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OncologyJournal of
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Oxidative Medicine and Cellular Longevity
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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
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Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
14 Oxidative Medicine and Cellular Longevity
minus212GG (98 rs67006649 previously minus684minus651) minus178Callele (73) and GCC
4(53) was predicted to increase
respiratory failure development (hazard ratio = 095 CI091ndash099) in German COPD patients [32] Significant inter-action was also identified between an intronic SNP Gallele (rs6726395 g178103229AgtG 884 frequency) andsmoking status on FEV
1decline relative to the reference
AA allele in the above Japanese cohort [34] Siedlinskiet al [39] reported that the CC genotype of anotherintronic SNP (rs2364723 g178126546GgtC) was associatedwith a lower FEV
1level compared to the wild-type geno-
type (GG) in two Netherland cohorts (CI minus636minus178frequency = 0525 and pooled cohort size = 2542) ThisSNP alone or as a haplotype with 4 more intronic SNPs(rs13001694Grs1806649Trs4243387Trs6726395G)was alsoassociated with high FEV
1levels in individuals that ever
smoked [39] In a Hungarian population of childhoodasthma SNPs at minus178 (CA) and 31015840 flanking (rs2588882TG)loci were inversely associated with infection-induced asthma(OR 0437 CI 028ndash080 OR 0290 CI 013ndash062 resp) andthese SNPs significantly influenced an asthma-environmentalpollution interaction [38]The intronic SNP rs1806649 (CgtT)was associated but not significantly with an increased risk ofhospitalization during high-level particulate matter (PM
10)
periods in asthma or COPD patients (119899 = 209) of the UnitedKingdom (UK) [41] Asthma andCOPDadmission rateswererelated to the increase in environmental PM
10concentration
Importantly effects of interaction between prenatal stressand NRF2 SNPs on descendant pulmonary health wereinvestigated by the Avalon Longitudinal Study in the UKmaternal smoking during pregnancy was not associated withlung function change determined by maximum mild expi-ratory flow (FEF
25ndash75) or with asthma incidence in school-aged children and this relation was not modified by NRF2SNP genotypes [42]However early gestation acetaminophenexposure significantly influenced the risk of asthma andwheezing at the age of 7 years in gt4000 mothers and gt5000children [33] When maternal copies of the minus212A allele werepresent association with asthma (11374891 OR 173 CI122ndash245) and wheezing (11494949 OR 153 95 CI 106ndash220) was significantly increased [33]
Gastrointestinal Disorders While there was no evidence inlung cancer cases studies in Japanese populations suggesteda potential association of NRF2 variations with gastro-intestinal tumorigenesisHelicobacter pylori (H pylori) causesgastritis which can lead to gastric atrophy and cancer Ingastric epithelium from the Japanese cancer cohorts (39gastric cancers 46 controls) H pylori infection was posi-tively correlated with aberrant CpG island methylation oftumor suppressor genes (eg p14) and minus214Gminus212G orminus214Aminus212G NRF2 haplotype was significantly associatedwith increased (OR 290 95 CI 114ndash736) or decreased(OR 033 95 CI 013ndash088) risk of the CpG methyla-tion respectively in the H pylori-infected patients [29]Further study from the same investigators determined thatminus214Aminus212G allele carriers had significantly (119875 = 0022)reduced risk of gastric cancer inH pylori-negative cases [30]The minus214AGminus212AG genotypes were negatively associated
(OR 045 CI 022ndash093) and the minus214Gminus212G genotypeswere positively associated (chronic continuous phenotypeOR 257 CI 101ndash660) with ulcerative colitis (89 patients 141controls) in a Japanese population [31]
Autoimmune Disorders Systemic lupus erythematosus (SLE)is a long-term autoimmune disease more frequently found infemales than in males It affects organs including skin jointskidneys and brain and nephritis is an aggressive character-istic in some patients Genome-wide association studies inhumans identified a suggestive quantitative trait locus nearNRF2 [43] A study of a Mexican Mestizo population (362patients with childhood-onset SLE 379 controls and 212nephritis diagnosed) determined that lupus with nephritiswas significantly (OR 181 CI 104ndash312) associated with theminus214GA SNP in females [25] The same SNPs were notclosely associated with SLE risk in a Japanese cohort [22]Vitiligo is a skin condition in which there is a loss of browncolor (pigment) from areas of skin resulting in irregularwhite patches It is thought to be an autoimmune diseasecaused by loss of cells (melanocytes) that produce brownpigment A study indicated that theminus178A allele increased therisk of vitiligo dose-dependently (OR 1724 95 CI 135ndash221for CA OR 2902 CI 162ndash519 for AA) [35]
Female Disorders It is well known that estrogen metabolites(eg catechols) cause ROS formation suggesting correla-tion of NRF2 and downstream effectors in postmenopausalmammary cancer In a study of a Finish population (KuopioBreast Cancer Project 119899 = 452 patients 370 controls)the minus178AA homozygous genotype (OR 4656 CI = 135ndash1606) and 31015840 flanking rs2706110 (TT OR 2079 CI 118ndash368) genotype were associated with increased risk of breastcancer while the 51015840 flanking minus3306TT homozygous allelewas significantly associated with lower survival (frequency =71219 OR 1687 CI 1105ndash275) [27] suggesting that NRF2genetic polymorphisms affect susceptibility and outcome ofthe patients The minus178A allele carriers together with intronicrs1962142A allele carriers were associated with lowered tissuelevels of NRF2 proteins [27] In postmenopausal women theminus178A allele (OR 179 95CI 370ndash8570) appeared tomodifythe risk of venous thromboembolism caused by oral estrogentherapy (AA or AC frequency = 333) as demonstratedby the French ESTHER study (161 cases 474 controls) [36]An intronic rs1806649CgtT SNP did not associate with breastcancer risk in postmenopausal women [40] However whenthis SNP and other at-risk alleles of ARE-responsive genes(NQO1 HO-1 NOS3) were combined there was a significantgene-dose effect on the breast cancer risk [40] Althoughcoding region SNPs in NRF2 and KEAP1 were identifiedin the Japanese endometrial adenocarcinoma patients noassociation of NRF2 SNPs with the disease was found [44]
Neurodegenerative Diseases Oxidative stress is known to beinvolved in Parkinsonrsquos disease (PD) presumably due toproduction of ROS fromhigh-dopaminemetabolism and lowlevels of antioxidants in the substantia nigra of the brainInvestigators found a protective NRF2 haplotype consistingof four 51015840 flanking SNPs (minus5238Gminus214Aminus212Gminus178C)
Oxidative Medicine and Cellular Longevity 15
and 4 intronic SNPs (rs2886161Ars1806649Ars2001350Ars10183914A) from Swedish (OR 09 CI 060ndash140) and Polish(OR 04 CI 030ndash060) populations (total 165+192 PD cases190+192 controls) [26]The investigators also suggested thatNRF2 haplotype alleles were associated with 2 years earlierage of Alzheimerrsquos disease (AD) onset 4 years earlier age ofposterior subcapsular cataract surgery and 4 years later ageof cortical cataract surgery while they were not significantlyrelated to AD or age-related cataract risk [45]
34 Genetic Mutations in Mouse Nrf2 Tsang et al [46]compiled 673 SNPs in 55 mouse strains and constructedtheir phylogenetic tree to correlate and clarify the originsof strains based on the assembled mouse genome sequenceand SNP data [20 47 48] Recently using the completegenome sequence of C57BL6J (B6) mouse as a referencehigh-density SNP screening in other laboratory strains orin panels of strains has been published (see [17]) Althoughmillions of mouse SNPs (gt10089892 as of December 2012)and haplotype mappings from more than 120 strains havebeen published as valuable references for dissecting thegenetic basis of complex traits [49ndash51] little attention hasbeen paid to polymorphisms of Nrf2 and their correlationwith disease phenotypes
Figure 3 demonstrates the proximal promoter region(minus1 to minus950)51015840-UTR (exon 1 up to TSS) and proteinsequence of mouse Nrf2 based on GRCm38p1 PrimaryAssembly (75704641ndash75675513 bp) mRNA variant1 and protein (NP 035032 encoded by NM 010902234ndash2027 bp) sequences Genetic variations in the Nrf2genome of inbred strains collected from public databasesare listed in Tables S2 and 5 (See Supplementary TableS2) Overall 968 genetic mutations are compiled forNrf2 gene and 5 kb upstream2 kb downstream regions785 SNPs between B6 and another 16 strains wereacquired from the Mouse Phenome Database (MPDhttpphenomejaxorg dbqrtn=snpret1) and additionalSNPs and other mutations were acquired from NCBI dbSNP(httpwwwncbinlmnihgovsnpterm=mus+musculus20nfe2l2) In total 132 mutations are in the promoter (37in proximal 1 kb) 49 in exons (38 in coding region 19 Cns)727 in introns and 60 in the 31015840 flanking region Excludingmutations in the 51015840 and 31015840 flanking sequences murine Nrf2sequences appear to be more highly variable (1 variationper 375 bp) than much of the mouse genome which has anapproximate frequency of one SNP per every 245 bp (httpwwwinformaticsjaxorgmgihomehomepagesstatsallstatsshtmlallstats snp)
Nrf2 was found to be a susceptibility gene from genome-wide linkage analysis in a murine model of hyperoxia-induced ALI [52] A promoter SNP minus103TgtC (previouslypublished as minus336TgtC) in Nrf2 was found and predicted toadd an additional Sp1 binding site in hyperoxia-susceptibleB6 mice but not in resistant C3HHeJ mice [52] Genotypesfrom the SNP and from simple-sequence length polymor-phism markers of the Nrf2 locus (D2Mit248 and D2Mit94)cosegregated in the B6C3F
2mouse cohort [52] and Nrf2
deficient mice were significantly more susceptible to ALI
sub-phenotypes caused by hyperoxia than similarly exposedwild-type mice supporting Nrf2 as a contributor to thephenotypic traits [53] Although no other functional analyseson Nrf2 SNPs or haplotype association studies have beenconducted in inbred mice strains bearing haplotypes suchas multiple Cns in functional domains (eg F71L L451VH543Q and L575M) may be useful to elucidate the role ofNrf2 in differential susceptibility to oxidative diseases
4 Oncogenic Somatic Mutations in HumanNF-E2-Related Factor 2
Somatic mutation is a change in the DNA of somatic cellsthat affects derived cells but is not inherited by offspringEfforts to discover somatic mutations have provided insightinto mutagenesis and cancer development Lung cancerparticularly non-small cell lung cancer (NSCLC) is theleading cause of cancer death worldwide Somatic mutationsof NRF2 and KEAP1 discovered in lung cancer patients havedetermined the oncogenic potential ofNRF2 [54 55] KEAP1somatic mutations were associated with its reduced proteinlevels in lung cancer tissues and cells [56 57] Investigationsof NSCLC in various ethnic populations as well as cancersin gastrointestine breast and prostate have coordinatelydemonstrated that multiple Cns somatic mutations in KEAP1cause dysfunction of the translated protein and in turnconstitutive activation of NRF2 increasing risk of neoplasiaand chemoresistance [12 55 58 59] Somatic mutations ofNRF2 have been detected in various cancer tissues (largelysquamous cell carcinomas) in Asian populations (Table 6)NRF2 mutations were significantly associated with NSCLCcases (squamous cell lung carcinoma adenocarcinoma) ofthe Japanese (107 [54]) the Chinese (23 [60]) andthe Koreans (8 [61]) as well as with lung cancer celllines Smoking history was also correlated with mutationoccurrence in all of the studies [54 60 61] In addition tolung cancers laryngeal squamous carcinoma (13 in [61])esophageal squamous cancer (ESC 22 in [60] 114 in[61]) head and neck cancers (25 in [54]) skin (117 casein [61]) and oral cancer cell lines had somatic changesin NRF2 In contrast to wide-spread KEAP1 mutationsmutations in NRF2 were clustered in DLGETGE motifs ofthe Neh2 domain which are critical in the ldquohinge and latchrdquomodel of KEAP1 binding [12] Similar to KEAP1 somaticmutations it has been postulated that NRF2 mutations incancer cells lead to NRF2 accumulation by suppressing itsubiquitination or KEAP1 binding which eventually confersmalignant potential and resistance to chemotherapy
Most variable sites in NRF2 included aa residues 29 (AspD) 31 (Gly G) 77 (Asp D) and 79 (Glu E) (Table 6)Residue 33 (Ser S) in the Neh2 domain is mutated by eithergenetic or somatic processes (Figure 4) Cns in the EDGFmotif of NRF2 was experimentally determined to impairrecognition of KEAP1 [54] NRF2 mutations were signif-icantly correlated with increased (25-fold) copy number(31 of mutants versus 3 wild types) in Japanese NSCLCcases [63] Aberrant mutation of NRF2 also led to increasedexpression of downstream effectors includingRagD known to
16 Oxidative Medicine and Cellular Longevity
pL575M
pF71L
pH543QpL451V
pA151P pA151V pN159D pQ198delpV105D
pA204T pS224I225S226del pT253S pS278ApD305G
pM409V pR410HpP388L pA389G pT395I
Genomic DNA (reversed contig GRCm38 p1 C57BL6JNM 0109023)
Protein (NP 035032)1
51101151201251301351401451501551
minus400
minus500
minus50
minus100
minus200
minus300
minus450
minus350
minus250
minus150
minus950
minus900
minus850
minus800
minus750
minus700
151
101151201
2001
g minus470gg minusg minus
c minus41c minus
c
c
c c
gg minus 6C
g minus125insTTC g minus118insC
g minus281delC
g minus63delCg minus1 (75704642 c minus234)
Translation start site (c +1)Termination codon (c +2025 +2027)
c minus136 minus134in-frame stop codonc minus8585
g minus41
246
440
minus381
minus11
minus91 minus90
minus332
minus153
minus98
c minus197c minus209 minus202
gminus942 minus899 (cminus1175 minus1132) CpG islands [71]
g 460CgtT
g minus103CCgtT [51]
CgtTCgtG
g minus320CgtT
CgtT
CgtT
CgtTg minus426GgtA
GgtACgtA
CgtA
GgtA
g minus238GgtTTgtC
TgtC
TgtC
TgtCTgtG
g minus229AgtG
CgtG
CgtG
GgtC
gminus826 minus654 (cminus1059 ) CpG islands [71]minus887
Figure 3 DNA (partial promoter and exons 1 and 5) and protein sequence ofmouseNrf2 SNPs and amino acid residues for non-synonymousSNPs are marked Promoter regions bearing 5CpG islands are underlined
151
201251301351401451501551601
Genetic mutation (Table 3 and Figure 2)Somatic mutation (Table 6)
Neh2
Neh3
Neh4Neh5
Neh6
Neh1
Protein isoform 1 (NP 006155)
101151
Figure 4 Genetic and somatic mutation loci in human NRF2 protein
Oxidative Medicine and Cellular Longevity 17
Table5Mou
seup
stream
andexon
varia
tions
ofNfe2
l2locusin17
inbred
strainsR
eference
sequ
ence
isC5
7BL6J
(B6strain
1)geno
me(GI14933824975547698ndash75513576)SNPalleleand
geno
typesareshow
nas
chromosom
econ
tigsequ
enceStrains
2129S1S
vlmJ3AJ
4AKR
J5BA
LBcJ6C3
HH
eJ7C
57BL
6NJ8CA
STEiJ9CB
AJ
10D
BA2J11FVBNJ12LPJ
13N
ODShiLtJ14N
ZOH
ILtJ
15P
WK
PhJ16SPR
ETEiJ
17W
SBEiJ
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs2566
08517
75705565
GA
(CT)
51015840Flankingminus924
NC
GG
GG
GG
GG
GG
GG
GG
AA
Grs47274959
75705562
AT
(TA
)51015840Flankingminus921
NC
AT
TA
TT
AA
TT
AT
AT
TT
Ars216940398
75705554ndash75705555
Tadd(A
add)
51015840Flankingminus913minus914
NC
CGrs239114134
75705540
CT(G
A)
51015840Flankingminus899
NC
CT
TC
TT
CC
TT
CT
CT
CT
Crs46
461765
75705528
TG(A
C)oA
C
51015840Flankingminus887
NC
TG
GT
GG
TT
GG
TG
TG
GG
Trs5144
9853
75705525
CA(G
T)
51015840Flankingminus884
NC
CA
AC
AA
CC
AA
CA
CA
CC
Crs249093111
75705512
CT(G
A)
51015840Flankingminus871
NC
CT
TC
TT
CC
TT
CT
CT
TT
Crs263745200
75705510
CG(G
C)
51015840Flankingminus869
NC
GG
GG
GG
GG
GG
GG
GG
CG
Grs45651867
75705498
TG(A
C)
51015840Flankingminus857
NC
TG
GT
GG
TT
GG
TG
TG
GG
Trs219997531
75705495
GA
(CT)
51015840Flankingminus854
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs251918379
75705451
TA(A
T)
51015840Flankingminus810
NC
TT
TT
TT
TT
TT
TT
TT
TA
Trs27978306
75705449
TA(A
T)
51015840Flankingminus808
NC
TA
AT
AA
TT
AA
TA
TA
TT
Trs216087412
75705429
TG(A
C)lowast
51015840Flankingminus788
NC
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
GTlowast
Tlowast
rs261229914
75705423ndash75705424
AGdel(CT
del)
51015840Flankingminus783minus784
NC
AGrs27978307
75705400
CT(G
A)
51015840Flankingminus759
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs243167395
75705359
GA
(CT)
51015840Flankingminus718
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs27978308
75705307
GA
(CT)
51015840Flankingminus66
6NC
GG
GG
GG
GG
GG
GG
GG
AG
Grs254744
09875705294
AT
(TA
)51015840Flankingminus653
NC
AA
AA
AA
AA
AA
AA
AA
AT
Ars228133419
75705179
GT
(CA
)51015840Flankingminus538
NC
GG
GG
GG
GG
GG
GG
GG
GT
Grs214719520
75705111
GA
(CT)
51015840Flankingminus470
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs2440
87730
75705101
GA
(CT)
51015840Flankingminus46
0NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs227781699
75705081
CG(G
C)
51015840Flankingminus44
0NC
CC
CC
CC
CC
CC
CC
CC
GC
Crs257870353
75705067
CT(G
A)
51015840Flankingminus426
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs24487744
075705022
AG
(TC)
51015840Flankingminus381
NC
AA
AA
AA
AA
AA
AA
AA
AG
Ars234628138
75704973
AC
(TG
)51015840Flankingminus332
NC
AA
AA
AA
AA
AA
AA
AA
AC
A
18 Oxidative Medicine and Cellular Longevity
Table5Con
tinued
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs247275247
75704961
GA
(CT)
51015840Flankingminus320
NC
GG
GG
GG
GG
GG
GG
AG
GG
Grs247519047
75704922
Gdel(Cdel)
51015840Flankingminus281
NC
Grs27978309
(rs51915758)
75704887
GA
C(C
TG
)51015840Flankingminus246
NC
GA
AG
AA
GG
AA
GA
GA
CC
G
rs218139102
75704879
CA(G
T)
51015840Flankingminus238
NC
CC
CC
CC
CC
CC
CC
CC
AC
Crs251990355
75704870
TC(A
G)
51015840Flankingminus229
NC
TT
TT
TT
TT
TT
TT
TT
TC
Trs238746955
75704794
AG
(TC)
51015840Flankingminus153
NC
AA
AA
AA
AA
AA
AA
AA
GA
Ars221664
405
75704786
AG
(TC)
51015840Flankingminus145
NC
AA
AA
AA
AA
AA
AA
AA
AG
Ars217054035
75704766
ndash75704767
GAAadd(TTC
add)
51015840Flankingminus125minus126
NC
GA
rs248182931
75704759ndash75704760
Gadd(C
add)
51015840Flankingminus118
minus119
NC
AGrs217197904
75704744
GA
(CT)
51015840Flankingminus103
(+)S
p1[23]
GA
AG
AA
GG
AA
GA
AA
GG
Ars2644
9364975704704
Gdel(Cdel)
51015840Flankingminus63
NC
Grs2404
81112
757046
82CT(G
A)
51015840Flankingminus41
NC
CC
CC
CC
CC
CC
CC
CC
CT
Crs27978310
75704617
AG
(TC)
UTR
-525
NC
AA
AA
AA
AA
AA
AA
AA
GG
Ars27978311
75704610
CG(G
C)
UTR
-532
NC
GG
GG
GG
GG
GG
GG
GG
GG
Crs27978312
757046
05GA
(CT)
UTR
-537
NC
GA
AG
AA
GG
AA
GA
GA
GG
Grs214784220
757044
99AG
(TC)
UTR
-5143
NC
AA
AA
AA
AA
AA
AA
AA
GG
Ars244146318
757044
98GT
(CA
)UTR
-5144
NC
GG
GG
GG
GG
GG
GG
GG
GT
Grs27978313
757044
97GC
(CG
)UTR
-5145
NC
GC
CG
CC
GC
CC
GC
CC
CC
Crs215431944
757044
93CT(G
A)
UTR
-5149
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs257015697
757044
49GA
(CT)
UTR
-5193
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs252234782
757044
19GT
(CA
)UTR
-5223
NC
Grs1346
0861
75679262
AG
(TC)
Exon
244
6Cn
sF71L
AG
GA
GG
AG
GG
AG
AG
GG
Grs1346
0859
75679202
GA
(CT)lowast
Exon
2506
CsH91=
GA
AG
AA
GG
AA
GA
GA
GG
Grs27978436
75679187
GA
(CT)lowast
Exon
2521
CsT9
6=G
GG
GG
GG
AG
GG
GG
GA
GG
rs226131070
75679178
GA
(CT)
Exon
2530
CsS99=
GG
GG
GG
GG
GG
GG
GG
GA
Grs227683834
75678576
AT
(TA
)Ex
on3
547
CnsV
105D
AA
AA
AA
AA
AA
AA
AA
AT
Ars257321187
75678506
TC(A
G)
Exon
3617
CsP128=
TT
TT
TT
TC
TT
TT
TT
TT
Trs27978444
75678500
TA(A
T)
Exon
3623
CsV130=
TT
TT
TT
TA
TT
TT
TT
AT
Trs227743136
75677686
GA
(CT)
Exon
4662
CsH143=
GG
GG
GG
GG
GG
GG
GG
GA
Grs215327202
75677664
CG(G
C)
Exon
4684
CnsA
151P
CC
CC
CC
CG
CC
CC
CC
CC
Crs247539755
75677663
GA
(CT)
Exon
4685
CnsA
151V
GG
GG
GG
GA
GG
GG
GG
GG
Grs233164
668
75677640
TC(A
G)
Exon
4708
CnsN
159D
TT
TT
TT
TT
TT
TT
TT
TC
T
rs2340
95816
75677162ndash75677160
GCT
del
(AGCdel)
Exon
5826minus
828
Cds-in
delQ
198
GCT
Oxidative Medicine and Cellular Longevity 19
Table5Con
tinued
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs27978452
75677145
CT(G
A)
Exon
5843
CnsA
204T
CC
CC
CC
CC
CC
CC
CC
CC
T
rs225047937
75677086ndash75677078
GAG
ATCG
ATdel
(ATG
CATC
TCdel)
Exon
5902minus
910
Cds-in
del
(S224I225S226)
GAG
ATC
GAT
rs241469868
75676998
TA(G
T)
Exon
5990
CnT2
53S
TT
TT
TT
TT
TT
TT
TT
TA
Trs221602571
75676923
AC
(TG
)Ex
on5
1065
CnsS
278A
AA
AA
AA
AA
AA
AA
AA
AC
Ars252576472
75676876
GA
(CT)
Exon
51112
CsS293=
GG
GG
GG
GG
GG
GG
GG
GA
Grs250802933
75676841
TC(A
G)
Exon
5114
7Cn
sD305G
TT
TT
TT
TT
TT
TT
TT
TC
Trs225425698
75676792
CT(G
A)
Exon
5119
6Cs
P321=
CC
CC
CC
CC
CC
CC
CC
CT
Crs13459064
75676732
CT(G
A)
Exon
51256
CsT3
41=
CT
TC
TT
CT
TT
CT
CT
TC
Crs27978453
75676717
CT(G
A)
Exon
51271
CsA346=
CC
CC
CC
CC
CC
CC
CC
TC
Crs214335034
75676636
AG
(TC)
Exon
51352
CsD373=
AA
AA
AA
AA
AA
AA
AA
AG
Ars24958364
4756766
03AC
(TG
)Ex
on5
1385
CsP3
84=
AA
AA
AA
AA
AA
AA
AA
AC
Ars27978454
75676592
GA
(CT)
Exon
51396
CnsP
388L
GG
GG
GG
GG
GG
GG
GG
AG
Grs215384328
75676589
GC
(CG
)Ex
on5
1399
CnsA
389G
GG
GG
GG
GG
GG
GG
GG
GC
Grs251728286
75676571
GA
(CT)
Exon
51417
CnsT
395I
Grs247602334
75676567
TC(A
G)
Exon
51421
CsV396=
TT
TT
TT
TT
TT
TT
TT
TC
Trs234216231
75676530
TC(A
G)
Exon
51458
CnsM
409V
TT
TT
TT
TT
TT
TT
TT
TC
Trs212904337
75676526
CT(G
A)
Exon
51462
CnsR
410H
CC
CC
CC
CC
CC
CC
CC
CT
Crs252650779
75676522
TC(A
G)
Exon
51466
CsE4
11=
TT
TT
TT
TT
TT
TT
TT
TC
Trs231273560
75676516
TC(A
G)
Exon
51472
CsQ413=
TT
TT
TT
TT
TT
TT
TT
TC
Trs257886949
756764
13GT
(CA
)Ex
on5
1575
CsR4
48=
GG
GG
GG
GG
GG
GG
GG
GT
Grs241537608
756764
04GC
(CG
)Ex
on5
1584
CnsL
451V
GG
GG
GG
GG
GG
GG
GG
GC
Grs227619071
75676312
TC(A
G)
Exon
51676
CsQ481=
TT
TT
TT
TT
TT
TT
TT
TC
Trs258913831
75676290
AG
(TC)
Exon
51698
CsL4
89=
AA
AA
AA
AA
AA
AA
AA
AG
Ars225593319
75676126
AT
(TA
)Ex
on5
1862
CnsH
543Q
Ars4223233
75676105
GA
(CT)
Exon
51883
CsS550=
GG
GG
GG
GA
GG
GG
GG
GG
Grs4223232
75676032
GT
(CA
)Ex
on5
1956
CnsL
575M
Grs4223231
75675816
CG
UTR
-32172
NC
GG
GG
GG
GG
GG
GG
GG
GC
Grs1346
860
75675682
AT
UTR
-32306
NC
ASequ
ence
varia
tions
inmou
seNr
f2wereob
tained
from
Mou
sePh
enom
eDatabase(http
ph
enom
ejaxorgSNP)
andNCB
ISNPdatabase
(http
wwwncbinlm
nihgovSNP)N
CBIreference
sequ
ence
isMus
Musculusstra
inC5
7BL6J
chromosom
e2GRC
m38p1(NC
0000
687
GI37209910875704641ndash756755192
9123
bp)To
tal968
genetic
mutations
arerepo
rted
inNr
f2gene
and5k
bup
stream
(ge75704
642minus
1)and2k
bdo
wnstre
am(le75675512)sequences
asof
Janu
ary2003A
llSN
Psandallelesa
representedas
geno
miccontig
(reversedsequ
ence
indicated)E
xon175704
641ndash75704364(278
bpT
TS=75704
408)
exon
275679431ndash756791
65(267
bp)exon
375678579ndash75678490(90b
p)exon475677713ndash756775
46(168
bp)andexon
57567718
4ndash75678849
(1666
bp)Proteinam
inoacid
(aa)
resid
uesinNP00
61552
(597
aa)Cs
cod
ing-syno
nymou
sCn
scoding
-non
syno
nymou
sAminoacid
A-alanineD-aspartic
acidE
-glutamicacidF-phenylalanineG
-glycineH
-histidineI-iso
leucineL-leu
cineand
M-m
ethion
ineN-
asparagineP-prolin
eQ-glutamineR-arginineS-serineT-threon
ineandV-valin
elowastErrorsin
databasesfi
xedNC
notcon
firmed
20 Oxidative Medicine and Cellular Longevity
Table 6 NRF2 somatic mutations revealed in various human cancers
Domain Amino acid residues DNA mutation Cancer types (cases) ReferencesLocus Wild type Mutant
DLG motif
24 W (Trp) C (Cys) c72GgtCGgtT NSCLC neck ESC [54 62]K (Lys) c72TgtC ESC [62]
26 Q (Gln) E (Glu) c76CgtG NSCLC ESC [54 62]
27 D (Asp) G (Gly)lowast c80GgtA NSCLC [60]Y (Tyr) c79GgtT ESC [61]
28 I (Ile) T (Thr) c83CgtT NSCLC [54]
29 D (Asp) G (Gly) c86AgtG Head and neck ESC [54 62]H (His) c85GgtC NSCLC laynx [60 61]
30 L (Leu) F (Phe) c88CgtT NSCLC ESC [54 62]
31 G (Gly) A (Ala) c92GgtC NSCLC ESC skin [54 60ndash62]
32 V (Val) T (Thr) c95TgtG NSCLC [54]del c93 95delAGT ESC [61]
33ndash36 S-R-E-V S-R-E-V-S-R-E-Vlowast c97 108dupAGTCGAGCCGTA ESC [61]
34 R (Arg) Q (Gln) c101GgtA NCSCL [54 60 61]P (Pro) c101GgtC NSCLC [63]
ETGF motif
75 Q (Gln) H (His) c225AgtC Head and neck ESC [54 62]
77 D (Asp)
V (Val) c230AgtT NSCLC ESC [54 60 62]G (Gly) c230AgtG ESC [62]A (Ala) c230AgtC NSCLC [61]N (Asn) c229GgtA Larynx [61]
78 E (Glu) K (Lys) c232GgtA NSCLC ESC [54 62]
79 E (Glu)
K (Lys) c235GgtA NSCLC ESC [54 61 62]Q (Gln) c235GgtC NSCLC ESC [54 60ndash62]G (Gly) c236AgtG Larynx [61]E-E c234 236dupAGA ESC [61]
80 T (Thr) K (Lys) c239CgtACgtG NSCLC ESC [54 61 62]
80 T (Thr)P (Phe) c238AgtC ESC [62]I (Ile) c239CgtT Head and neck [54]A (Ala) c238AgtG NSCLC [63]
81 G (Gly) V (Val) c242GgtT ESC [61]D (Asp) c242GgtA NSCLC ESC [60ndash62]
82 E (Glu)
D (Asp) c246AgtT ESC oral cancer cell line [54 62]G (Gly) c245AgtG NSCLC [54]Q (Gln)lowast c244GgtC NSCLC ESC [60 61]V (Val)lowast c245AgtT ESC [61]
83 F (Phe) L (Leu)lowast c247TgtC NSCLC [60]NSCLC non-small cell lung cancer ESC esophageal squamous cancer and lowasterrors in reference fixed Number of cases 82 NSCLC and 10 ESC in [62] 125NSCLC 70 ESC 23 larynx and 17 skin in [61] 103 NSCLC and 12 head and neck in [54] 90 NSCLC in [63] 103 NSCLC in [60]
be involved in squamous lung cancer cell proliferation [64]suggesting that the mutation is functional and overcomesKEAP1 inhibition Singh et al [65] determined in vitrothat RNAi-mediated depletion of NRF2 in lung cancer cellsenhanced ROS production and susceptibility to cell deathby ionizing radiation These studies support the concept thatelevated NRF2 and ARE responsiveness provides cancer cellswith proliferative advantage for malignant transformation
and undue protection from anti-cancer therapy Onco-genic epidermal growth factor receptor (EGFR) signaling isrecently found to be critical in NRF2-mediated proliferationof NSCLC cells [66]
Collectively ldquogain of functionrdquo mutations in NRF2 thatreduce KEAP1 recognition are suggested to be predictivemarkers for poor responsiveness to chemotherapy and radi-ation therapy Although NRF2-mediated cellular defense
Oxidative Medicine and Cellular Longevity 21
processes are essential in the initiation stage enhancedNRF2-ARE activity in advanced stages of cancer developmentmay create a favorable intracellular environment for tumorcell growth and survival [67 68] In this context NRF2may be a potential molecular target for the treatment ofradio-resistance cancers especially those that have ldquoloss offunctionrdquo mutations in EGFR KRAS or KEAP1 as well asldquogain of functionrdquo mutations in NRF2
5 Epigenetic Alterations of NF-E2-RelatedFactor 2
Epigenetic modifications are alterations of molecules inter-acting with genes without changes to the primary DNAsequence They include post-translational modification ofhistones DNA methylation events chromatin conforma-tional changes and alterations to noncoding regulatoryRNAs Epigenetic alterations are stable and often inheritablebut are reversible and may affect expression of the geneDysregulation or defects in epigenetic processes particularlyhypermethylation of tumor suppressor gene promoters (egCpG islands) or histone modifications are thought to beassociated with carcinogenesis Investigators have reportedhypermethylation in CpG islands of KEAP1 which wereassociated with reduced KEAP1 expression in human cancersfrom lung prostate colon and so forth [69ndash72] Similar tosomatic mutations epigenetic changes on KEAP1 impairedthe function of its encoded protein leading to constitutiveNRF2 activation
Supporting the role of ldquopathogenic mutationsrdquo in NRF2expression of Nrf2 and downstream Nqo1 was suppressedin prostate tumors of mice (transgenic adenocarcinomaof mouse prostate TRAMP) Among 15 promoter CpGislands located between minus942 and minus654 (cminus1175 cminus1132and cminus1059 cminus887 gap in cminus1131 cminus1060 see Figure 3)hypermethylation of the first 5 CpG islands (minus942 minus899 andcminus1175 cminus1132) was significantly associated with tumorige-nesis [73] Moreover treatment with inhibitors for DNAmethyltransferase and histone deacetylase restored Nrf2expression in these tumor cells [73] A dietary phytochemicalcurcumin known as a DNA hypomethylation agent restoredepigenetically silent Nrf2 expression through CpG demethy-lation in carcinogen-induced mouse tumor cells [74]
The whole genome epigenetic datasets for 5 species arepublicly accessible at NCBI Epigenomics [75 76]The humanNRF2 epigenome of primary cells (breast penis) and H1stem cell line as well as mouse Nrf2 CpG island methylationdata for sperm blood and cerebellum are currently avail-able (httpwwwncbinlmnihgovepigenomics) Althoughno direct evidence of disease-associated epigenetic modu-lation has been identified in human NRF2 various phyto-chemical NRF2 agonists such as sulforaphane and curcuminhave shown their roles in DNA methylation and histonemodification (see reviews by Lee and colleagues eg [77])Taken together epigenetic modifications of theNRF2KEAP1axle are predicted to cause dysregulation of ARE-mediatedcellular defense leading to deleterious health effects and
phytochemical antioxidants as epigenetic modulators forNRF2 are suggested to be useful in cancer prevention
6 Conclusions
NRF2 is evolutionally conserved with high-sequence homol-ogy in many species However it is a highly mutable geneand numerous genetic variants have been discovered inhuman ethnic groups Importantly certain SNPs or haplo-types have been identified in various diseases as ldquoat-riskrdquoalleles and are related to functional alterations In additionto genetic variations multiple somatic mutations identifiedin the KEAP1 recognition domain of NRF2 in cancer cellshave been found to be oncogenic due to dysregulation ofNRF2 homeostasis by its excess ldquogain of functionrdquo Epigeneticalteration of theNRF2 is under investigation and is predictedto have pathogenic influences as learned from mouse andphytochemical agonist studies Continuous updates of Nrf2allelic variants in inbred mouse strains will provide a usefultool for effective experimental designs formodels of oxidativedisorders to provide insight into the disease mechanisms andintervention strategies
Abbreviations
AD Alzheimerrsquos diseaseALI acute lung injuryARE antioxidant response elementbZIP basic leucine zippercds coding DNA sequencesCI confidence intervalCOPD chronic obstructive pulmonary diseaseESC esophageal squamous cancerFEV1 forced expiratory volume in one second
Gbp giga base pairsGST glutathione S-transferaseGWAS genome-wide association studyHGVS Human Genome Variation SocietyHO-1 heme oxygenase-1119867 119901119910119897119900119903119894 119867119890119897119894119888119900119887119886119888119905119890119903 119901119910119897119900119903119894KEAP1 Kelch-like ECH activating protein 1MPD Mouse Phenome DatabaseMRP multidrug resistance proteinNCBI National Center for Biotechnology InformationNeh NRF2-ECH homologyNfe2l2 nuclear factor (erythroid-derived 2)-like 2Nrf2 NF-E2-related factor 2NSCLC nonsmall cell lung cancerNQO1 NAD(P)Hquinone oxidoreductase 1OR odds ratioPD Parkinsonrsquos diseasePM particulate matterROS reactive oxygen speciesSLE systemic lupus erythematosusSNP single nucleotide polymorphismSOD superoxide dismutaseURT untranslated region
22 Oxidative Medicine and Cellular Longevity
Disclosure
Authorrsquos contribution to the Work was done as part of theAuthorrsquos official duties as a NIH employee and is a Work ofthe United States Government Therefore copyright may notbe established in the United States
Conflict of Interests
The author declares that there is no conflict of interests
Acknowledgments
The research related to this paper was supported by theIntramural Research Program of the National Institutes ofHealth of the National Institute of Environmental HealthSciences (NIEHS) Drs Steven Kleeberger and StephanieLondon at the NIEHS provided excellent critical review ofthis paper The author thanks Mrs Jacqui Marzec for herhelpful comments and English editing
References
[1] J Y Chan M C Cheung P Moi K Chan and Y W KanldquoChromosomal localization of the humanNF-E2 family of bZIPtranscription factors by fluorescence in situ hybridizationrdquoHuman Genetics vol 95 no 3 pp 265ndash269 1995
[2] K Itoh K Igarashi N Hayashi M Nishizawa and MYamamoto ldquoCloning and characterization of a novel erythroidcell-derived CNC family transcription factor heterodimerizingwith the small Maf family proteinsrdquo Molecular and CellularBiology vol 15 no 8 pp 4184ndash4193 1995
[3] W O Osburn and T W Kensler ldquoNRF2 signaling an adaptiveresponse pathway for protection against environmental toxicinsultsrdquoMutation Research vol 659 no 1-2 pp 31ndash39 2008
[4] D Papp K Lenti D Modos D Fazekas Z Dul D Tureiet al ldquoThe NRF2-related interactome and regulome containmultifunctional proteins and fine-tuned autoregulatory loopsrdquoFEBS Letters vol 586 no 13 pp 1795ndash1802 2012
[5] X Wang D J Tomso B N Chorley et al ldquoIdentificationof polymorphic antioxidant response elements in the humangenomerdquo Human Molecular Genetics vol 16 no 10 pp 1188ndash1200 2007
[6] D Malhotra E Portales-Casamar A Singh et al ldquoGlobalmapping of binding sites for NRF2 identifies novel targets incell survival response through ChIP-Seq profiling and networkanalysisrdquo Nucleic Acids Research vol 38 no 17 pp 5718ndash57342010
[7] K Chan R Lu J C Chang and Y W Kan ldquoNRF2 a memberof the NFE2 family of transcription factors is not essential formurine erythropoiesis growth and developmentrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 93 no 24 pp 13943ndash13948 1996
[8] K Itoh T Chiba S Takahashi et al ldquoAn NRF2small Mafheterodimer mediates the induction of phase II detoxifyingenzyme genes through antioxidant response elementsrdquo Bio-chemical and Biophysical Research Communications vol 236no 2 pp 313ndash322 1997
[9] FMartin J M vanDeursen R A Shivdasani CW Jackson AG Troutman and P A Ney ldquoErythroid maturation and globin
gene expression inmicewith combined deficiency ofNF-E2 andNrf-2rdquo Blood vol 91 no 9 pp 3459ndash3466 1998
[10] K Itoh N Wakabayashi Y Katoh et al ldquoKEAP1 repressesnuclear activation of antioxidant responsive elements by NRF2through binding to the amino-terminal Neh2 domainrdquo Genesand Development vol 13 no 1 pp 76ndash86 1999
[11] A Kobayashi M I Kang H Okawa et al ldquoOxidative stresssensor KEAP1 functions as an adaptor for Cul3-based E3 ligaseto regulate proteasomal degradation of NRF2rdquo Molecular andCellular Biology vol 24 no 16 pp 7130ndash7139 2004
[12] K Taguchi H Motohashi and M Yamamoto ldquoMolecularmechanisms of the KEAP1-NRF2 pathway in stress responseand cancer evolutionrdquo Genes to Cells vol 16 no 2 pp 123ndash1402011
[13] K Mukaigasa L T Nguyen L Li H Nakajima M Yamamotoand M Kobayashi ldquoGenetic evidence of an evolutionarilyconserved role for NRF2 in the protection against oxidativestressrdquoMolecular and Cellular Biology vol 32 no 21 pp 4455ndash4461 2012
[14] JMaher andMYamamoto ldquoThe rise of antioxidant signalingmdashthe evolution and hormetic actions of NRF2rdquo Toxicology andApplied Pharmacology vol 244 no 1 pp 4ndash15 2010
[15] S C Lo X Li M T Henzl L J Beamer and M HanninkldquoStructure of the KEAP1NRF2 interface provides mechanisticinsight intoNRF2 signalingrdquoThe EMBO Journal vol 25 no 15pp 3605ndash3617 2006
[16] V A McKusick ldquoMendelian inheritance in man and its onlineversion OMIMrdquo American Journal of Human Genetics vol 80no 4 pp 588ndash604 2007
[17] B Yalcin D J Adams J Flint and T M Keane ldquoNext-generation sequencing of experimental mouse strainsrdquo Mam-malian Genome vol 23 no 9-10 pp 490ndash498 2012
[18] D R Bentley S Balasubramanian H P Swerdlow et alldquoAccurate whole human genome sequencing using reversibleterminator chemistryrdquo Nature vol 456 no 7218 pp 53ndash592008
[19] P S G Chain D V Grafham R S Fulton et al ldquoGenomeproject standards in a new era of sequencingrdquo Science vol 326no 5950 pp 236ndash237 2009
[20] RHWaterstonK Lindblad-Toh E Birney J Rogers J F AbrilP Agarwal et al ldquoInitial sequencing and comparative analysis ofthe mouse genomerdquo Nature vol 420 pp 520ndash562 2002
[21] T M Keane L Goodstadt P Danecek M A White K WongB Yalcin et al ldquoMouse genomic variation and its effect onphenotypes and gene regulationrdquoNature vol 477 pp 289ndash2942011
[22] T Yamamoto K Yoh A Kobayashi et al ldquoIdentification ofpolymorphisms in the promoter region of the human NRF2generdquo Biochemical and Biophysical Research Communicationsvol 321 no 1 pp 72ndash79 2004
[23] J M Marzec J D Christie S P Reddy et al ldquoFunctionalpolymorphisms in the transcription factor NRF2 in humansincrease the risk of acute lung injuryrdquo The FASEB Journal vol21 no 9 pp 2237ndash2246 2007
[24] H Fukushima-Uesaka Y Saito K Maekawa et al ldquoGeneticvariations and haplotype structures of transcriptional factorNRF2 and its cytosolic reservoir protein KEAP1 in JapaneserdquoDrug Metabolism and Pharmacokinetics vol 22 no 3 pp 212ndash219 2007
[25] E J Cordova R Velazquez-Cruz F Centeno V Baca and LOrozco ldquoThe NRF2 gene variant -653GA is associated with
Oxidative Medicine and Cellular Longevity 23
nephritis in childhood-onset systemic lupus erythematosusrdquoLupus vol 19 no 10 pp 1237ndash1242 2010
[26] M von Otter S Landgren S Nilsson et al ldquoAssociation ofNRF2-encoding NFE2L2 haplotypes with Parkinsonrsquos diseaserdquoBMCMedical Genetics vol 11 no 1 article 36 2010
[27] JMHartikainenM TengstromVMKosmaV L Kinnula AMannermaa andY Soini ldquoGenetic polymorphisms and proteinexpression of NRF2 and sulfiredoxin predict survival outcomesin breast cancerrdquo Cancer Research vol 72 pp 5537ndash5546 2012
[28] T Arisawa T Tahara T Shibata et al ldquoThe relationshipbetween Helicobacter pylori infection and promoter polymor-phism of the NRF2 gene in chronic gastritisrdquo InternationalJournal of Molecular Medicine vol 19 no 1 pp 143ndash148 2007
[29] T Arisawa T Tahara T Shibata et al ldquoThe influence ofpromoter polymorphism of nuclear factor-erythroid 2-relatedfactor 2 gene on the aberrant DNA methylation in gastricepitheliumrdquo Oncology Reports vol 19 no 1 pp 211ndash216 2008
[30] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene pro-moter polymorphism and gastric carcinogenesisrdquo Hepato-Gastroenterology vol 55 no 82-83 pp 750ndash754 2008
[31] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene promoterpolymorphism is associated with ulcerative colitis in a Japanesepopulationrdquo Hepato-Gastroenterology vol 55 no 82-83 pp394ndash397 2008
[32] C C Hua L C Chang J C Tseng C M Chu Y C Liu andW B Shieh ldquoFunctional haplotypes in the promoter region oftranscription factor NRF2 in chronic obstructive pulmonarydiseaserdquo Disease Markers vol 28 no 3 pp 185ndash193 2010
[33] S O Shaheen R B Newson S M Ring M J Rose-ZerilliJ W Holloway and A J Henderson ldquoPrenatal and infantacetaminophen exposure antioxidant gene polymorphismsand childhood asthmardquo The Journal of Allergy and ClinicalImmunology vol 126 no 6 pp 1141e7ndash1148e7 2010
[34] H Masuko T Sakamoto Y Kaneko et al ldquoLower FEV1 innon-COPD nonasthmatic subjects association with smokingannual decline in FEV1 total IgE levels and TSLP genotypesrdquoInternational Journal of Chronic Obstructive Pulmonary Diseasevol 6 pp 181ndash189 2011
[35] C P Guan M N Zhou A E Xu et al ldquoThe susceptibilityto vitiligo is associated with NF-E2-related factor 2 (NRF2)gene polymorphisms a study on Chinese Han populationrdquoExperimental Dermatology vol 17 no 12 pp 1059ndash1062 2008
[36] J Bouligand O Cabaret M Canonico et al ldquoEffect of NFE2L2genetic polymorphism on the association between oral estrogentherapy and the risk of venous thromboembolism in post-menopausal womenrdquo Clinical Pharmacology amp Therapeuticsvol 89 no 1 pp 60ndash64 2011
[37] D S OrsquoMahony B J Glavan T D Holden C Fong R A BlackG Rona et al ldquoInflammation and immune-related candidategene associations with acute lung injury susceptibility andseverity a validation studyrdquo PLoS ONE vol 7 no 12 Article IDe51104 2012
[38] I Ungvari E Hadadi V Virag et al ldquoRelationship between airpollutionNFE2L2 gene polymorphisms and childhood asthmain aHungarian populationrdquo Journal of CommunityGenetics vol3 no 1 pp 25ndash33 2012
[39] M SiedlinskiD S Postma JMA Boer et al ldquoLevel and courseof FEV1 in relation to polymorphisms inNFE2L2 andKEAP1 inthe general populationrdquo Respiratory Research vol 10 article 732009
[40] C C Hong C B Ambrosone J Ahn et al ldquoGenetic variabilityin iron-related oxidative stress pathways (NRF2 NQ01 NOS3
and HO-1) iron intake and risk of postmenopausal breastcancerrdquo Cancer Epidemiology Biomarkers and Prevention vol16 no 9 pp 1784ndash1794 2007
[41] C Canova C Dunster F J Kelly C Minelli P L Shah CCaneja et al ldquoPM10-induced hospital admissions for asthmaand chronic obstructive pulmonary disease the modifyingeffect of individual characteristicsrdquo Epidemiology vol 23 no 4pp 607ndash615 2012
[42] A J Henderson R B Newson M Rose-Zerilli S M RingJ W Holloway and S O Shaheen ldquoMaternal NRF2 andgluthathione-S-transferase polymorphisms donotmodify asso-ciations of prenatal tobacco smoke exposure with asthma andlung function in school-aged childrenrdquo Thorax vol 65 no 10pp 897ndash902 2010
[43] C Xing A L Sestak J A Kelly et al ldquoLocalization andreplication of the systemic lupus erythematosus linkage signalat 4p16 interaction with 2p11 12q24 and 19q13 in EuropeanAmericansrdquo Human Genetics vol 120 no 5 pp 623ndash631 2007
[44] T F Wong K Yoshinaga Y Monma K Ito H NiikuraS Nagase et al ldquoAssociation of KEAP1 and NRF2 geneticmutations and polymorphisms with endometrioid endometrialadenocarcinoma survivalrdquo International Journal of Gynecologi-cal Cancer vol 21 no 8 pp 1428ndash1435 2011
[45] M von Otter S Landgren S Nilsson et al ldquoNRF2-encodingNFE2L2 haplotypes influence disease progression but not riskin Alzheimerrsquos disease and age-related cataractrdquoMechanisms ofAgeing and Development vol 131 no 2 pp 105ndash110 2010
[46] S Tsang Z Sun B Luke et al ldquoA comprehensive SNP-basedgenetic analysis of inbred mouse strainsrdquoMammalian Genomevol 16 no 7 pp 476ndash480 2005
[47] C M Wade E J Kulbokas III A W Kirby et al ldquoThe mosaicstructure of variation in the laboratory mouse genomerdquoNaturevol 420 no 6915 pp 574ndash578 2002
[48] T Wiltshire M T Pletcher S Batalov et al ldquoGenome-widesingle-nucleotide polymorphism analysis defines haplotypepatterns in mouserdquo Proceedings of the National Academy ofSciences of the United States of America vol 100 no 6 pp 3380ndash3385 2003
[49] K A Frazer E Eskin H M Kang et al ldquoA sequence-basedvariation map of 827 million SNPs in inbred mouse strainsrdquoNature vol 448 no 7157 pp 1050ndash1053 2007
[50] T P Maddatu S C Grubb C J Bult andM A Bogue ldquoMousephenome database (MPD)rdquo Nucleic Acids Research vol 40 ppD887ndashD894 2007
[51] A Kirby H M Kang C M Wade et al ldquoFine mapping in 94inbred mouse strains using a high-density haplotype resourcerdquoGenetics vol 185 no 3 pp 1081ndash1095 2010
[52] H Y Cho A E Jedlicka S P M Reddy L Y Zhang T WKensler and S R Kleeberger ldquoLinkage analysis of susceptibilityto hyperoxia NRF2 is a candidate generdquo American Journal ofRespiratory Cell and Molecular Biology vol 26 no 1 pp 42ndash512002
[53] H Y Cho A E Jedlicka S P M Reddy et al ldquoRole of NRF2in protection against hyperoxic lung injury in micerdquo AmericanJournal of Respiratory Cell and Molecular Biology vol 26 no 2pp 175ndash182 2002
[54] T Shibata T Ohta K I Tong et al ldquoCancer related mutationsin NRF2 impair its recognition by KEAP1-Cul3 E3 ligase andpromote malignancyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 36 pp13568ndash13573 2008
24 Oxidative Medicine and Cellular Longevity
[55] A Singh V Misra R K Thimmulappa et al ldquoDysfunctionalKEAP1-NRF2 interaction in non-small-cell lung cancerrdquo PLoSMedicine vol 3 no 10 article e420 2006
[56] T Ohta K Iijima M Miyamoto et al ldquoLoss of KEAP1 functionactivates NRF2 and provides advantages for lung cancer cellgrowthrdquo Cancer Research vol 68 no 5 pp 1303ndash1309 2008
[57] B Padmanabhan K I Tong T Ohta et al ldquoStructural basisfor defects of KEAP1 activity provoked by its point mutationsin lung cancerrdquoMolecular Cell vol 21 no 5 pp 689ndash700 2006
[58] T Shibata A Kokubu M Gotoh et al ldquoGenetic alteration ofKEAP1 confers constitutive NRF2 activation and resistance tochemotherapy in gallbladder cancerrdquoGastroenterology vol 135no 4 pp 1358e4ndash1368e4 2008
[59] N J Yoo H R Kim Y R Kim C H An and S H LeeldquoSomatic mutations of the KEAP1 gene in common solidcancersrdquo Histopathology vol 60 no 6 pp 943ndash952 2012
[60] Y Hu Y Ju D Lin Z Wang Y Huang S Zhang et alldquoMutation of the NRF2 gene in non-small cell lung cancerrdquoMolecular Biology Reports vol 39 no 4 pp 4743ndash4747 2012
[61] Y R Kim J EOhM S Kim et al ldquoOncogenicNRF2mutationsin squamous cell carcinomas of oesophagus and skinrdquo TheJournal of Pathology vol 220 no 4 pp 446ndash451 2010
[62] T Shibata A Kokubu S Saito et al ldquoNRF2 mutation confersmalignant potential and resistance to chemoradiation therapyin advanced esophageal squamous cancerrdquo Neoplasia vol 13pp 864ndash873 2011
[63] H Sasaki M Shitara K Yokota Y Hikosaka S MoriyamaM Yano et al ldquoIncreased NRF2 gene (NFE2L2) copy numbercorrelates with mutations in lung squamous cell carcinomasrdquoMolecular Medicine Reports vol 6 no 2 pp 391ndash394 2012
[64] H Sasaki M Shitara K Yokota Y Hikosaka S Moriyama MYano et al ldquoRagD gene expression andNRF2mutations in lungsquamous cell carcinomasrdquo Oncology Letters vol 4 pp 1167ndash1170 2012
[65] A Singh M Bodas N Wakabayashi F Bunz and S BiswalldquoGain of NRF2 function in non-small-cell lung cancer cellsconfers radioresistancerdquo Antioxidants and Redox Signaling vol13 no 11 pp 1627ndash1637 2010
[66] T Yamadori Y Ishii SHommaYMorishimaKKurishimaKItoh et al ldquoMolecular mechanisms for the regulation of NRF2-mediated cell proliferation in non-small-cell lung cancersrdquoOncogene vol 31 pp 4768ndash4777 2012
[67] A K Bauer H Y Cho L Miller-Degraff C Walker K HelmsJ Fostel et al ldquoTargeted deletion of NRF2 reduces urethane-induced lung tumor development in micerdquo PLoS ONE vol 6no 10 Article ID e26590 2011
[68] H Y Cho and S R Kleeberger ldquoNRF2 protects against airwaydisordersrdquo Toxicology and Applied Pharmacology vol 244 no1 pp 43ndash56 2010
[69] R Wang J An F Ji H Jiao H Sun and D Zhou ldquoHyperme-thylation of the KEAP1 gene in human lung cancer cell linesand lung cancer tissuesrdquo Biochemical and Biophysical ResearchCommunications vol 373 no 1 pp 151ndash154 2008
[70] P Zhang A Singh S Yegnasubramanian et al ldquoLoss ofkelch-like ECH-associated protein 1 function in prostate cancercells causes chemoresistance and radioresistance and promotestumor growthrdquoMolecular Cancer Therapeutics vol 9 no 2 pp336ndash346 2010
[71] N Hanada T Takahata Q Zhou et al ldquoMethylation of theKEAP1 gene promoter region in human colorectal cancerrdquoBMCCancer vol 12 article 66 2012
[72] L A Muscarella R Barbano V DrsquoAngelo et al ldquoRegulationof KEAP1 expression by promoter methylation in malignantgliomas and association with patientrsquos outcomerdquo Epigeneticsvol 6 no 3 pp 317ndash325 2011
[73] S Yu T O Khor K L Cheung et al ldquoNRF2 expressionis regulated by epigenetic mechanisms in prostate cancer ofTRAMP micerdquo PLoS ONE vol 5 no 1 Article ID e8579 2010
[74] T O Khor Y Huang T Y Wu L Shu J Lee and A N KongldquoPharmacodynamics of curcumin as DNA hypomethylationagent in restoring the expression of NRF2 via promoter CpGsdemethylationrdquo Biochemical Pharmacology vol 82 no 9 pp1073ndash1078 2011
[75] I M Fingerman L McDaniel X Zhang et al ldquoNCBI epige-nomics a new public resource for exploring epigenomic datasetsrdquo Nucleic Acids Research vol 39 supplemen 1 pp D908ndashD912 2011
[76] I M Fingerman X Zhang W Ratzat N Husain R F Cohenand G D Schuler ldquoNCBI epigenomics whatrsquos new for 2013rdquoNucleic Acids Research vol 41 no 1 pp D221ndashD225 2013
[77] J H Lee T O Khor L Shu Z Y Su F Fuentes andA N Kong ldquoDietary phytochemicals and cancer preventionNRF2 signaling epigenetics and cell death mechanisms inblocking cancer initiation and progressionrdquo Pharmacology ampTherapeutics vol 137 no 2 pp 153ndash171 2012
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
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Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
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Computational and Mathematical Methods in Medicine
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Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Oxidative Medicine and Cellular Longevity 15
and 4 intronic SNPs (rs2886161Ars1806649Ars2001350Ars10183914A) from Swedish (OR 09 CI 060ndash140) and Polish(OR 04 CI 030ndash060) populations (total 165+192 PD cases190+192 controls) [26]The investigators also suggested thatNRF2 haplotype alleles were associated with 2 years earlierage of Alzheimerrsquos disease (AD) onset 4 years earlier age ofposterior subcapsular cataract surgery and 4 years later ageof cortical cataract surgery while they were not significantlyrelated to AD or age-related cataract risk [45]
34 Genetic Mutations in Mouse Nrf2 Tsang et al [46]compiled 673 SNPs in 55 mouse strains and constructedtheir phylogenetic tree to correlate and clarify the originsof strains based on the assembled mouse genome sequenceand SNP data [20 47 48] Recently using the completegenome sequence of C57BL6J (B6) mouse as a referencehigh-density SNP screening in other laboratory strains orin panels of strains has been published (see [17]) Althoughmillions of mouse SNPs (gt10089892 as of December 2012)and haplotype mappings from more than 120 strains havebeen published as valuable references for dissecting thegenetic basis of complex traits [49ndash51] little attention hasbeen paid to polymorphisms of Nrf2 and their correlationwith disease phenotypes
Figure 3 demonstrates the proximal promoter region(minus1 to minus950)51015840-UTR (exon 1 up to TSS) and proteinsequence of mouse Nrf2 based on GRCm38p1 PrimaryAssembly (75704641ndash75675513 bp) mRNA variant1 and protein (NP 035032 encoded by NM 010902234ndash2027 bp) sequences Genetic variations in the Nrf2genome of inbred strains collected from public databasesare listed in Tables S2 and 5 (See Supplementary TableS2) Overall 968 genetic mutations are compiled forNrf2 gene and 5 kb upstream2 kb downstream regions785 SNPs between B6 and another 16 strains wereacquired from the Mouse Phenome Database (MPDhttpphenomejaxorg dbqrtn=snpret1) and additionalSNPs and other mutations were acquired from NCBI dbSNP(httpwwwncbinlmnihgovsnpterm=mus+musculus20nfe2l2) In total 132 mutations are in the promoter (37in proximal 1 kb) 49 in exons (38 in coding region 19 Cns)727 in introns and 60 in the 31015840 flanking region Excludingmutations in the 51015840 and 31015840 flanking sequences murine Nrf2sequences appear to be more highly variable (1 variationper 375 bp) than much of the mouse genome which has anapproximate frequency of one SNP per every 245 bp (httpwwwinformaticsjaxorgmgihomehomepagesstatsallstatsshtmlallstats snp)
Nrf2 was found to be a susceptibility gene from genome-wide linkage analysis in a murine model of hyperoxia-induced ALI [52] A promoter SNP minus103TgtC (previouslypublished as minus336TgtC) in Nrf2 was found and predicted toadd an additional Sp1 binding site in hyperoxia-susceptibleB6 mice but not in resistant C3HHeJ mice [52] Genotypesfrom the SNP and from simple-sequence length polymor-phism markers of the Nrf2 locus (D2Mit248 and D2Mit94)cosegregated in the B6C3F
2mouse cohort [52] and Nrf2
deficient mice were significantly more susceptible to ALI
sub-phenotypes caused by hyperoxia than similarly exposedwild-type mice supporting Nrf2 as a contributor to thephenotypic traits [53] Although no other functional analyseson Nrf2 SNPs or haplotype association studies have beenconducted in inbred mice strains bearing haplotypes suchas multiple Cns in functional domains (eg F71L L451VH543Q and L575M) may be useful to elucidate the role ofNrf2 in differential susceptibility to oxidative diseases
4 Oncogenic Somatic Mutations in HumanNF-E2-Related Factor 2
Somatic mutation is a change in the DNA of somatic cellsthat affects derived cells but is not inherited by offspringEfforts to discover somatic mutations have provided insightinto mutagenesis and cancer development Lung cancerparticularly non-small cell lung cancer (NSCLC) is theleading cause of cancer death worldwide Somatic mutationsof NRF2 and KEAP1 discovered in lung cancer patients havedetermined the oncogenic potential ofNRF2 [54 55] KEAP1somatic mutations were associated with its reduced proteinlevels in lung cancer tissues and cells [56 57] Investigationsof NSCLC in various ethnic populations as well as cancersin gastrointestine breast and prostate have coordinatelydemonstrated that multiple Cns somatic mutations in KEAP1cause dysfunction of the translated protein and in turnconstitutive activation of NRF2 increasing risk of neoplasiaand chemoresistance [12 55 58 59] Somatic mutations ofNRF2 have been detected in various cancer tissues (largelysquamous cell carcinomas) in Asian populations (Table 6)NRF2 mutations were significantly associated with NSCLCcases (squamous cell lung carcinoma adenocarcinoma) ofthe Japanese (107 [54]) the Chinese (23 [60]) andthe Koreans (8 [61]) as well as with lung cancer celllines Smoking history was also correlated with mutationoccurrence in all of the studies [54 60 61] In addition tolung cancers laryngeal squamous carcinoma (13 in [61])esophageal squamous cancer (ESC 22 in [60] 114 in[61]) head and neck cancers (25 in [54]) skin (117 casein [61]) and oral cancer cell lines had somatic changesin NRF2 In contrast to wide-spread KEAP1 mutationsmutations in NRF2 were clustered in DLGETGE motifs ofthe Neh2 domain which are critical in the ldquohinge and latchrdquomodel of KEAP1 binding [12] Similar to KEAP1 somaticmutations it has been postulated that NRF2 mutations incancer cells lead to NRF2 accumulation by suppressing itsubiquitination or KEAP1 binding which eventually confersmalignant potential and resistance to chemotherapy
Most variable sites in NRF2 included aa residues 29 (AspD) 31 (Gly G) 77 (Asp D) and 79 (Glu E) (Table 6)Residue 33 (Ser S) in the Neh2 domain is mutated by eithergenetic or somatic processes (Figure 4) Cns in the EDGFmotif of NRF2 was experimentally determined to impairrecognition of KEAP1 [54] NRF2 mutations were signif-icantly correlated with increased (25-fold) copy number(31 of mutants versus 3 wild types) in Japanese NSCLCcases [63] Aberrant mutation of NRF2 also led to increasedexpression of downstream effectors includingRagD known to
16 Oxidative Medicine and Cellular Longevity
pL575M
pF71L
pH543QpL451V
pA151P pA151V pN159D pQ198delpV105D
pA204T pS224I225S226del pT253S pS278ApD305G
pM409V pR410HpP388L pA389G pT395I
Genomic DNA (reversed contig GRCm38 p1 C57BL6JNM 0109023)
Protein (NP 035032)1
51101151201251301351401451501551
minus400
minus500
minus50
minus100
minus200
minus300
minus450
minus350
minus250
minus150
minus950
minus900
minus850
minus800
minus750
minus700
151
101151201
2001
g minus470gg minusg minus
c minus41c minus
c
c
c c
gg minus 6C
g minus125insTTC g minus118insC
g minus281delC
g minus63delCg minus1 (75704642 c minus234)
Translation start site (c +1)Termination codon (c +2025 +2027)
c minus136 minus134in-frame stop codonc minus8585
g minus41
246
440
minus381
minus11
minus91 minus90
minus332
minus153
minus98
c minus197c minus209 minus202
gminus942 minus899 (cminus1175 minus1132) CpG islands [71]
g 460CgtT
g minus103CCgtT [51]
CgtTCgtG
g minus320CgtT
CgtT
CgtT
CgtTg minus426GgtA
GgtACgtA
CgtA
GgtA
g minus238GgtTTgtC
TgtC
TgtC
TgtCTgtG
g minus229AgtG
CgtG
CgtG
GgtC
gminus826 minus654 (cminus1059 ) CpG islands [71]minus887
Figure 3 DNA (partial promoter and exons 1 and 5) and protein sequence ofmouseNrf2 SNPs and amino acid residues for non-synonymousSNPs are marked Promoter regions bearing 5CpG islands are underlined
151
201251301351401451501551601
Genetic mutation (Table 3 and Figure 2)Somatic mutation (Table 6)
Neh2
Neh3
Neh4Neh5
Neh6
Neh1
Protein isoform 1 (NP 006155)
101151
Figure 4 Genetic and somatic mutation loci in human NRF2 protein
Oxidative Medicine and Cellular Longevity 17
Table5Mou
seup
stream
andexon
varia
tions
ofNfe2
l2locusin17
inbred
strainsR
eference
sequ
ence
isC5
7BL6J
(B6strain
1)geno
me(GI14933824975547698ndash75513576)SNPalleleand
geno
typesareshow
nas
chromosom
econ
tigsequ
enceStrains
2129S1S
vlmJ3AJ
4AKR
J5BA
LBcJ6C3
HH
eJ7C
57BL
6NJ8CA
STEiJ9CB
AJ
10D
BA2J11FVBNJ12LPJ
13N
ODShiLtJ14N
ZOH
ILtJ
15P
WK
PhJ16SPR
ETEiJ
17W
SBEiJ
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs2566
08517
75705565
GA
(CT)
51015840Flankingminus924
NC
GG
GG
GG
GG
GG
GG
GG
AA
Grs47274959
75705562
AT
(TA
)51015840Flankingminus921
NC
AT
TA
TT
AA
TT
AT
AT
TT
Ars216940398
75705554ndash75705555
Tadd(A
add)
51015840Flankingminus913minus914
NC
CGrs239114134
75705540
CT(G
A)
51015840Flankingminus899
NC
CT
TC
TT
CC
TT
CT
CT
CT
Crs46
461765
75705528
TG(A
C)oA
C
51015840Flankingminus887
NC
TG
GT
GG
TT
GG
TG
TG
GG
Trs5144
9853
75705525
CA(G
T)
51015840Flankingminus884
NC
CA
AC
AA
CC
AA
CA
CA
CC
Crs249093111
75705512
CT(G
A)
51015840Flankingminus871
NC
CT
TC
TT
CC
TT
CT
CT
TT
Crs263745200
75705510
CG(G
C)
51015840Flankingminus869
NC
GG
GG
GG
GG
GG
GG
GG
CG
Grs45651867
75705498
TG(A
C)
51015840Flankingminus857
NC
TG
GT
GG
TT
GG
TG
TG
GG
Trs219997531
75705495
GA
(CT)
51015840Flankingminus854
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs251918379
75705451
TA(A
T)
51015840Flankingminus810
NC
TT
TT
TT
TT
TT
TT
TT
TA
Trs27978306
75705449
TA(A
T)
51015840Flankingminus808
NC
TA
AT
AA
TT
AA
TA
TA
TT
Trs216087412
75705429
TG(A
C)lowast
51015840Flankingminus788
NC
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
GTlowast
Tlowast
rs261229914
75705423ndash75705424
AGdel(CT
del)
51015840Flankingminus783minus784
NC
AGrs27978307
75705400
CT(G
A)
51015840Flankingminus759
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs243167395
75705359
GA
(CT)
51015840Flankingminus718
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs27978308
75705307
GA
(CT)
51015840Flankingminus66
6NC
GG
GG
GG
GG
GG
GG
GG
AG
Grs254744
09875705294
AT
(TA
)51015840Flankingminus653
NC
AA
AA
AA
AA
AA
AA
AA
AT
Ars228133419
75705179
GT
(CA
)51015840Flankingminus538
NC
GG
GG
GG
GG
GG
GG
GG
GT
Grs214719520
75705111
GA
(CT)
51015840Flankingminus470
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs2440
87730
75705101
GA
(CT)
51015840Flankingminus46
0NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs227781699
75705081
CG(G
C)
51015840Flankingminus44
0NC
CC
CC
CC
CC
CC
CC
CC
GC
Crs257870353
75705067
CT(G
A)
51015840Flankingminus426
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs24487744
075705022
AG
(TC)
51015840Flankingminus381
NC
AA
AA
AA
AA
AA
AA
AA
AG
Ars234628138
75704973
AC
(TG
)51015840Flankingminus332
NC
AA
AA
AA
AA
AA
AA
AA
AC
A
18 Oxidative Medicine and Cellular Longevity
Table5Con
tinued
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs247275247
75704961
GA
(CT)
51015840Flankingminus320
NC
GG
GG
GG
GG
GG
GG
AG
GG
Grs247519047
75704922
Gdel(Cdel)
51015840Flankingminus281
NC
Grs27978309
(rs51915758)
75704887
GA
C(C
TG
)51015840Flankingminus246
NC
GA
AG
AA
GG
AA
GA
GA
CC
G
rs218139102
75704879
CA(G
T)
51015840Flankingminus238
NC
CC
CC
CC
CC
CC
CC
CC
AC
Crs251990355
75704870
TC(A
G)
51015840Flankingminus229
NC
TT
TT
TT
TT
TT
TT
TT
TC
Trs238746955
75704794
AG
(TC)
51015840Flankingminus153
NC
AA
AA
AA
AA
AA
AA
AA
GA
Ars221664
405
75704786
AG
(TC)
51015840Flankingminus145
NC
AA
AA
AA
AA
AA
AA
AA
AG
Ars217054035
75704766
ndash75704767
GAAadd(TTC
add)
51015840Flankingminus125minus126
NC
GA
rs248182931
75704759ndash75704760
Gadd(C
add)
51015840Flankingminus118
minus119
NC
AGrs217197904
75704744
GA
(CT)
51015840Flankingminus103
(+)S
p1[23]
GA
AG
AA
GG
AA
GA
AA
GG
Ars2644
9364975704704
Gdel(Cdel)
51015840Flankingminus63
NC
Grs2404
81112
757046
82CT(G
A)
51015840Flankingminus41
NC
CC
CC
CC
CC
CC
CC
CC
CT
Crs27978310
75704617
AG
(TC)
UTR
-525
NC
AA
AA
AA
AA
AA
AA
AA
GG
Ars27978311
75704610
CG(G
C)
UTR
-532
NC
GG
GG
GG
GG
GG
GG
GG
GG
Crs27978312
757046
05GA
(CT)
UTR
-537
NC
GA
AG
AA
GG
AA
GA
GA
GG
Grs214784220
757044
99AG
(TC)
UTR
-5143
NC
AA
AA
AA
AA
AA
AA
AA
GG
Ars244146318
757044
98GT
(CA
)UTR
-5144
NC
GG
GG
GG
GG
GG
GG
GG
GT
Grs27978313
757044
97GC
(CG
)UTR
-5145
NC
GC
CG
CC
GC
CC
GC
CC
CC
Crs215431944
757044
93CT(G
A)
UTR
-5149
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs257015697
757044
49GA
(CT)
UTR
-5193
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs252234782
757044
19GT
(CA
)UTR
-5223
NC
Grs1346
0861
75679262
AG
(TC)
Exon
244
6Cn
sF71L
AG
GA
GG
AG
GG
AG
AG
GG
Grs1346
0859
75679202
GA
(CT)lowast
Exon
2506
CsH91=
GA
AG
AA
GG
AA
GA
GA
GG
Grs27978436
75679187
GA
(CT)lowast
Exon
2521
CsT9
6=G
GG
GG
GG
AG
GG
GG
GA
GG
rs226131070
75679178
GA
(CT)
Exon
2530
CsS99=
GG
GG
GG
GG
GG
GG
GG
GA
Grs227683834
75678576
AT
(TA
)Ex
on3
547
CnsV
105D
AA
AA
AA
AA
AA
AA
AA
AT
Ars257321187
75678506
TC(A
G)
Exon
3617
CsP128=
TT
TT
TT
TC
TT
TT
TT
TT
Trs27978444
75678500
TA(A
T)
Exon
3623
CsV130=
TT
TT
TT
TA
TT
TT
TT
AT
Trs227743136
75677686
GA
(CT)
Exon
4662
CsH143=
GG
GG
GG
GG
GG
GG
GG
GA
Grs215327202
75677664
CG(G
C)
Exon
4684
CnsA
151P
CC
CC
CC
CG
CC
CC
CC
CC
Crs247539755
75677663
GA
(CT)
Exon
4685
CnsA
151V
GG
GG
GG
GA
GG
GG
GG
GG
Grs233164
668
75677640
TC(A
G)
Exon
4708
CnsN
159D
TT
TT
TT
TT
TT
TT
TT
TC
T
rs2340
95816
75677162ndash75677160
GCT
del
(AGCdel)
Exon
5826minus
828
Cds-in
delQ
198
GCT
Oxidative Medicine and Cellular Longevity 19
Table5Con
tinued
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs27978452
75677145
CT(G
A)
Exon
5843
CnsA
204T
CC
CC
CC
CC
CC
CC
CC
CC
T
rs225047937
75677086ndash75677078
GAG
ATCG
ATdel
(ATG
CATC
TCdel)
Exon
5902minus
910
Cds-in
del
(S224I225S226)
GAG
ATC
GAT
rs241469868
75676998
TA(G
T)
Exon
5990
CnT2
53S
TT
TT
TT
TT
TT
TT
TT
TA
Trs221602571
75676923
AC
(TG
)Ex
on5
1065
CnsS
278A
AA
AA
AA
AA
AA
AA
AA
AC
Ars252576472
75676876
GA
(CT)
Exon
51112
CsS293=
GG
GG
GG
GG
GG
GG
GG
GA
Grs250802933
75676841
TC(A
G)
Exon
5114
7Cn
sD305G
TT
TT
TT
TT
TT
TT
TT
TC
Trs225425698
75676792
CT(G
A)
Exon
5119
6Cs
P321=
CC
CC
CC
CC
CC
CC
CC
CT
Crs13459064
75676732
CT(G
A)
Exon
51256
CsT3
41=
CT
TC
TT
CT
TT
CT
CT
TC
Crs27978453
75676717
CT(G
A)
Exon
51271
CsA346=
CC
CC
CC
CC
CC
CC
CC
TC
Crs214335034
75676636
AG
(TC)
Exon
51352
CsD373=
AA
AA
AA
AA
AA
AA
AA
AG
Ars24958364
4756766
03AC
(TG
)Ex
on5
1385
CsP3
84=
AA
AA
AA
AA
AA
AA
AA
AC
Ars27978454
75676592
GA
(CT)
Exon
51396
CnsP
388L
GG
GG
GG
GG
GG
GG
GG
AG
Grs215384328
75676589
GC
(CG
)Ex
on5
1399
CnsA
389G
GG
GG
GG
GG
GG
GG
GG
GC
Grs251728286
75676571
GA
(CT)
Exon
51417
CnsT
395I
Grs247602334
75676567
TC(A
G)
Exon
51421
CsV396=
TT
TT
TT
TT
TT
TT
TT
TC
Trs234216231
75676530
TC(A
G)
Exon
51458
CnsM
409V
TT
TT
TT
TT
TT
TT
TT
TC
Trs212904337
75676526
CT(G
A)
Exon
51462
CnsR
410H
CC
CC
CC
CC
CC
CC
CC
CT
Crs252650779
75676522
TC(A
G)
Exon
51466
CsE4
11=
TT
TT
TT
TT
TT
TT
TT
TC
Trs231273560
75676516
TC(A
G)
Exon
51472
CsQ413=
TT
TT
TT
TT
TT
TT
TT
TC
Trs257886949
756764
13GT
(CA
)Ex
on5
1575
CsR4
48=
GG
GG
GG
GG
GG
GG
GG
GT
Grs241537608
756764
04GC
(CG
)Ex
on5
1584
CnsL
451V
GG
GG
GG
GG
GG
GG
GG
GC
Grs227619071
75676312
TC(A
G)
Exon
51676
CsQ481=
TT
TT
TT
TT
TT
TT
TT
TC
Trs258913831
75676290
AG
(TC)
Exon
51698
CsL4
89=
AA
AA
AA
AA
AA
AA
AA
AG
Ars225593319
75676126
AT
(TA
)Ex
on5
1862
CnsH
543Q
Ars4223233
75676105
GA
(CT)
Exon
51883
CsS550=
GG
GG
GG
GA
GG
GG
GG
GG
Grs4223232
75676032
GT
(CA
)Ex
on5
1956
CnsL
575M
Grs4223231
75675816
CG
UTR
-32172
NC
GG
GG
GG
GG
GG
GG
GG
GC
Grs1346
860
75675682
AT
UTR
-32306
NC
ASequ
ence
varia
tions
inmou
seNr
f2wereob
tained
from
Mou
sePh
enom
eDatabase(http
ph
enom
ejaxorgSNP)
andNCB
ISNPdatabase
(http
wwwncbinlm
nihgovSNP)N
CBIreference
sequ
ence
isMus
Musculusstra
inC5
7BL6J
chromosom
e2GRC
m38p1(NC
0000
687
GI37209910875704641ndash756755192
9123
bp)To
tal968
genetic
mutations
arerepo
rted
inNr
f2gene
and5k
bup
stream
(ge75704
642minus
1)and2k
bdo
wnstre
am(le75675512)sequences
asof
Janu
ary2003A
llSN
Psandallelesa
representedas
geno
miccontig
(reversedsequ
ence
indicated)E
xon175704
641ndash75704364(278
bpT
TS=75704
408)
exon
275679431ndash756791
65(267
bp)exon
375678579ndash75678490(90b
p)exon475677713ndash756775
46(168
bp)andexon
57567718
4ndash75678849
(1666
bp)Proteinam
inoacid
(aa)
resid
uesinNP00
61552
(597
aa)Cs
cod
ing-syno
nymou
sCn
scoding
-non
syno
nymou
sAminoacid
A-alanineD-aspartic
acidE
-glutamicacidF-phenylalanineG
-glycineH
-histidineI-iso
leucineL-leu
cineand
M-m
ethion
ineN-
asparagineP-prolin
eQ-glutamineR-arginineS-serineT-threon
ineandV-valin
elowastErrorsin
databasesfi
xedNC
notcon
firmed
20 Oxidative Medicine and Cellular Longevity
Table 6 NRF2 somatic mutations revealed in various human cancers
Domain Amino acid residues DNA mutation Cancer types (cases) ReferencesLocus Wild type Mutant
DLG motif
24 W (Trp) C (Cys) c72GgtCGgtT NSCLC neck ESC [54 62]K (Lys) c72TgtC ESC [62]
26 Q (Gln) E (Glu) c76CgtG NSCLC ESC [54 62]
27 D (Asp) G (Gly)lowast c80GgtA NSCLC [60]Y (Tyr) c79GgtT ESC [61]
28 I (Ile) T (Thr) c83CgtT NSCLC [54]
29 D (Asp) G (Gly) c86AgtG Head and neck ESC [54 62]H (His) c85GgtC NSCLC laynx [60 61]
30 L (Leu) F (Phe) c88CgtT NSCLC ESC [54 62]
31 G (Gly) A (Ala) c92GgtC NSCLC ESC skin [54 60ndash62]
32 V (Val) T (Thr) c95TgtG NSCLC [54]del c93 95delAGT ESC [61]
33ndash36 S-R-E-V S-R-E-V-S-R-E-Vlowast c97 108dupAGTCGAGCCGTA ESC [61]
34 R (Arg) Q (Gln) c101GgtA NCSCL [54 60 61]P (Pro) c101GgtC NSCLC [63]
ETGF motif
75 Q (Gln) H (His) c225AgtC Head and neck ESC [54 62]
77 D (Asp)
V (Val) c230AgtT NSCLC ESC [54 60 62]G (Gly) c230AgtG ESC [62]A (Ala) c230AgtC NSCLC [61]N (Asn) c229GgtA Larynx [61]
78 E (Glu) K (Lys) c232GgtA NSCLC ESC [54 62]
79 E (Glu)
K (Lys) c235GgtA NSCLC ESC [54 61 62]Q (Gln) c235GgtC NSCLC ESC [54 60ndash62]G (Gly) c236AgtG Larynx [61]E-E c234 236dupAGA ESC [61]
80 T (Thr) K (Lys) c239CgtACgtG NSCLC ESC [54 61 62]
80 T (Thr)P (Phe) c238AgtC ESC [62]I (Ile) c239CgtT Head and neck [54]A (Ala) c238AgtG NSCLC [63]
81 G (Gly) V (Val) c242GgtT ESC [61]D (Asp) c242GgtA NSCLC ESC [60ndash62]
82 E (Glu)
D (Asp) c246AgtT ESC oral cancer cell line [54 62]G (Gly) c245AgtG NSCLC [54]Q (Gln)lowast c244GgtC NSCLC ESC [60 61]V (Val)lowast c245AgtT ESC [61]
83 F (Phe) L (Leu)lowast c247TgtC NSCLC [60]NSCLC non-small cell lung cancer ESC esophageal squamous cancer and lowasterrors in reference fixed Number of cases 82 NSCLC and 10 ESC in [62] 125NSCLC 70 ESC 23 larynx and 17 skin in [61] 103 NSCLC and 12 head and neck in [54] 90 NSCLC in [63] 103 NSCLC in [60]
be involved in squamous lung cancer cell proliferation [64]suggesting that the mutation is functional and overcomesKEAP1 inhibition Singh et al [65] determined in vitrothat RNAi-mediated depletion of NRF2 in lung cancer cellsenhanced ROS production and susceptibility to cell deathby ionizing radiation These studies support the concept thatelevated NRF2 and ARE responsiveness provides cancer cellswith proliferative advantage for malignant transformation
and undue protection from anti-cancer therapy Onco-genic epidermal growth factor receptor (EGFR) signaling isrecently found to be critical in NRF2-mediated proliferationof NSCLC cells [66]
Collectively ldquogain of functionrdquo mutations in NRF2 thatreduce KEAP1 recognition are suggested to be predictivemarkers for poor responsiveness to chemotherapy and radi-ation therapy Although NRF2-mediated cellular defense
Oxidative Medicine and Cellular Longevity 21
processes are essential in the initiation stage enhancedNRF2-ARE activity in advanced stages of cancer developmentmay create a favorable intracellular environment for tumorcell growth and survival [67 68] In this context NRF2may be a potential molecular target for the treatment ofradio-resistance cancers especially those that have ldquoloss offunctionrdquo mutations in EGFR KRAS or KEAP1 as well asldquogain of functionrdquo mutations in NRF2
5 Epigenetic Alterations of NF-E2-RelatedFactor 2
Epigenetic modifications are alterations of molecules inter-acting with genes without changes to the primary DNAsequence They include post-translational modification ofhistones DNA methylation events chromatin conforma-tional changes and alterations to noncoding regulatoryRNAs Epigenetic alterations are stable and often inheritablebut are reversible and may affect expression of the geneDysregulation or defects in epigenetic processes particularlyhypermethylation of tumor suppressor gene promoters (egCpG islands) or histone modifications are thought to beassociated with carcinogenesis Investigators have reportedhypermethylation in CpG islands of KEAP1 which wereassociated with reduced KEAP1 expression in human cancersfrom lung prostate colon and so forth [69ndash72] Similar tosomatic mutations epigenetic changes on KEAP1 impairedthe function of its encoded protein leading to constitutiveNRF2 activation
Supporting the role of ldquopathogenic mutationsrdquo in NRF2expression of Nrf2 and downstream Nqo1 was suppressedin prostate tumors of mice (transgenic adenocarcinomaof mouse prostate TRAMP) Among 15 promoter CpGislands located between minus942 and minus654 (cminus1175 cminus1132and cminus1059 cminus887 gap in cminus1131 cminus1060 see Figure 3)hypermethylation of the first 5 CpG islands (minus942 minus899 andcminus1175 cminus1132) was significantly associated with tumorige-nesis [73] Moreover treatment with inhibitors for DNAmethyltransferase and histone deacetylase restored Nrf2expression in these tumor cells [73] A dietary phytochemicalcurcumin known as a DNA hypomethylation agent restoredepigenetically silent Nrf2 expression through CpG demethy-lation in carcinogen-induced mouse tumor cells [74]
The whole genome epigenetic datasets for 5 species arepublicly accessible at NCBI Epigenomics [75 76]The humanNRF2 epigenome of primary cells (breast penis) and H1stem cell line as well as mouse Nrf2 CpG island methylationdata for sperm blood and cerebellum are currently avail-able (httpwwwncbinlmnihgovepigenomics) Althoughno direct evidence of disease-associated epigenetic modu-lation has been identified in human NRF2 various phyto-chemical NRF2 agonists such as sulforaphane and curcuminhave shown their roles in DNA methylation and histonemodification (see reviews by Lee and colleagues eg [77])Taken together epigenetic modifications of theNRF2KEAP1axle are predicted to cause dysregulation of ARE-mediatedcellular defense leading to deleterious health effects and
phytochemical antioxidants as epigenetic modulators forNRF2 are suggested to be useful in cancer prevention
6 Conclusions
NRF2 is evolutionally conserved with high-sequence homol-ogy in many species However it is a highly mutable geneand numerous genetic variants have been discovered inhuman ethnic groups Importantly certain SNPs or haplo-types have been identified in various diseases as ldquoat-riskrdquoalleles and are related to functional alterations In additionto genetic variations multiple somatic mutations identifiedin the KEAP1 recognition domain of NRF2 in cancer cellshave been found to be oncogenic due to dysregulation ofNRF2 homeostasis by its excess ldquogain of functionrdquo Epigeneticalteration of theNRF2 is under investigation and is predictedto have pathogenic influences as learned from mouse andphytochemical agonist studies Continuous updates of Nrf2allelic variants in inbred mouse strains will provide a usefultool for effective experimental designs formodels of oxidativedisorders to provide insight into the disease mechanisms andintervention strategies
Abbreviations
AD Alzheimerrsquos diseaseALI acute lung injuryARE antioxidant response elementbZIP basic leucine zippercds coding DNA sequencesCI confidence intervalCOPD chronic obstructive pulmonary diseaseESC esophageal squamous cancerFEV1 forced expiratory volume in one second
Gbp giga base pairsGST glutathione S-transferaseGWAS genome-wide association studyHGVS Human Genome Variation SocietyHO-1 heme oxygenase-1119867 119901119910119897119900119903119894 119867119890119897119894119888119900119887119886119888119905119890119903 119901119910119897119900119903119894KEAP1 Kelch-like ECH activating protein 1MPD Mouse Phenome DatabaseMRP multidrug resistance proteinNCBI National Center for Biotechnology InformationNeh NRF2-ECH homologyNfe2l2 nuclear factor (erythroid-derived 2)-like 2Nrf2 NF-E2-related factor 2NSCLC nonsmall cell lung cancerNQO1 NAD(P)Hquinone oxidoreductase 1OR odds ratioPD Parkinsonrsquos diseasePM particulate matterROS reactive oxygen speciesSLE systemic lupus erythematosusSNP single nucleotide polymorphismSOD superoxide dismutaseURT untranslated region
22 Oxidative Medicine and Cellular Longevity
Disclosure
Authorrsquos contribution to the Work was done as part of theAuthorrsquos official duties as a NIH employee and is a Work ofthe United States Government Therefore copyright may notbe established in the United States
Conflict of Interests
The author declares that there is no conflict of interests
Acknowledgments
The research related to this paper was supported by theIntramural Research Program of the National Institutes ofHealth of the National Institute of Environmental HealthSciences (NIEHS) Drs Steven Kleeberger and StephanieLondon at the NIEHS provided excellent critical review ofthis paper The author thanks Mrs Jacqui Marzec for herhelpful comments and English editing
References
[1] J Y Chan M C Cheung P Moi K Chan and Y W KanldquoChromosomal localization of the humanNF-E2 family of bZIPtranscription factors by fluorescence in situ hybridizationrdquoHuman Genetics vol 95 no 3 pp 265ndash269 1995
[2] K Itoh K Igarashi N Hayashi M Nishizawa and MYamamoto ldquoCloning and characterization of a novel erythroidcell-derived CNC family transcription factor heterodimerizingwith the small Maf family proteinsrdquo Molecular and CellularBiology vol 15 no 8 pp 4184ndash4193 1995
[3] W O Osburn and T W Kensler ldquoNRF2 signaling an adaptiveresponse pathway for protection against environmental toxicinsultsrdquoMutation Research vol 659 no 1-2 pp 31ndash39 2008
[4] D Papp K Lenti D Modos D Fazekas Z Dul D Tureiet al ldquoThe NRF2-related interactome and regulome containmultifunctional proteins and fine-tuned autoregulatory loopsrdquoFEBS Letters vol 586 no 13 pp 1795ndash1802 2012
[5] X Wang D J Tomso B N Chorley et al ldquoIdentificationof polymorphic antioxidant response elements in the humangenomerdquo Human Molecular Genetics vol 16 no 10 pp 1188ndash1200 2007
[6] D Malhotra E Portales-Casamar A Singh et al ldquoGlobalmapping of binding sites for NRF2 identifies novel targets incell survival response through ChIP-Seq profiling and networkanalysisrdquo Nucleic Acids Research vol 38 no 17 pp 5718ndash57342010
[7] K Chan R Lu J C Chang and Y W Kan ldquoNRF2 a memberof the NFE2 family of transcription factors is not essential formurine erythropoiesis growth and developmentrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 93 no 24 pp 13943ndash13948 1996
[8] K Itoh T Chiba S Takahashi et al ldquoAn NRF2small Mafheterodimer mediates the induction of phase II detoxifyingenzyme genes through antioxidant response elementsrdquo Bio-chemical and Biophysical Research Communications vol 236no 2 pp 313ndash322 1997
[9] FMartin J M vanDeursen R A Shivdasani CW Jackson AG Troutman and P A Ney ldquoErythroid maturation and globin
gene expression inmicewith combined deficiency ofNF-E2 andNrf-2rdquo Blood vol 91 no 9 pp 3459ndash3466 1998
[10] K Itoh N Wakabayashi Y Katoh et al ldquoKEAP1 repressesnuclear activation of antioxidant responsive elements by NRF2through binding to the amino-terminal Neh2 domainrdquo Genesand Development vol 13 no 1 pp 76ndash86 1999
[11] A Kobayashi M I Kang H Okawa et al ldquoOxidative stresssensor KEAP1 functions as an adaptor for Cul3-based E3 ligaseto regulate proteasomal degradation of NRF2rdquo Molecular andCellular Biology vol 24 no 16 pp 7130ndash7139 2004
[12] K Taguchi H Motohashi and M Yamamoto ldquoMolecularmechanisms of the KEAP1-NRF2 pathway in stress responseand cancer evolutionrdquo Genes to Cells vol 16 no 2 pp 123ndash1402011
[13] K Mukaigasa L T Nguyen L Li H Nakajima M Yamamotoand M Kobayashi ldquoGenetic evidence of an evolutionarilyconserved role for NRF2 in the protection against oxidativestressrdquoMolecular and Cellular Biology vol 32 no 21 pp 4455ndash4461 2012
[14] JMaher andMYamamoto ldquoThe rise of antioxidant signalingmdashthe evolution and hormetic actions of NRF2rdquo Toxicology andApplied Pharmacology vol 244 no 1 pp 4ndash15 2010
[15] S C Lo X Li M T Henzl L J Beamer and M HanninkldquoStructure of the KEAP1NRF2 interface provides mechanisticinsight intoNRF2 signalingrdquoThe EMBO Journal vol 25 no 15pp 3605ndash3617 2006
[16] V A McKusick ldquoMendelian inheritance in man and its onlineversion OMIMrdquo American Journal of Human Genetics vol 80no 4 pp 588ndash604 2007
[17] B Yalcin D J Adams J Flint and T M Keane ldquoNext-generation sequencing of experimental mouse strainsrdquo Mam-malian Genome vol 23 no 9-10 pp 490ndash498 2012
[18] D R Bentley S Balasubramanian H P Swerdlow et alldquoAccurate whole human genome sequencing using reversibleterminator chemistryrdquo Nature vol 456 no 7218 pp 53ndash592008
[19] P S G Chain D V Grafham R S Fulton et al ldquoGenomeproject standards in a new era of sequencingrdquo Science vol 326no 5950 pp 236ndash237 2009
[20] RHWaterstonK Lindblad-Toh E Birney J Rogers J F AbrilP Agarwal et al ldquoInitial sequencing and comparative analysis ofthe mouse genomerdquo Nature vol 420 pp 520ndash562 2002
[21] T M Keane L Goodstadt P Danecek M A White K WongB Yalcin et al ldquoMouse genomic variation and its effect onphenotypes and gene regulationrdquoNature vol 477 pp 289ndash2942011
[22] T Yamamoto K Yoh A Kobayashi et al ldquoIdentification ofpolymorphisms in the promoter region of the human NRF2generdquo Biochemical and Biophysical Research Communicationsvol 321 no 1 pp 72ndash79 2004
[23] J M Marzec J D Christie S P Reddy et al ldquoFunctionalpolymorphisms in the transcription factor NRF2 in humansincrease the risk of acute lung injuryrdquo The FASEB Journal vol21 no 9 pp 2237ndash2246 2007
[24] H Fukushima-Uesaka Y Saito K Maekawa et al ldquoGeneticvariations and haplotype structures of transcriptional factorNRF2 and its cytosolic reservoir protein KEAP1 in JapaneserdquoDrug Metabolism and Pharmacokinetics vol 22 no 3 pp 212ndash219 2007
[25] E J Cordova R Velazquez-Cruz F Centeno V Baca and LOrozco ldquoThe NRF2 gene variant -653GA is associated with
Oxidative Medicine and Cellular Longevity 23
nephritis in childhood-onset systemic lupus erythematosusrdquoLupus vol 19 no 10 pp 1237ndash1242 2010
[26] M von Otter S Landgren S Nilsson et al ldquoAssociation ofNRF2-encoding NFE2L2 haplotypes with Parkinsonrsquos diseaserdquoBMCMedical Genetics vol 11 no 1 article 36 2010
[27] JMHartikainenM TengstromVMKosmaV L Kinnula AMannermaa andY Soini ldquoGenetic polymorphisms and proteinexpression of NRF2 and sulfiredoxin predict survival outcomesin breast cancerrdquo Cancer Research vol 72 pp 5537ndash5546 2012
[28] T Arisawa T Tahara T Shibata et al ldquoThe relationshipbetween Helicobacter pylori infection and promoter polymor-phism of the NRF2 gene in chronic gastritisrdquo InternationalJournal of Molecular Medicine vol 19 no 1 pp 143ndash148 2007
[29] T Arisawa T Tahara T Shibata et al ldquoThe influence ofpromoter polymorphism of nuclear factor-erythroid 2-relatedfactor 2 gene on the aberrant DNA methylation in gastricepitheliumrdquo Oncology Reports vol 19 no 1 pp 211ndash216 2008
[30] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene pro-moter polymorphism and gastric carcinogenesisrdquo Hepato-Gastroenterology vol 55 no 82-83 pp 750ndash754 2008
[31] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene promoterpolymorphism is associated with ulcerative colitis in a Japanesepopulationrdquo Hepato-Gastroenterology vol 55 no 82-83 pp394ndash397 2008
[32] C C Hua L C Chang J C Tseng C M Chu Y C Liu andW B Shieh ldquoFunctional haplotypes in the promoter region oftranscription factor NRF2 in chronic obstructive pulmonarydiseaserdquo Disease Markers vol 28 no 3 pp 185ndash193 2010
[33] S O Shaheen R B Newson S M Ring M J Rose-ZerilliJ W Holloway and A J Henderson ldquoPrenatal and infantacetaminophen exposure antioxidant gene polymorphismsand childhood asthmardquo The Journal of Allergy and ClinicalImmunology vol 126 no 6 pp 1141e7ndash1148e7 2010
[34] H Masuko T Sakamoto Y Kaneko et al ldquoLower FEV1 innon-COPD nonasthmatic subjects association with smokingannual decline in FEV1 total IgE levels and TSLP genotypesrdquoInternational Journal of Chronic Obstructive Pulmonary Diseasevol 6 pp 181ndash189 2011
[35] C P Guan M N Zhou A E Xu et al ldquoThe susceptibilityto vitiligo is associated with NF-E2-related factor 2 (NRF2)gene polymorphisms a study on Chinese Han populationrdquoExperimental Dermatology vol 17 no 12 pp 1059ndash1062 2008
[36] J Bouligand O Cabaret M Canonico et al ldquoEffect of NFE2L2genetic polymorphism on the association between oral estrogentherapy and the risk of venous thromboembolism in post-menopausal womenrdquo Clinical Pharmacology amp Therapeuticsvol 89 no 1 pp 60ndash64 2011
[37] D S OrsquoMahony B J Glavan T D Holden C Fong R A BlackG Rona et al ldquoInflammation and immune-related candidategene associations with acute lung injury susceptibility andseverity a validation studyrdquo PLoS ONE vol 7 no 12 Article IDe51104 2012
[38] I Ungvari E Hadadi V Virag et al ldquoRelationship between airpollutionNFE2L2 gene polymorphisms and childhood asthmain aHungarian populationrdquo Journal of CommunityGenetics vol3 no 1 pp 25ndash33 2012
[39] M SiedlinskiD S Postma JMA Boer et al ldquoLevel and courseof FEV1 in relation to polymorphisms inNFE2L2 andKEAP1 inthe general populationrdquo Respiratory Research vol 10 article 732009
[40] C C Hong C B Ambrosone J Ahn et al ldquoGenetic variabilityin iron-related oxidative stress pathways (NRF2 NQ01 NOS3
and HO-1) iron intake and risk of postmenopausal breastcancerrdquo Cancer Epidemiology Biomarkers and Prevention vol16 no 9 pp 1784ndash1794 2007
[41] C Canova C Dunster F J Kelly C Minelli P L Shah CCaneja et al ldquoPM10-induced hospital admissions for asthmaand chronic obstructive pulmonary disease the modifyingeffect of individual characteristicsrdquo Epidemiology vol 23 no 4pp 607ndash615 2012
[42] A J Henderson R B Newson M Rose-Zerilli S M RingJ W Holloway and S O Shaheen ldquoMaternal NRF2 andgluthathione-S-transferase polymorphisms donotmodify asso-ciations of prenatal tobacco smoke exposure with asthma andlung function in school-aged childrenrdquo Thorax vol 65 no 10pp 897ndash902 2010
[43] C Xing A L Sestak J A Kelly et al ldquoLocalization andreplication of the systemic lupus erythematosus linkage signalat 4p16 interaction with 2p11 12q24 and 19q13 in EuropeanAmericansrdquo Human Genetics vol 120 no 5 pp 623ndash631 2007
[44] T F Wong K Yoshinaga Y Monma K Ito H NiikuraS Nagase et al ldquoAssociation of KEAP1 and NRF2 geneticmutations and polymorphisms with endometrioid endometrialadenocarcinoma survivalrdquo International Journal of Gynecologi-cal Cancer vol 21 no 8 pp 1428ndash1435 2011
[45] M von Otter S Landgren S Nilsson et al ldquoNRF2-encodingNFE2L2 haplotypes influence disease progression but not riskin Alzheimerrsquos disease and age-related cataractrdquoMechanisms ofAgeing and Development vol 131 no 2 pp 105ndash110 2010
[46] S Tsang Z Sun B Luke et al ldquoA comprehensive SNP-basedgenetic analysis of inbred mouse strainsrdquoMammalian Genomevol 16 no 7 pp 476ndash480 2005
[47] C M Wade E J Kulbokas III A W Kirby et al ldquoThe mosaicstructure of variation in the laboratory mouse genomerdquoNaturevol 420 no 6915 pp 574ndash578 2002
[48] T Wiltshire M T Pletcher S Batalov et al ldquoGenome-widesingle-nucleotide polymorphism analysis defines haplotypepatterns in mouserdquo Proceedings of the National Academy ofSciences of the United States of America vol 100 no 6 pp 3380ndash3385 2003
[49] K A Frazer E Eskin H M Kang et al ldquoA sequence-basedvariation map of 827 million SNPs in inbred mouse strainsrdquoNature vol 448 no 7157 pp 1050ndash1053 2007
[50] T P Maddatu S C Grubb C J Bult andM A Bogue ldquoMousephenome database (MPD)rdquo Nucleic Acids Research vol 40 ppD887ndashD894 2007
[51] A Kirby H M Kang C M Wade et al ldquoFine mapping in 94inbred mouse strains using a high-density haplotype resourcerdquoGenetics vol 185 no 3 pp 1081ndash1095 2010
[52] H Y Cho A E Jedlicka S P M Reddy L Y Zhang T WKensler and S R Kleeberger ldquoLinkage analysis of susceptibilityto hyperoxia NRF2 is a candidate generdquo American Journal ofRespiratory Cell and Molecular Biology vol 26 no 1 pp 42ndash512002
[53] H Y Cho A E Jedlicka S P M Reddy et al ldquoRole of NRF2in protection against hyperoxic lung injury in micerdquo AmericanJournal of Respiratory Cell and Molecular Biology vol 26 no 2pp 175ndash182 2002
[54] T Shibata T Ohta K I Tong et al ldquoCancer related mutationsin NRF2 impair its recognition by KEAP1-Cul3 E3 ligase andpromote malignancyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 36 pp13568ndash13573 2008
24 Oxidative Medicine and Cellular Longevity
[55] A Singh V Misra R K Thimmulappa et al ldquoDysfunctionalKEAP1-NRF2 interaction in non-small-cell lung cancerrdquo PLoSMedicine vol 3 no 10 article e420 2006
[56] T Ohta K Iijima M Miyamoto et al ldquoLoss of KEAP1 functionactivates NRF2 and provides advantages for lung cancer cellgrowthrdquo Cancer Research vol 68 no 5 pp 1303ndash1309 2008
[57] B Padmanabhan K I Tong T Ohta et al ldquoStructural basisfor defects of KEAP1 activity provoked by its point mutationsin lung cancerrdquoMolecular Cell vol 21 no 5 pp 689ndash700 2006
[58] T Shibata A Kokubu M Gotoh et al ldquoGenetic alteration ofKEAP1 confers constitutive NRF2 activation and resistance tochemotherapy in gallbladder cancerrdquoGastroenterology vol 135no 4 pp 1358e4ndash1368e4 2008
[59] N J Yoo H R Kim Y R Kim C H An and S H LeeldquoSomatic mutations of the KEAP1 gene in common solidcancersrdquo Histopathology vol 60 no 6 pp 943ndash952 2012
[60] Y Hu Y Ju D Lin Z Wang Y Huang S Zhang et alldquoMutation of the NRF2 gene in non-small cell lung cancerrdquoMolecular Biology Reports vol 39 no 4 pp 4743ndash4747 2012
[61] Y R Kim J EOhM S Kim et al ldquoOncogenicNRF2mutationsin squamous cell carcinomas of oesophagus and skinrdquo TheJournal of Pathology vol 220 no 4 pp 446ndash451 2010
[62] T Shibata A Kokubu S Saito et al ldquoNRF2 mutation confersmalignant potential and resistance to chemoradiation therapyin advanced esophageal squamous cancerrdquo Neoplasia vol 13pp 864ndash873 2011
[63] H Sasaki M Shitara K Yokota Y Hikosaka S MoriyamaM Yano et al ldquoIncreased NRF2 gene (NFE2L2) copy numbercorrelates with mutations in lung squamous cell carcinomasrdquoMolecular Medicine Reports vol 6 no 2 pp 391ndash394 2012
[64] H Sasaki M Shitara K Yokota Y Hikosaka S Moriyama MYano et al ldquoRagD gene expression andNRF2mutations in lungsquamous cell carcinomasrdquo Oncology Letters vol 4 pp 1167ndash1170 2012
[65] A Singh M Bodas N Wakabayashi F Bunz and S BiswalldquoGain of NRF2 function in non-small-cell lung cancer cellsconfers radioresistancerdquo Antioxidants and Redox Signaling vol13 no 11 pp 1627ndash1637 2010
[66] T Yamadori Y Ishii SHommaYMorishimaKKurishimaKItoh et al ldquoMolecular mechanisms for the regulation of NRF2-mediated cell proliferation in non-small-cell lung cancersrdquoOncogene vol 31 pp 4768ndash4777 2012
[67] A K Bauer H Y Cho L Miller-Degraff C Walker K HelmsJ Fostel et al ldquoTargeted deletion of NRF2 reduces urethane-induced lung tumor development in micerdquo PLoS ONE vol 6no 10 Article ID e26590 2011
[68] H Y Cho and S R Kleeberger ldquoNRF2 protects against airwaydisordersrdquo Toxicology and Applied Pharmacology vol 244 no1 pp 43ndash56 2010
[69] R Wang J An F Ji H Jiao H Sun and D Zhou ldquoHyperme-thylation of the KEAP1 gene in human lung cancer cell linesand lung cancer tissuesrdquo Biochemical and Biophysical ResearchCommunications vol 373 no 1 pp 151ndash154 2008
[70] P Zhang A Singh S Yegnasubramanian et al ldquoLoss ofkelch-like ECH-associated protein 1 function in prostate cancercells causes chemoresistance and radioresistance and promotestumor growthrdquoMolecular Cancer Therapeutics vol 9 no 2 pp336ndash346 2010
[71] N Hanada T Takahata Q Zhou et al ldquoMethylation of theKEAP1 gene promoter region in human colorectal cancerrdquoBMCCancer vol 12 article 66 2012
[72] L A Muscarella R Barbano V DrsquoAngelo et al ldquoRegulationof KEAP1 expression by promoter methylation in malignantgliomas and association with patientrsquos outcomerdquo Epigeneticsvol 6 no 3 pp 317ndash325 2011
[73] S Yu T O Khor K L Cheung et al ldquoNRF2 expressionis regulated by epigenetic mechanisms in prostate cancer ofTRAMP micerdquo PLoS ONE vol 5 no 1 Article ID e8579 2010
[74] T O Khor Y Huang T Y Wu L Shu J Lee and A N KongldquoPharmacodynamics of curcumin as DNA hypomethylationagent in restoring the expression of NRF2 via promoter CpGsdemethylationrdquo Biochemical Pharmacology vol 82 no 9 pp1073ndash1078 2011
[75] I M Fingerman L McDaniel X Zhang et al ldquoNCBI epige-nomics a new public resource for exploring epigenomic datasetsrdquo Nucleic Acids Research vol 39 supplemen 1 pp D908ndashD912 2011
[76] I M Fingerman X Zhang W Ratzat N Husain R F Cohenand G D Schuler ldquoNCBI epigenomics whatrsquos new for 2013rdquoNucleic Acids Research vol 41 no 1 pp D221ndashD225 2013
[77] J H Lee T O Khor L Shu Z Y Su F Fuentes andA N Kong ldquoDietary phytochemicals and cancer preventionNRF2 signaling epigenetics and cell death mechanisms inblocking cancer initiation and progressionrdquo Pharmacology ampTherapeutics vol 137 no 2 pp 153ndash171 2012
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
16 Oxidative Medicine and Cellular Longevity
pL575M
pF71L
pH543QpL451V
pA151P pA151V pN159D pQ198delpV105D
pA204T pS224I225S226del pT253S pS278ApD305G
pM409V pR410HpP388L pA389G pT395I
Genomic DNA (reversed contig GRCm38 p1 C57BL6JNM 0109023)
Protein (NP 035032)1
51101151201251301351401451501551
minus400
minus500
minus50
minus100
minus200
minus300
minus450
minus350
minus250
minus150
minus950
minus900
minus850
minus800
minus750
minus700
151
101151201
2001
g minus470gg minusg minus
c minus41c minus
c
c
c c
gg minus 6C
g minus125insTTC g minus118insC
g minus281delC
g minus63delCg minus1 (75704642 c minus234)
Translation start site (c +1)Termination codon (c +2025 +2027)
c minus136 minus134in-frame stop codonc minus8585
g minus41
246
440
minus381
minus11
minus91 minus90
minus332
minus153
minus98
c minus197c minus209 minus202
gminus942 minus899 (cminus1175 minus1132) CpG islands [71]
g 460CgtT
g minus103CCgtT [51]
CgtTCgtG
g minus320CgtT
CgtT
CgtT
CgtTg minus426GgtA
GgtACgtA
CgtA
GgtA
g minus238GgtTTgtC
TgtC
TgtC
TgtCTgtG
g minus229AgtG
CgtG
CgtG
GgtC
gminus826 minus654 (cminus1059 ) CpG islands [71]minus887
Figure 3 DNA (partial promoter and exons 1 and 5) and protein sequence ofmouseNrf2 SNPs and amino acid residues for non-synonymousSNPs are marked Promoter regions bearing 5CpG islands are underlined
151
201251301351401451501551601
Genetic mutation (Table 3 and Figure 2)Somatic mutation (Table 6)
Neh2
Neh3
Neh4Neh5
Neh6
Neh1
Protein isoform 1 (NP 006155)
101151
Figure 4 Genetic and somatic mutation loci in human NRF2 protein
Oxidative Medicine and Cellular Longevity 17
Table5Mou
seup
stream
andexon
varia
tions
ofNfe2
l2locusin17
inbred
strainsR
eference
sequ
ence
isC5
7BL6J
(B6strain
1)geno
me(GI14933824975547698ndash75513576)SNPalleleand
geno
typesareshow
nas
chromosom
econ
tigsequ
enceStrains
2129S1S
vlmJ3AJ
4AKR
J5BA
LBcJ6C3
HH
eJ7C
57BL
6NJ8CA
STEiJ9CB
AJ
10D
BA2J11FVBNJ12LPJ
13N
ODShiLtJ14N
ZOH
ILtJ
15P
WK
PhJ16SPR
ETEiJ
17W
SBEiJ
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs2566
08517
75705565
GA
(CT)
51015840Flankingminus924
NC
GG
GG
GG
GG
GG
GG
GG
AA
Grs47274959
75705562
AT
(TA
)51015840Flankingminus921
NC
AT
TA
TT
AA
TT
AT
AT
TT
Ars216940398
75705554ndash75705555
Tadd(A
add)
51015840Flankingminus913minus914
NC
CGrs239114134
75705540
CT(G
A)
51015840Flankingminus899
NC
CT
TC
TT
CC
TT
CT
CT
CT
Crs46
461765
75705528
TG(A
C)oA
C
51015840Flankingminus887
NC
TG
GT
GG
TT
GG
TG
TG
GG
Trs5144
9853
75705525
CA(G
T)
51015840Flankingminus884
NC
CA
AC
AA
CC
AA
CA
CA
CC
Crs249093111
75705512
CT(G
A)
51015840Flankingminus871
NC
CT
TC
TT
CC
TT
CT
CT
TT
Crs263745200
75705510
CG(G
C)
51015840Flankingminus869
NC
GG
GG
GG
GG
GG
GG
GG
CG
Grs45651867
75705498
TG(A
C)
51015840Flankingminus857
NC
TG
GT
GG
TT
GG
TG
TG
GG
Trs219997531
75705495
GA
(CT)
51015840Flankingminus854
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs251918379
75705451
TA(A
T)
51015840Flankingminus810
NC
TT
TT
TT
TT
TT
TT
TT
TA
Trs27978306
75705449
TA(A
T)
51015840Flankingminus808
NC
TA
AT
AA
TT
AA
TA
TA
TT
Trs216087412
75705429
TG(A
C)lowast
51015840Flankingminus788
NC
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
GTlowast
Tlowast
rs261229914
75705423ndash75705424
AGdel(CT
del)
51015840Flankingminus783minus784
NC
AGrs27978307
75705400
CT(G
A)
51015840Flankingminus759
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs243167395
75705359
GA
(CT)
51015840Flankingminus718
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs27978308
75705307
GA
(CT)
51015840Flankingminus66
6NC
GG
GG
GG
GG
GG
GG
GG
AG
Grs254744
09875705294
AT
(TA
)51015840Flankingminus653
NC
AA
AA
AA
AA
AA
AA
AA
AT
Ars228133419
75705179
GT
(CA
)51015840Flankingminus538
NC
GG
GG
GG
GG
GG
GG
GG
GT
Grs214719520
75705111
GA
(CT)
51015840Flankingminus470
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs2440
87730
75705101
GA
(CT)
51015840Flankingminus46
0NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs227781699
75705081
CG(G
C)
51015840Flankingminus44
0NC
CC
CC
CC
CC
CC
CC
CC
GC
Crs257870353
75705067
CT(G
A)
51015840Flankingminus426
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs24487744
075705022
AG
(TC)
51015840Flankingminus381
NC
AA
AA
AA
AA
AA
AA
AA
AG
Ars234628138
75704973
AC
(TG
)51015840Flankingminus332
NC
AA
AA
AA
AA
AA
AA
AA
AC
A
18 Oxidative Medicine and Cellular Longevity
Table5Con
tinued
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs247275247
75704961
GA
(CT)
51015840Flankingminus320
NC
GG
GG
GG
GG
GG
GG
AG
GG
Grs247519047
75704922
Gdel(Cdel)
51015840Flankingminus281
NC
Grs27978309
(rs51915758)
75704887
GA
C(C
TG
)51015840Flankingminus246
NC
GA
AG
AA
GG
AA
GA
GA
CC
G
rs218139102
75704879
CA(G
T)
51015840Flankingminus238
NC
CC
CC
CC
CC
CC
CC
CC
AC
Crs251990355
75704870
TC(A
G)
51015840Flankingminus229
NC
TT
TT
TT
TT
TT
TT
TT
TC
Trs238746955
75704794
AG
(TC)
51015840Flankingminus153
NC
AA
AA
AA
AA
AA
AA
AA
GA
Ars221664
405
75704786
AG
(TC)
51015840Flankingminus145
NC
AA
AA
AA
AA
AA
AA
AA
AG
Ars217054035
75704766
ndash75704767
GAAadd(TTC
add)
51015840Flankingminus125minus126
NC
GA
rs248182931
75704759ndash75704760
Gadd(C
add)
51015840Flankingminus118
minus119
NC
AGrs217197904
75704744
GA
(CT)
51015840Flankingminus103
(+)S
p1[23]
GA
AG
AA
GG
AA
GA
AA
GG
Ars2644
9364975704704
Gdel(Cdel)
51015840Flankingminus63
NC
Grs2404
81112
757046
82CT(G
A)
51015840Flankingminus41
NC
CC
CC
CC
CC
CC
CC
CC
CT
Crs27978310
75704617
AG
(TC)
UTR
-525
NC
AA
AA
AA
AA
AA
AA
AA
GG
Ars27978311
75704610
CG(G
C)
UTR
-532
NC
GG
GG
GG
GG
GG
GG
GG
GG
Crs27978312
757046
05GA
(CT)
UTR
-537
NC
GA
AG
AA
GG
AA
GA
GA
GG
Grs214784220
757044
99AG
(TC)
UTR
-5143
NC
AA
AA
AA
AA
AA
AA
AA
GG
Ars244146318
757044
98GT
(CA
)UTR
-5144
NC
GG
GG
GG
GG
GG
GG
GG
GT
Grs27978313
757044
97GC
(CG
)UTR
-5145
NC
GC
CG
CC
GC
CC
GC
CC
CC
Crs215431944
757044
93CT(G
A)
UTR
-5149
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs257015697
757044
49GA
(CT)
UTR
-5193
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs252234782
757044
19GT
(CA
)UTR
-5223
NC
Grs1346
0861
75679262
AG
(TC)
Exon
244
6Cn
sF71L
AG
GA
GG
AG
GG
AG
AG
GG
Grs1346
0859
75679202
GA
(CT)lowast
Exon
2506
CsH91=
GA
AG
AA
GG
AA
GA
GA
GG
Grs27978436
75679187
GA
(CT)lowast
Exon
2521
CsT9
6=G
GG
GG
GG
AG
GG
GG
GA
GG
rs226131070
75679178
GA
(CT)
Exon
2530
CsS99=
GG
GG
GG
GG
GG
GG
GG
GA
Grs227683834
75678576
AT
(TA
)Ex
on3
547
CnsV
105D
AA
AA
AA
AA
AA
AA
AA
AT
Ars257321187
75678506
TC(A
G)
Exon
3617
CsP128=
TT
TT
TT
TC
TT
TT
TT
TT
Trs27978444
75678500
TA(A
T)
Exon
3623
CsV130=
TT
TT
TT
TA
TT
TT
TT
AT
Trs227743136
75677686
GA
(CT)
Exon
4662
CsH143=
GG
GG
GG
GG
GG
GG
GG
GA
Grs215327202
75677664
CG(G
C)
Exon
4684
CnsA
151P
CC
CC
CC
CG
CC
CC
CC
CC
Crs247539755
75677663
GA
(CT)
Exon
4685
CnsA
151V
GG
GG
GG
GA
GG
GG
GG
GG
Grs233164
668
75677640
TC(A
G)
Exon
4708
CnsN
159D
TT
TT
TT
TT
TT
TT
TT
TC
T
rs2340
95816
75677162ndash75677160
GCT
del
(AGCdel)
Exon
5826minus
828
Cds-in
delQ
198
GCT
Oxidative Medicine and Cellular Longevity 19
Table5Con
tinued
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs27978452
75677145
CT(G
A)
Exon
5843
CnsA
204T
CC
CC
CC
CC
CC
CC
CC
CC
T
rs225047937
75677086ndash75677078
GAG
ATCG
ATdel
(ATG
CATC
TCdel)
Exon
5902minus
910
Cds-in
del
(S224I225S226)
GAG
ATC
GAT
rs241469868
75676998
TA(G
T)
Exon
5990
CnT2
53S
TT
TT
TT
TT
TT
TT
TT
TA
Trs221602571
75676923
AC
(TG
)Ex
on5
1065
CnsS
278A
AA
AA
AA
AA
AA
AA
AA
AC
Ars252576472
75676876
GA
(CT)
Exon
51112
CsS293=
GG
GG
GG
GG
GG
GG
GG
GA
Grs250802933
75676841
TC(A
G)
Exon
5114
7Cn
sD305G
TT
TT
TT
TT
TT
TT
TT
TC
Trs225425698
75676792
CT(G
A)
Exon
5119
6Cs
P321=
CC
CC
CC
CC
CC
CC
CC
CT
Crs13459064
75676732
CT(G
A)
Exon
51256
CsT3
41=
CT
TC
TT
CT
TT
CT
CT
TC
Crs27978453
75676717
CT(G
A)
Exon
51271
CsA346=
CC
CC
CC
CC
CC
CC
CC
TC
Crs214335034
75676636
AG
(TC)
Exon
51352
CsD373=
AA
AA
AA
AA
AA
AA
AA
AG
Ars24958364
4756766
03AC
(TG
)Ex
on5
1385
CsP3
84=
AA
AA
AA
AA
AA
AA
AA
AC
Ars27978454
75676592
GA
(CT)
Exon
51396
CnsP
388L
GG
GG
GG
GG
GG
GG
GG
AG
Grs215384328
75676589
GC
(CG
)Ex
on5
1399
CnsA
389G
GG
GG
GG
GG
GG
GG
GG
GC
Grs251728286
75676571
GA
(CT)
Exon
51417
CnsT
395I
Grs247602334
75676567
TC(A
G)
Exon
51421
CsV396=
TT
TT
TT
TT
TT
TT
TT
TC
Trs234216231
75676530
TC(A
G)
Exon
51458
CnsM
409V
TT
TT
TT
TT
TT
TT
TT
TC
Trs212904337
75676526
CT(G
A)
Exon
51462
CnsR
410H
CC
CC
CC
CC
CC
CC
CC
CT
Crs252650779
75676522
TC(A
G)
Exon
51466
CsE4
11=
TT
TT
TT
TT
TT
TT
TT
TC
Trs231273560
75676516
TC(A
G)
Exon
51472
CsQ413=
TT
TT
TT
TT
TT
TT
TT
TC
Trs257886949
756764
13GT
(CA
)Ex
on5
1575
CsR4
48=
GG
GG
GG
GG
GG
GG
GG
GT
Grs241537608
756764
04GC
(CG
)Ex
on5
1584
CnsL
451V
GG
GG
GG
GG
GG
GG
GG
GC
Grs227619071
75676312
TC(A
G)
Exon
51676
CsQ481=
TT
TT
TT
TT
TT
TT
TT
TC
Trs258913831
75676290
AG
(TC)
Exon
51698
CsL4
89=
AA
AA
AA
AA
AA
AA
AA
AG
Ars225593319
75676126
AT
(TA
)Ex
on5
1862
CnsH
543Q
Ars4223233
75676105
GA
(CT)
Exon
51883
CsS550=
GG
GG
GG
GA
GG
GG
GG
GG
Grs4223232
75676032
GT
(CA
)Ex
on5
1956
CnsL
575M
Grs4223231
75675816
CG
UTR
-32172
NC
GG
GG
GG
GG
GG
GG
GG
GC
Grs1346
860
75675682
AT
UTR
-32306
NC
ASequ
ence
varia
tions
inmou
seNr
f2wereob
tained
from
Mou
sePh
enom
eDatabase(http
ph
enom
ejaxorgSNP)
andNCB
ISNPdatabase
(http
wwwncbinlm
nihgovSNP)N
CBIreference
sequ
ence
isMus
Musculusstra
inC5
7BL6J
chromosom
e2GRC
m38p1(NC
0000
687
GI37209910875704641ndash756755192
9123
bp)To
tal968
genetic
mutations
arerepo
rted
inNr
f2gene
and5k
bup
stream
(ge75704
642minus
1)and2k
bdo
wnstre
am(le75675512)sequences
asof
Janu
ary2003A
llSN
Psandallelesa
representedas
geno
miccontig
(reversedsequ
ence
indicated)E
xon175704
641ndash75704364(278
bpT
TS=75704
408)
exon
275679431ndash756791
65(267
bp)exon
375678579ndash75678490(90b
p)exon475677713ndash756775
46(168
bp)andexon
57567718
4ndash75678849
(1666
bp)Proteinam
inoacid
(aa)
resid
uesinNP00
61552
(597
aa)Cs
cod
ing-syno
nymou
sCn
scoding
-non
syno
nymou
sAminoacid
A-alanineD-aspartic
acidE
-glutamicacidF-phenylalanineG
-glycineH
-histidineI-iso
leucineL-leu
cineand
M-m
ethion
ineN-
asparagineP-prolin
eQ-glutamineR-arginineS-serineT-threon
ineandV-valin
elowastErrorsin
databasesfi
xedNC
notcon
firmed
20 Oxidative Medicine and Cellular Longevity
Table 6 NRF2 somatic mutations revealed in various human cancers
Domain Amino acid residues DNA mutation Cancer types (cases) ReferencesLocus Wild type Mutant
DLG motif
24 W (Trp) C (Cys) c72GgtCGgtT NSCLC neck ESC [54 62]K (Lys) c72TgtC ESC [62]
26 Q (Gln) E (Glu) c76CgtG NSCLC ESC [54 62]
27 D (Asp) G (Gly)lowast c80GgtA NSCLC [60]Y (Tyr) c79GgtT ESC [61]
28 I (Ile) T (Thr) c83CgtT NSCLC [54]
29 D (Asp) G (Gly) c86AgtG Head and neck ESC [54 62]H (His) c85GgtC NSCLC laynx [60 61]
30 L (Leu) F (Phe) c88CgtT NSCLC ESC [54 62]
31 G (Gly) A (Ala) c92GgtC NSCLC ESC skin [54 60ndash62]
32 V (Val) T (Thr) c95TgtG NSCLC [54]del c93 95delAGT ESC [61]
33ndash36 S-R-E-V S-R-E-V-S-R-E-Vlowast c97 108dupAGTCGAGCCGTA ESC [61]
34 R (Arg) Q (Gln) c101GgtA NCSCL [54 60 61]P (Pro) c101GgtC NSCLC [63]
ETGF motif
75 Q (Gln) H (His) c225AgtC Head and neck ESC [54 62]
77 D (Asp)
V (Val) c230AgtT NSCLC ESC [54 60 62]G (Gly) c230AgtG ESC [62]A (Ala) c230AgtC NSCLC [61]N (Asn) c229GgtA Larynx [61]
78 E (Glu) K (Lys) c232GgtA NSCLC ESC [54 62]
79 E (Glu)
K (Lys) c235GgtA NSCLC ESC [54 61 62]Q (Gln) c235GgtC NSCLC ESC [54 60ndash62]G (Gly) c236AgtG Larynx [61]E-E c234 236dupAGA ESC [61]
80 T (Thr) K (Lys) c239CgtACgtG NSCLC ESC [54 61 62]
80 T (Thr)P (Phe) c238AgtC ESC [62]I (Ile) c239CgtT Head and neck [54]A (Ala) c238AgtG NSCLC [63]
81 G (Gly) V (Val) c242GgtT ESC [61]D (Asp) c242GgtA NSCLC ESC [60ndash62]
82 E (Glu)
D (Asp) c246AgtT ESC oral cancer cell line [54 62]G (Gly) c245AgtG NSCLC [54]Q (Gln)lowast c244GgtC NSCLC ESC [60 61]V (Val)lowast c245AgtT ESC [61]
83 F (Phe) L (Leu)lowast c247TgtC NSCLC [60]NSCLC non-small cell lung cancer ESC esophageal squamous cancer and lowasterrors in reference fixed Number of cases 82 NSCLC and 10 ESC in [62] 125NSCLC 70 ESC 23 larynx and 17 skin in [61] 103 NSCLC and 12 head and neck in [54] 90 NSCLC in [63] 103 NSCLC in [60]
be involved in squamous lung cancer cell proliferation [64]suggesting that the mutation is functional and overcomesKEAP1 inhibition Singh et al [65] determined in vitrothat RNAi-mediated depletion of NRF2 in lung cancer cellsenhanced ROS production and susceptibility to cell deathby ionizing radiation These studies support the concept thatelevated NRF2 and ARE responsiveness provides cancer cellswith proliferative advantage for malignant transformation
and undue protection from anti-cancer therapy Onco-genic epidermal growth factor receptor (EGFR) signaling isrecently found to be critical in NRF2-mediated proliferationof NSCLC cells [66]
Collectively ldquogain of functionrdquo mutations in NRF2 thatreduce KEAP1 recognition are suggested to be predictivemarkers for poor responsiveness to chemotherapy and radi-ation therapy Although NRF2-mediated cellular defense
Oxidative Medicine and Cellular Longevity 21
processes are essential in the initiation stage enhancedNRF2-ARE activity in advanced stages of cancer developmentmay create a favorable intracellular environment for tumorcell growth and survival [67 68] In this context NRF2may be a potential molecular target for the treatment ofradio-resistance cancers especially those that have ldquoloss offunctionrdquo mutations in EGFR KRAS or KEAP1 as well asldquogain of functionrdquo mutations in NRF2
5 Epigenetic Alterations of NF-E2-RelatedFactor 2
Epigenetic modifications are alterations of molecules inter-acting with genes without changes to the primary DNAsequence They include post-translational modification ofhistones DNA methylation events chromatin conforma-tional changes and alterations to noncoding regulatoryRNAs Epigenetic alterations are stable and often inheritablebut are reversible and may affect expression of the geneDysregulation or defects in epigenetic processes particularlyhypermethylation of tumor suppressor gene promoters (egCpG islands) or histone modifications are thought to beassociated with carcinogenesis Investigators have reportedhypermethylation in CpG islands of KEAP1 which wereassociated with reduced KEAP1 expression in human cancersfrom lung prostate colon and so forth [69ndash72] Similar tosomatic mutations epigenetic changes on KEAP1 impairedthe function of its encoded protein leading to constitutiveNRF2 activation
Supporting the role of ldquopathogenic mutationsrdquo in NRF2expression of Nrf2 and downstream Nqo1 was suppressedin prostate tumors of mice (transgenic adenocarcinomaof mouse prostate TRAMP) Among 15 promoter CpGislands located between minus942 and minus654 (cminus1175 cminus1132and cminus1059 cminus887 gap in cminus1131 cminus1060 see Figure 3)hypermethylation of the first 5 CpG islands (minus942 minus899 andcminus1175 cminus1132) was significantly associated with tumorige-nesis [73] Moreover treatment with inhibitors for DNAmethyltransferase and histone deacetylase restored Nrf2expression in these tumor cells [73] A dietary phytochemicalcurcumin known as a DNA hypomethylation agent restoredepigenetically silent Nrf2 expression through CpG demethy-lation in carcinogen-induced mouse tumor cells [74]
The whole genome epigenetic datasets for 5 species arepublicly accessible at NCBI Epigenomics [75 76]The humanNRF2 epigenome of primary cells (breast penis) and H1stem cell line as well as mouse Nrf2 CpG island methylationdata for sperm blood and cerebellum are currently avail-able (httpwwwncbinlmnihgovepigenomics) Althoughno direct evidence of disease-associated epigenetic modu-lation has been identified in human NRF2 various phyto-chemical NRF2 agonists such as sulforaphane and curcuminhave shown their roles in DNA methylation and histonemodification (see reviews by Lee and colleagues eg [77])Taken together epigenetic modifications of theNRF2KEAP1axle are predicted to cause dysregulation of ARE-mediatedcellular defense leading to deleterious health effects and
phytochemical antioxidants as epigenetic modulators forNRF2 are suggested to be useful in cancer prevention
6 Conclusions
NRF2 is evolutionally conserved with high-sequence homol-ogy in many species However it is a highly mutable geneand numerous genetic variants have been discovered inhuman ethnic groups Importantly certain SNPs or haplo-types have been identified in various diseases as ldquoat-riskrdquoalleles and are related to functional alterations In additionto genetic variations multiple somatic mutations identifiedin the KEAP1 recognition domain of NRF2 in cancer cellshave been found to be oncogenic due to dysregulation ofNRF2 homeostasis by its excess ldquogain of functionrdquo Epigeneticalteration of theNRF2 is under investigation and is predictedto have pathogenic influences as learned from mouse andphytochemical agonist studies Continuous updates of Nrf2allelic variants in inbred mouse strains will provide a usefultool for effective experimental designs formodels of oxidativedisorders to provide insight into the disease mechanisms andintervention strategies
Abbreviations
AD Alzheimerrsquos diseaseALI acute lung injuryARE antioxidant response elementbZIP basic leucine zippercds coding DNA sequencesCI confidence intervalCOPD chronic obstructive pulmonary diseaseESC esophageal squamous cancerFEV1 forced expiratory volume in one second
Gbp giga base pairsGST glutathione S-transferaseGWAS genome-wide association studyHGVS Human Genome Variation SocietyHO-1 heme oxygenase-1119867 119901119910119897119900119903119894 119867119890119897119894119888119900119887119886119888119905119890119903 119901119910119897119900119903119894KEAP1 Kelch-like ECH activating protein 1MPD Mouse Phenome DatabaseMRP multidrug resistance proteinNCBI National Center for Biotechnology InformationNeh NRF2-ECH homologyNfe2l2 nuclear factor (erythroid-derived 2)-like 2Nrf2 NF-E2-related factor 2NSCLC nonsmall cell lung cancerNQO1 NAD(P)Hquinone oxidoreductase 1OR odds ratioPD Parkinsonrsquos diseasePM particulate matterROS reactive oxygen speciesSLE systemic lupus erythematosusSNP single nucleotide polymorphismSOD superoxide dismutaseURT untranslated region
22 Oxidative Medicine and Cellular Longevity
Disclosure
Authorrsquos contribution to the Work was done as part of theAuthorrsquos official duties as a NIH employee and is a Work ofthe United States Government Therefore copyright may notbe established in the United States
Conflict of Interests
The author declares that there is no conflict of interests
Acknowledgments
The research related to this paper was supported by theIntramural Research Program of the National Institutes ofHealth of the National Institute of Environmental HealthSciences (NIEHS) Drs Steven Kleeberger and StephanieLondon at the NIEHS provided excellent critical review ofthis paper The author thanks Mrs Jacqui Marzec for herhelpful comments and English editing
References
[1] J Y Chan M C Cheung P Moi K Chan and Y W KanldquoChromosomal localization of the humanNF-E2 family of bZIPtranscription factors by fluorescence in situ hybridizationrdquoHuman Genetics vol 95 no 3 pp 265ndash269 1995
[2] K Itoh K Igarashi N Hayashi M Nishizawa and MYamamoto ldquoCloning and characterization of a novel erythroidcell-derived CNC family transcription factor heterodimerizingwith the small Maf family proteinsrdquo Molecular and CellularBiology vol 15 no 8 pp 4184ndash4193 1995
[3] W O Osburn and T W Kensler ldquoNRF2 signaling an adaptiveresponse pathway for protection against environmental toxicinsultsrdquoMutation Research vol 659 no 1-2 pp 31ndash39 2008
[4] D Papp K Lenti D Modos D Fazekas Z Dul D Tureiet al ldquoThe NRF2-related interactome and regulome containmultifunctional proteins and fine-tuned autoregulatory loopsrdquoFEBS Letters vol 586 no 13 pp 1795ndash1802 2012
[5] X Wang D J Tomso B N Chorley et al ldquoIdentificationof polymorphic antioxidant response elements in the humangenomerdquo Human Molecular Genetics vol 16 no 10 pp 1188ndash1200 2007
[6] D Malhotra E Portales-Casamar A Singh et al ldquoGlobalmapping of binding sites for NRF2 identifies novel targets incell survival response through ChIP-Seq profiling and networkanalysisrdquo Nucleic Acids Research vol 38 no 17 pp 5718ndash57342010
[7] K Chan R Lu J C Chang and Y W Kan ldquoNRF2 a memberof the NFE2 family of transcription factors is not essential formurine erythropoiesis growth and developmentrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 93 no 24 pp 13943ndash13948 1996
[8] K Itoh T Chiba S Takahashi et al ldquoAn NRF2small Mafheterodimer mediates the induction of phase II detoxifyingenzyme genes through antioxidant response elementsrdquo Bio-chemical and Biophysical Research Communications vol 236no 2 pp 313ndash322 1997
[9] FMartin J M vanDeursen R A Shivdasani CW Jackson AG Troutman and P A Ney ldquoErythroid maturation and globin
gene expression inmicewith combined deficiency ofNF-E2 andNrf-2rdquo Blood vol 91 no 9 pp 3459ndash3466 1998
[10] K Itoh N Wakabayashi Y Katoh et al ldquoKEAP1 repressesnuclear activation of antioxidant responsive elements by NRF2through binding to the amino-terminal Neh2 domainrdquo Genesand Development vol 13 no 1 pp 76ndash86 1999
[11] A Kobayashi M I Kang H Okawa et al ldquoOxidative stresssensor KEAP1 functions as an adaptor for Cul3-based E3 ligaseto regulate proteasomal degradation of NRF2rdquo Molecular andCellular Biology vol 24 no 16 pp 7130ndash7139 2004
[12] K Taguchi H Motohashi and M Yamamoto ldquoMolecularmechanisms of the KEAP1-NRF2 pathway in stress responseand cancer evolutionrdquo Genes to Cells vol 16 no 2 pp 123ndash1402011
[13] K Mukaigasa L T Nguyen L Li H Nakajima M Yamamotoand M Kobayashi ldquoGenetic evidence of an evolutionarilyconserved role for NRF2 in the protection against oxidativestressrdquoMolecular and Cellular Biology vol 32 no 21 pp 4455ndash4461 2012
[14] JMaher andMYamamoto ldquoThe rise of antioxidant signalingmdashthe evolution and hormetic actions of NRF2rdquo Toxicology andApplied Pharmacology vol 244 no 1 pp 4ndash15 2010
[15] S C Lo X Li M T Henzl L J Beamer and M HanninkldquoStructure of the KEAP1NRF2 interface provides mechanisticinsight intoNRF2 signalingrdquoThe EMBO Journal vol 25 no 15pp 3605ndash3617 2006
[16] V A McKusick ldquoMendelian inheritance in man and its onlineversion OMIMrdquo American Journal of Human Genetics vol 80no 4 pp 588ndash604 2007
[17] B Yalcin D J Adams J Flint and T M Keane ldquoNext-generation sequencing of experimental mouse strainsrdquo Mam-malian Genome vol 23 no 9-10 pp 490ndash498 2012
[18] D R Bentley S Balasubramanian H P Swerdlow et alldquoAccurate whole human genome sequencing using reversibleterminator chemistryrdquo Nature vol 456 no 7218 pp 53ndash592008
[19] P S G Chain D V Grafham R S Fulton et al ldquoGenomeproject standards in a new era of sequencingrdquo Science vol 326no 5950 pp 236ndash237 2009
[20] RHWaterstonK Lindblad-Toh E Birney J Rogers J F AbrilP Agarwal et al ldquoInitial sequencing and comparative analysis ofthe mouse genomerdquo Nature vol 420 pp 520ndash562 2002
[21] T M Keane L Goodstadt P Danecek M A White K WongB Yalcin et al ldquoMouse genomic variation and its effect onphenotypes and gene regulationrdquoNature vol 477 pp 289ndash2942011
[22] T Yamamoto K Yoh A Kobayashi et al ldquoIdentification ofpolymorphisms in the promoter region of the human NRF2generdquo Biochemical and Biophysical Research Communicationsvol 321 no 1 pp 72ndash79 2004
[23] J M Marzec J D Christie S P Reddy et al ldquoFunctionalpolymorphisms in the transcription factor NRF2 in humansincrease the risk of acute lung injuryrdquo The FASEB Journal vol21 no 9 pp 2237ndash2246 2007
[24] H Fukushima-Uesaka Y Saito K Maekawa et al ldquoGeneticvariations and haplotype structures of transcriptional factorNRF2 and its cytosolic reservoir protein KEAP1 in JapaneserdquoDrug Metabolism and Pharmacokinetics vol 22 no 3 pp 212ndash219 2007
[25] E J Cordova R Velazquez-Cruz F Centeno V Baca and LOrozco ldquoThe NRF2 gene variant -653GA is associated with
Oxidative Medicine and Cellular Longevity 23
nephritis in childhood-onset systemic lupus erythematosusrdquoLupus vol 19 no 10 pp 1237ndash1242 2010
[26] M von Otter S Landgren S Nilsson et al ldquoAssociation ofNRF2-encoding NFE2L2 haplotypes with Parkinsonrsquos diseaserdquoBMCMedical Genetics vol 11 no 1 article 36 2010
[27] JMHartikainenM TengstromVMKosmaV L Kinnula AMannermaa andY Soini ldquoGenetic polymorphisms and proteinexpression of NRF2 and sulfiredoxin predict survival outcomesin breast cancerrdquo Cancer Research vol 72 pp 5537ndash5546 2012
[28] T Arisawa T Tahara T Shibata et al ldquoThe relationshipbetween Helicobacter pylori infection and promoter polymor-phism of the NRF2 gene in chronic gastritisrdquo InternationalJournal of Molecular Medicine vol 19 no 1 pp 143ndash148 2007
[29] T Arisawa T Tahara T Shibata et al ldquoThe influence ofpromoter polymorphism of nuclear factor-erythroid 2-relatedfactor 2 gene on the aberrant DNA methylation in gastricepitheliumrdquo Oncology Reports vol 19 no 1 pp 211ndash216 2008
[30] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene pro-moter polymorphism and gastric carcinogenesisrdquo Hepato-Gastroenterology vol 55 no 82-83 pp 750ndash754 2008
[31] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene promoterpolymorphism is associated with ulcerative colitis in a Japanesepopulationrdquo Hepato-Gastroenterology vol 55 no 82-83 pp394ndash397 2008
[32] C C Hua L C Chang J C Tseng C M Chu Y C Liu andW B Shieh ldquoFunctional haplotypes in the promoter region oftranscription factor NRF2 in chronic obstructive pulmonarydiseaserdquo Disease Markers vol 28 no 3 pp 185ndash193 2010
[33] S O Shaheen R B Newson S M Ring M J Rose-ZerilliJ W Holloway and A J Henderson ldquoPrenatal and infantacetaminophen exposure antioxidant gene polymorphismsand childhood asthmardquo The Journal of Allergy and ClinicalImmunology vol 126 no 6 pp 1141e7ndash1148e7 2010
[34] H Masuko T Sakamoto Y Kaneko et al ldquoLower FEV1 innon-COPD nonasthmatic subjects association with smokingannual decline in FEV1 total IgE levels and TSLP genotypesrdquoInternational Journal of Chronic Obstructive Pulmonary Diseasevol 6 pp 181ndash189 2011
[35] C P Guan M N Zhou A E Xu et al ldquoThe susceptibilityto vitiligo is associated with NF-E2-related factor 2 (NRF2)gene polymorphisms a study on Chinese Han populationrdquoExperimental Dermatology vol 17 no 12 pp 1059ndash1062 2008
[36] J Bouligand O Cabaret M Canonico et al ldquoEffect of NFE2L2genetic polymorphism on the association between oral estrogentherapy and the risk of venous thromboembolism in post-menopausal womenrdquo Clinical Pharmacology amp Therapeuticsvol 89 no 1 pp 60ndash64 2011
[37] D S OrsquoMahony B J Glavan T D Holden C Fong R A BlackG Rona et al ldquoInflammation and immune-related candidategene associations with acute lung injury susceptibility andseverity a validation studyrdquo PLoS ONE vol 7 no 12 Article IDe51104 2012
[38] I Ungvari E Hadadi V Virag et al ldquoRelationship between airpollutionNFE2L2 gene polymorphisms and childhood asthmain aHungarian populationrdquo Journal of CommunityGenetics vol3 no 1 pp 25ndash33 2012
[39] M SiedlinskiD S Postma JMA Boer et al ldquoLevel and courseof FEV1 in relation to polymorphisms inNFE2L2 andKEAP1 inthe general populationrdquo Respiratory Research vol 10 article 732009
[40] C C Hong C B Ambrosone J Ahn et al ldquoGenetic variabilityin iron-related oxidative stress pathways (NRF2 NQ01 NOS3
and HO-1) iron intake and risk of postmenopausal breastcancerrdquo Cancer Epidemiology Biomarkers and Prevention vol16 no 9 pp 1784ndash1794 2007
[41] C Canova C Dunster F J Kelly C Minelli P L Shah CCaneja et al ldquoPM10-induced hospital admissions for asthmaand chronic obstructive pulmonary disease the modifyingeffect of individual characteristicsrdquo Epidemiology vol 23 no 4pp 607ndash615 2012
[42] A J Henderson R B Newson M Rose-Zerilli S M RingJ W Holloway and S O Shaheen ldquoMaternal NRF2 andgluthathione-S-transferase polymorphisms donotmodify asso-ciations of prenatal tobacco smoke exposure with asthma andlung function in school-aged childrenrdquo Thorax vol 65 no 10pp 897ndash902 2010
[43] C Xing A L Sestak J A Kelly et al ldquoLocalization andreplication of the systemic lupus erythematosus linkage signalat 4p16 interaction with 2p11 12q24 and 19q13 in EuropeanAmericansrdquo Human Genetics vol 120 no 5 pp 623ndash631 2007
[44] T F Wong K Yoshinaga Y Monma K Ito H NiikuraS Nagase et al ldquoAssociation of KEAP1 and NRF2 geneticmutations and polymorphisms with endometrioid endometrialadenocarcinoma survivalrdquo International Journal of Gynecologi-cal Cancer vol 21 no 8 pp 1428ndash1435 2011
[45] M von Otter S Landgren S Nilsson et al ldquoNRF2-encodingNFE2L2 haplotypes influence disease progression but not riskin Alzheimerrsquos disease and age-related cataractrdquoMechanisms ofAgeing and Development vol 131 no 2 pp 105ndash110 2010
[46] S Tsang Z Sun B Luke et al ldquoA comprehensive SNP-basedgenetic analysis of inbred mouse strainsrdquoMammalian Genomevol 16 no 7 pp 476ndash480 2005
[47] C M Wade E J Kulbokas III A W Kirby et al ldquoThe mosaicstructure of variation in the laboratory mouse genomerdquoNaturevol 420 no 6915 pp 574ndash578 2002
[48] T Wiltshire M T Pletcher S Batalov et al ldquoGenome-widesingle-nucleotide polymorphism analysis defines haplotypepatterns in mouserdquo Proceedings of the National Academy ofSciences of the United States of America vol 100 no 6 pp 3380ndash3385 2003
[49] K A Frazer E Eskin H M Kang et al ldquoA sequence-basedvariation map of 827 million SNPs in inbred mouse strainsrdquoNature vol 448 no 7157 pp 1050ndash1053 2007
[50] T P Maddatu S C Grubb C J Bult andM A Bogue ldquoMousephenome database (MPD)rdquo Nucleic Acids Research vol 40 ppD887ndashD894 2007
[51] A Kirby H M Kang C M Wade et al ldquoFine mapping in 94inbred mouse strains using a high-density haplotype resourcerdquoGenetics vol 185 no 3 pp 1081ndash1095 2010
[52] H Y Cho A E Jedlicka S P M Reddy L Y Zhang T WKensler and S R Kleeberger ldquoLinkage analysis of susceptibilityto hyperoxia NRF2 is a candidate generdquo American Journal ofRespiratory Cell and Molecular Biology vol 26 no 1 pp 42ndash512002
[53] H Y Cho A E Jedlicka S P M Reddy et al ldquoRole of NRF2in protection against hyperoxic lung injury in micerdquo AmericanJournal of Respiratory Cell and Molecular Biology vol 26 no 2pp 175ndash182 2002
[54] T Shibata T Ohta K I Tong et al ldquoCancer related mutationsin NRF2 impair its recognition by KEAP1-Cul3 E3 ligase andpromote malignancyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 36 pp13568ndash13573 2008
24 Oxidative Medicine and Cellular Longevity
[55] A Singh V Misra R K Thimmulappa et al ldquoDysfunctionalKEAP1-NRF2 interaction in non-small-cell lung cancerrdquo PLoSMedicine vol 3 no 10 article e420 2006
[56] T Ohta K Iijima M Miyamoto et al ldquoLoss of KEAP1 functionactivates NRF2 and provides advantages for lung cancer cellgrowthrdquo Cancer Research vol 68 no 5 pp 1303ndash1309 2008
[57] B Padmanabhan K I Tong T Ohta et al ldquoStructural basisfor defects of KEAP1 activity provoked by its point mutationsin lung cancerrdquoMolecular Cell vol 21 no 5 pp 689ndash700 2006
[58] T Shibata A Kokubu M Gotoh et al ldquoGenetic alteration ofKEAP1 confers constitutive NRF2 activation and resistance tochemotherapy in gallbladder cancerrdquoGastroenterology vol 135no 4 pp 1358e4ndash1368e4 2008
[59] N J Yoo H R Kim Y R Kim C H An and S H LeeldquoSomatic mutations of the KEAP1 gene in common solidcancersrdquo Histopathology vol 60 no 6 pp 943ndash952 2012
[60] Y Hu Y Ju D Lin Z Wang Y Huang S Zhang et alldquoMutation of the NRF2 gene in non-small cell lung cancerrdquoMolecular Biology Reports vol 39 no 4 pp 4743ndash4747 2012
[61] Y R Kim J EOhM S Kim et al ldquoOncogenicNRF2mutationsin squamous cell carcinomas of oesophagus and skinrdquo TheJournal of Pathology vol 220 no 4 pp 446ndash451 2010
[62] T Shibata A Kokubu S Saito et al ldquoNRF2 mutation confersmalignant potential and resistance to chemoradiation therapyin advanced esophageal squamous cancerrdquo Neoplasia vol 13pp 864ndash873 2011
[63] H Sasaki M Shitara K Yokota Y Hikosaka S MoriyamaM Yano et al ldquoIncreased NRF2 gene (NFE2L2) copy numbercorrelates with mutations in lung squamous cell carcinomasrdquoMolecular Medicine Reports vol 6 no 2 pp 391ndash394 2012
[64] H Sasaki M Shitara K Yokota Y Hikosaka S Moriyama MYano et al ldquoRagD gene expression andNRF2mutations in lungsquamous cell carcinomasrdquo Oncology Letters vol 4 pp 1167ndash1170 2012
[65] A Singh M Bodas N Wakabayashi F Bunz and S BiswalldquoGain of NRF2 function in non-small-cell lung cancer cellsconfers radioresistancerdquo Antioxidants and Redox Signaling vol13 no 11 pp 1627ndash1637 2010
[66] T Yamadori Y Ishii SHommaYMorishimaKKurishimaKItoh et al ldquoMolecular mechanisms for the regulation of NRF2-mediated cell proliferation in non-small-cell lung cancersrdquoOncogene vol 31 pp 4768ndash4777 2012
[67] A K Bauer H Y Cho L Miller-Degraff C Walker K HelmsJ Fostel et al ldquoTargeted deletion of NRF2 reduces urethane-induced lung tumor development in micerdquo PLoS ONE vol 6no 10 Article ID e26590 2011
[68] H Y Cho and S R Kleeberger ldquoNRF2 protects against airwaydisordersrdquo Toxicology and Applied Pharmacology vol 244 no1 pp 43ndash56 2010
[69] R Wang J An F Ji H Jiao H Sun and D Zhou ldquoHyperme-thylation of the KEAP1 gene in human lung cancer cell linesand lung cancer tissuesrdquo Biochemical and Biophysical ResearchCommunications vol 373 no 1 pp 151ndash154 2008
[70] P Zhang A Singh S Yegnasubramanian et al ldquoLoss ofkelch-like ECH-associated protein 1 function in prostate cancercells causes chemoresistance and radioresistance and promotestumor growthrdquoMolecular Cancer Therapeutics vol 9 no 2 pp336ndash346 2010
[71] N Hanada T Takahata Q Zhou et al ldquoMethylation of theKEAP1 gene promoter region in human colorectal cancerrdquoBMCCancer vol 12 article 66 2012
[72] L A Muscarella R Barbano V DrsquoAngelo et al ldquoRegulationof KEAP1 expression by promoter methylation in malignantgliomas and association with patientrsquos outcomerdquo Epigeneticsvol 6 no 3 pp 317ndash325 2011
[73] S Yu T O Khor K L Cheung et al ldquoNRF2 expressionis regulated by epigenetic mechanisms in prostate cancer ofTRAMP micerdquo PLoS ONE vol 5 no 1 Article ID e8579 2010
[74] T O Khor Y Huang T Y Wu L Shu J Lee and A N KongldquoPharmacodynamics of curcumin as DNA hypomethylationagent in restoring the expression of NRF2 via promoter CpGsdemethylationrdquo Biochemical Pharmacology vol 82 no 9 pp1073ndash1078 2011
[75] I M Fingerman L McDaniel X Zhang et al ldquoNCBI epige-nomics a new public resource for exploring epigenomic datasetsrdquo Nucleic Acids Research vol 39 supplemen 1 pp D908ndashD912 2011
[76] I M Fingerman X Zhang W Ratzat N Husain R F Cohenand G D Schuler ldquoNCBI epigenomics whatrsquos new for 2013rdquoNucleic Acids Research vol 41 no 1 pp D221ndashD225 2013
[77] J H Lee T O Khor L Shu Z Y Su F Fuentes andA N Kong ldquoDietary phytochemicals and cancer preventionNRF2 signaling epigenetics and cell death mechanisms inblocking cancer initiation and progressionrdquo Pharmacology ampTherapeutics vol 137 no 2 pp 153ndash171 2012
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
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EndocrinologyInternational Journal of
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Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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OncologyJournal of
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Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Oxidative Medicine and Cellular Longevity 17
Table5Mou
seup
stream
andexon
varia
tions
ofNfe2
l2locusin17
inbred
strainsR
eference
sequ
ence
isC5
7BL6J
(B6strain
1)geno
me(GI14933824975547698ndash75513576)SNPalleleand
geno
typesareshow
nas
chromosom
econ
tigsequ
enceStrains
2129S1S
vlmJ3AJ
4AKR
J5BA
LBcJ6C3
HH
eJ7C
57BL
6NJ8CA
STEiJ9CB
AJ
10D
BA2J11FVBNJ12LPJ
13N
ODShiLtJ14N
ZOH
ILtJ
15P
WK
PhJ16SPR
ETEiJ
17W
SBEiJ
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs2566
08517
75705565
GA
(CT)
51015840Flankingminus924
NC
GG
GG
GG
GG
GG
GG
GG
AA
Grs47274959
75705562
AT
(TA
)51015840Flankingminus921
NC
AT
TA
TT
AA
TT
AT
AT
TT
Ars216940398
75705554ndash75705555
Tadd(A
add)
51015840Flankingminus913minus914
NC
CGrs239114134
75705540
CT(G
A)
51015840Flankingminus899
NC
CT
TC
TT
CC
TT
CT
CT
CT
Crs46
461765
75705528
TG(A
C)oA
C
51015840Flankingminus887
NC
TG
GT
GG
TT
GG
TG
TG
GG
Trs5144
9853
75705525
CA(G
T)
51015840Flankingminus884
NC
CA
AC
AA
CC
AA
CA
CA
CC
Crs249093111
75705512
CT(G
A)
51015840Flankingminus871
NC
CT
TC
TT
CC
TT
CT
CT
TT
Crs263745200
75705510
CG(G
C)
51015840Flankingminus869
NC
GG
GG
GG
GG
GG
GG
GG
CG
Grs45651867
75705498
TG(A
C)
51015840Flankingminus857
NC
TG
GT
GG
TT
GG
TG
TG
GG
Trs219997531
75705495
GA
(CT)
51015840Flankingminus854
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs251918379
75705451
TA(A
T)
51015840Flankingminus810
NC
TT
TT
TT
TT
TT
TT
TT
TA
Trs27978306
75705449
TA(A
T)
51015840Flankingminus808
NC
TA
AT
AA
TT
AA
TA
TA
TT
Trs216087412
75705429
TG(A
C)lowast
51015840Flankingminus788
NC
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
TlowastTlowast
GTlowast
Tlowast
rs261229914
75705423ndash75705424
AGdel(CT
del)
51015840Flankingminus783minus784
NC
AGrs27978307
75705400
CT(G
A)
51015840Flankingminus759
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs243167395
75705359
GA
(CT)
51015840Flankingminus718
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs27978308
75705307
GA
(CT)
51015840Flankingminus66
6NC
GG
GG
GG
GG
GG
GG
GG
AG
Grs254744
09875705294
AT
(TA
)51015840Flankingminus653
NC
AA
AA
AA
AA
AA
AA
AA
AT
Ars228133419
75705179
GT
(CA
)51015840Flankingminus538
NC
GG
GG
GG
GG
GG
GG
GG
GT
Grs214719520
75705111
GA
(CT)
51015840Flankingminus470
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs2440
87730
75705101
GA
(CT)
51015840Flankingminus46
0NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs227781699
75705081
CG(G
C)
51015840Flankingminus44
0NC
CC
CC
CC
CC
CC
CC
CC
GC
Crs257870353
75705067
CT(G
A)
51015840Flankingminus426
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs24487744
075705022
AG
(TC)
51015840Flankingminus381
NC
AA
AA
AA
AA
AA
AA
AA
AG
Ars234628138
75704973
AC
(TG
)51015840Flankingminus332
NC
AA
AA
AA
AA
AA
AA
AA
AC
A
18 Oxidative Medicine and Cellular Longevity
Table5Con
tinued
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs247275247
75704961
GA
(CT)
51015840Flankingminus320
NC
GG
GG
GG
GG
GG
GG
AG
GG
Grs247519047
75704922
Gdel(Cdel)
51015840Flankingminus281
NC
Grs27978309
(rs51915758)
75704887
GA
C(C
TG
)51015840Flankingminus246
NC
GA
AG
AA
GG
AA
GA
GA
CC
G
rs218139102
75704879
CA(G
T)
51015840Flankingminus238
NC
CC
CC
CC
CC
CC
CC
CC
AC
Crs251990355
75704870
TC(A
G)
51015840Flankingminus229
NC
TT
TT
TT
TT
TT
TT
TT
TC
Trs238746955
75704794
AG
(TC)
51015840Flankingminus153
NC
AA
AA
AA
AA
AA
AA
AA
GA
Ars221664
405
75704786
AG
(TC)
51015840Flankingminus145
NC
AA
AA
AA
AA
AA
AA
AA
AG
Ars217054035
75704766
ndash75704767
GAAadd(TTC
add)
51015840Flankingminus125minus126
NC
GA
rs248182931
75704759ndash75704760
Gadd(C
add)
51015840Flankingminus118
minus119
NC
AGrs217197904
75704744
GA
(CT)
51015840Flankingminus103
(+)S
p1[23]
GA
AG
AA
GG
AA
GA
AA
GG
Ars2644
9364975704704
Gdel(Cdel)
51015840Flankingminus63
NC
Grs2404
81112
757046
82CT(G
A)
51015840Flankingminus41
NC
CC
CC
CC
CC
CC
CC
CC
CT
Crs27978310
75704617
AG
(TC)
UTR
-525
NC
AA
AA
AA
AA
AA
AA
AA
GG
Ars27978311
75704610
CG(G
C)
UTR
-532
NC
GG
GG
GG
GG
GG
GG
GG
GG
Crs27978312
757046
05GA
(CT)
UTR
-537
NC
GA
AG
AA
GG
AA
GA
GA
GG
Grs214784220
757044
99AG
(TC)
UTR
-5143
NC
AA
AA
AA
AA
AA
AA
AA
GG
Ars244146318
757044
98GT
(CA
)UTR
-5144
NC
GG
GG
GG
GG
GG
GG
GG
GT
Grs27978313
757044
97GC
(CG
)UTR
-5145
NC
GC
CG
CC
GC
CC
GC
CC
CC
Crs215431944
757044
93CT(G
A)
UTR
-5149
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs257015697
757044
49GA
(CT)
UTR
-5193
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs252234782
757044
19GT
(CA
)UTR
-5223
NC
Grs1346
0861
75679262
AG
(TC)
Exon
244
6Cn
sF71L
AG
GA
GG
AG
GG
AG
AG
GG
Grs1346
0859
75679202
GA
(CT)lowast
Exon
2506
CsH91=
GA
AG
AA
GG
AA
GA
GA
GG
Grs27978436
75679187
GA
(CT)lowast
Exon
2521
CsT9
6=G
GG
GG
GG
AG
GG
GG
GA
GG
rs226131070
75679178
GA
(CT)
Exon
2530
CsS99=
GG
GG
GG
GG
GG
GG
GG
GA
Grs227683834
75678576
AT
(TA
)Ex
on3
547
CnsV
105D
AA
AA
AA
AA
AA
AA
AA
AT
Ars257321187
75678506
TC(A
G)
Exon
3617
CsP128=
TT
TT
TT
TC
TT
TT
TT
TT
Trs27978444
75678500
TA(A
T)
Exon
3623
CsV130=
TT
TT
TT
TA
TT
TT
TT
AT
Trs227743136
75677686
GA
(CT)
Exon
4662
CsH143=
GG
GG
GG
GG
GG
GG
GG
GA
Grs215327202
75677664
CG(G
C)
Exon
4684
CnsA
151P
CC
CC
CC
CG
CC
CC
CC
CC
Crs247539755
75677663
GA
(CT)
Exon
4685
CnsA
151V
GG
GG
GG
GA
GG
GG
GG
GG
Grs233164
668
75677640
TC(A
G)
Exon
4708
CnsN
159D
TT
TT
TT
TT
TT
TT
TT
TC
T
rs2340
95816
75677162ndash75677160
GCT
del
(AGCdel)
Exon
5826minus
828
Cds-in
delQ
198
GCT
Oxidative Medicine and Cellular Longevity 19
Table5Con
tinued
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs27978452
75677145
CT(G
A)
Exon
5843
CnsA
204T
CC
CC
CC
CC
CC
CC
CC
CC
T
rs225047937
75677086ndash75677078
GAG
ATCG
ATdel
(ATG
CATC
TCdel)
Exon
5902minus
910
Cds-in
del
(S224I225S226)
GAG
ATC
GAT
rs241469868
75676998
TA(G
T)
Exon
5990
CnT2
53S
TT
TT
TT
TT
TT
TT
TT
TA
Trs221602571
75676923
AC
(TG
)Ex
on5
1065
CnsS
278A
AA
AA
AA
AA
AA
AA
AA
AC
Ars252576472
75676876
GA
(CT)
Exon
51112
CsS293=
GG
GG
GG
GG
GG
GG
GG
GA
Grs250802933
75676841
TC(A
G)
Exon
5114
7Cn
sD305G
TT
TT
TT
TT
TT
TT
TT
TC
Trs225425698
75676792
CT(G
A)
Exon
5119
6Cs
P321=
CC
CC
CC
CC
CC
CC
CC
CT
Crs13459064
75676732
CT(G
A)
Exon
51256
CsT3
41=
CT
TC
TT
CT
TT
CT
CT
TC
Crs27978453
75676717
CT(G
A)
Exon
51271
CsA346=
CC
CC
CC
CC
CC
CC
CC
TC
Crs214335034
75676636
AG
(TC)
Exon
51352
CsD373=
AA
AA
AA
AA
AA
AA
AA
AG
Ars24958364
4756766
03AC
(TG
)Ex
on5
1385
CsP3
84=
AA
AA
AA
AA
AA
AA
AA
AC
Ars27978454
75676592
GA
(CT)
Exon
51396
CnsP
388L
GG
GG
GG
GG
GG
GG
GG
AG
Grs215384328
75676589
GC
(CG
)Ex
on5
1399
CnsA
389G
GG
GG
GG
GG
GG
GG
GG
GC
Grs251728286
75676571
GA
(CT)
Exon
51417
CnsT
395I
Grs247602334
75676567
TC(A
G)
Exon
51421
CsV396=
TT
TT
TT
TT
TT
TT
TT
TC
Trs234216231
75676530
TC(A
G)
Exon
51458
CnsM
409V
TT
TT
TT
TT
TT
TT
TT
TC
Trs212904337
75676526
CT(G
A)
Exon
51462
CnsR
410H
CC
CC
CC
CC
CC
CC
CC
CT
Crs252650779
75676522
TC(A
G)
Exon
51466
CsE4
11=
TT
TT
TT
TT
TT
TT
TT
TC
Trs231273560
75676516
TC(A
G)
Exon
51472
CsQ413=
TT
TT
TT
TT
TT
TT
TT
TC
Trs257886949
756764
13GT
(CA
)Ex
on5
1575
CsR4
48=
GG
GG
GG
GG
GG
GG
GG
GT
Grs241537608
756764
04GC
(CG
)Ex
on5
1584
CnsL
451V
GG
GG
GG
GG
GG
GG
GG
GC
Grs227619071
75676312
TC(A
G)
Exon
51676
CsQ481=
TT
TT
TT
TT
TT
TT
TT
TC
Trs258913831
75676290
AG
(TC)
Exon
51698
CsL4
89=
AA
AA
AA
AA
AA
AA
AA
AG
Ars225593319
75676126
AT
(TA
)Ex
on5
1862
CnsH
543Q
Ars4223233
75676105
GA
(CT)
Exon
51883
CsS550=
GG
GG
GG
GA
GG
GG
GG
GG
Grs4223232
75676032
GT
(CA
)Ex
on5
1956
CnsL
575M
Grs4223231
75675816
CG
UTR
-32172
NC
GG
GG
GG
GG
GG
GG
GG
GC
Grs1346
860
75675682
AT
UTR
-32306
NC
ASequ
ence
varia
tions
inmou
seNr
f2wereob
tained
from
Mou
sePh
enom
eDatabase(http
ph
enom
ejaxorgSNP)
andNCB
ISNPdatabase
(http
wwwncbinlm
nihgovSNP)N
CBIreference
sequ
ence
isMus
Musculusstra
inC5
7BL6J
chromosom
e2GRC
m38p1(NC
0000
687
GI37209910875704641ndash756755192
9123
bp)To
tal968
genetic
mutations
arerepo
rted
inNr
f2gene
and5k
bup
stream
(ge75704
642minus
1)and2k
bdo
wnstre
am(le75675512)sequences
asof
Janu
ary2003A
llSN
Psandallelesa
representedas
geno
miccontig
(reversedsequ
ence
indicated)E
xon175704
641ndash75704364(278
bpT
TS=75704
408)
exon
275679431ndash756791
65(267
bp)exon
375678579ndash75678490(90b
p)exon475677713ndash756775
46(168
bp)andexon
57567718
4ndash75678849
(1666
bp)Proteinam
inoacid
(aa)
resid
uesinNP00
61552
(597
aa)Cs
cod
ing-syno
nymou
sCn
scoding
-non
syno
nymou
sAminoacid
A-alanineD-aspartic
acidE
-glutamicacidF-phenylalanineG
-glycineH
-histidineI-iso
leucineL-leu
cineand
M-m
ethion
ineN-
asparagineP-prolin
eQ-glutamineR-arginineS-serineT-threon
ineandV-valin
elowastErrorsin
databasesfi
xedNC
notcon
firmed
20 Oxidative Medicine and Cellular Longevity
Table 6 NRF2 somatic mutations revealed in various human cancers
Domain Amino acid residues DNA mutation Cancer types (cases) ReferencesLocus Wild type Mutant
DLG motif
24 W (Trp) C (Cys) c72GgtCGgtT NSCLC neck ESC [54 62]K (Lys) c72TgtC ESC [62]
26 Q (Gln) E (Glu) c76CgtG NSCLC ESC [54 62]
27 D (Asp) G (Gly)lowast c80GgtA NSCLC [60]Y (Tyr) c79GgtT ESC [61]
28 I (Ile) T (Thr) c83CgtT NSCLC [54]
29 D (Asp) G (Gly) c86AgtG Head and neck ESC [54 62]H (His) c85GgtC NSCLC laynx [60 61]
30 L (Leu) F (Phe) c88CgtT NSCLC ESC [54 62]
31 G (Gly) A (Ala) c92GgtC NSCLC ESC skin [54 60ndash62]
32 V (Val) T (Thr) c95TgtG NSCLC [54]del c93 95delAGT ESC [61]
33ndash36 S-R-E-V S-R-E-V-S-R-E-Vlowast c97 108dupAGTCGAGCCGTA ESC [61]
34 R (Arg) Q (Gln) c101GgtA NCSCL [54 60 61]P (Pro) c101GgtC NSCLC [63]
ETGF motif
75 Q (Gln) H (His) c225AgtC Head and neck ESC [54 62]
77 D (Asp)
V (Val) c230AgtT NSCLC ESC [54 60 62]G (Gly) c230AgtG ESC [62]A (Ala) c230AgtC NSCLC [61]N (Asn) c229GgtA Larynx [61]
78 E (Glu) K (Lys) c232GgtA NSCLC ESC [54 62]
79 E (Glu)
K (Lys) c235GgtA NSCLC ESC [54 61 62]Q (Gln) c235GgtC NSCLC ESC [54 60ndash62]G (Gly) c236AgtG Larynx [61]E-E c234 236dupAGA ESC [61]
80 T (Thr) K (Lys) c239CgtACgtG NSCLC ESC [54 61 62]
80 T (Thr)P (Phe) c238AgtC ESC [62]I (Ile) c239CgtT Head and neck [54]A (Ala) c238AgtG NSCLC [63]
81 G (Gly) V (Val) c242GgtT ESC [61]D (Asp) c242GgtA NSCLC ESC [60ndash62]
82 E (Glu)
D (Asp) c246AgtT ESC oral cancer cell line [54 62]G (Gly) c245AgtG NSCLC [54]Q (Gln)lowast c244GgtC NSCLC ESC [60 61]V (Val)lowast c245AgtT ESC [61]
83 F (Phe) L (Leu)lowast c247TgtC NSCLC [60]NSCLC non-small cell lung cancer ESC esophageal squamous cancer and lowasterrors in reference fixed Number of cases 82 NSCLC and 10 ESC in [62] 125NSCLC 70 ESC 23 larynx and 17 skin in [61] 103 NSCLC and 12 head and neck in [54] 90 NSCLC in [63] 103 NSCLC in [60]
be involved in squamous lung cancer cell proliferation [64]suggesting that the mutation is functional and overcomesKEAP1 inhibition Singh et al [65] determined in vitrothat RNAi-mediated depletion of NRF2 in lung cancer cellsenhanced ROS production and susceptibility to cell deathby ionizing radiation These studies support the concept thatelevated NRF2 and ARE responsiveness provides cancer cellswith proliferative advantage for malignant transformation
and undue protection from anti-cancer therapy Onco-genic epidermal growth factor receptor (EGFR) signaling isrecently found to be critical in NRF2-mediated proliferationof NSCLC cells [66]
Collectively ldquogain of functionrdquo mutations in NRF2 thatreduce KEAP1 recognition are suggested to be predictivemarkers for poor responsiveness to chemotherapy and radi-ation therapy Although NRF2-mediated cellular defense
Oxidative Medicine and Cellular Longevity 21
processes are essential in the initiation stage enhancedNRF2-ARE activity in advanced stages of cancer developmentmay create a favorable intracellular environment for tumorcell growth and survival [67 68] In this context NRF2may be a potential molecular target for the treatment ofradio-resistance cancers especially those that have ldquoloss offunctionrdquo mutations in EGFR KRAS or KEAP1 as well asldquogain of functionrdquo mutations in NRF2
5 Epigenetic Alterations of NF-E2-RelatedFactor 2
Epigenetic modifications are alterations of molecules inter-acting with genes without changes to the primary DNAsequence They include post-translational modification ofhistones DNA methylation events chromatin conforma-tional changes and alterations to noncoding regulatoryRNAs Epigenetic alterations are stable and often inheritablebut are reversible and may affect expression of the geneDysregulation or defects in epigenetic processes particularlyhypermethylation of tumor suppressor gene promoters (egCpG islands) or histone modifications are thought to beassociated with carcinogenesis Investigators have reportedhypermethylation in CpG islands of KEAP1 which wereassociated with reduced KEAP1 expression in human cancersfrom lung prostate colon and so forth [69ndash72] Similar tosomatic mutations epigenetic changes on KEAP1 impairedthe function of its encoded protein leading to constitutiveNRF2 activation
Supporting the role of ldquopathogenic mutationsrdquo in NRF2expression of Nrf2 and downstream Nqo1 was suppressedin prostate tumors of mice (transgenic adenocarcinomaof mouse prostate TRAMP) Among 15 promoter CpGislands located between minus942 and minus654 (cminus1175 cminus1132and cminus1059 cminus887 gap in cminus1131 cminus1060 see Figure 3)hypermethylation of the first 5 CpG islands (minus942 minus899 andcminus1175 cminus1132) was significantly associated with tumorige-nesis [73] Moreover treatment with inhibitors for DNAmethyltransferase and histone deacetylase restored Nrf2expression in these tumor cells [73] A dietary phytochemicalcurcumin known as a DNA hypomethylation agent restoredepigenetically silent Nrf2 expression through CpG demethy-lation in carcinogen-induced mouse tumor cells [74]
The whole genome epigenetic datasets for 5 species arepublicly accessible at NCBI Epigenomics [75 76]The humanNRF2 epigenome of primary cells (breast penis) and H1stem cell line as well as mouse Nrf2 CpG island methylationdata for sperm blood and cerebellum are currently avail-able (httpwwwncbinlmnihgovepigenomics) Althoughno direct evidence of disease-associated epigenetic modu-lation has been identified in human NRF2 various phyto-chemical NRF2 agonists such as sulforaphane and curcuminhave shown their roles in DNA methylation and histonemodification (see reviews by Lee and colleagues eg [77])Taken together epigenetic modifications of theNRF2KEAP1axle are predicted to cause dysregulation of ARE-mediatedcellular defense leading to deleterious health effects and
phytochemical antioxidants as epigenetic modulators forNRF2 are suggested to be useful in cancer prevention
6 Conclusions
NRF2 is evolutionally conserved with high-sequence homol-ogy in many species However it is a highly mutable geneand numerous genetic variants have been discovered inhuman ethnic groups Importantly certain SNPs or haplo-types have been identified in various diseases as ldquoat-riskrdquoalleles and are related to functional alterations In additionto genetic variations multiple somatic mutations identifiedin the KEAP1 recognition domain of NRF2 in cancer cellshave been found to be oncogenic due to dysregulation ofNRF2 homeostasis by its excess ldquogain of functionrdquo Epigeneticalteration of theNRF2 is under investigation and is predictedto have pathogenic influences as learned from mouse andphytochemical agonist studies Continuous updates of Nrf2allelic variants in inbred mouse strains will provide a usefultool for effective experimental designs formodels of oxidativedisorders to provide insight into the disease mechanisms andintervention strategies
Abbreviations
AD Alzheimerrsquos diseaseALI acute lung injuryARE antioxidant response elementbZIP basic leucine zippercds coding DNA sequencesCI confidence intervalCOPD chronic obstructive pulmonary diseaseESC esophageal squamous cancerFEV1 forced expiratory volume in one second
Gbp giga base pairsGST glutathione S-transferaseGWAS genome-wide association studyHGVS Human Genome Variation SocietyHO-1 heme oxygenase-1119867 119901119910119897119900119903119894 119867119890119897119894119888119900119887119886119888119905119890119903 119901119910119897119900119903119894KEAP1 Kelch-like ECH activating protein 1MPD Mouse Phenome DatabaseMRP multidrug resistance proteinNCBI National Center for Biotechnology InformationNeh NRF2-ECH homologyNfe2l2 nuclear factor (erythroid-derived 2)-like 2Nrf2 NF-E2-related factor 2NSCLC nonsmall cell lung cancerNQO1 NAD(P)Hquinone oxidoreductase 1OR odds ratioPD Parkinsonrsquos diseasePM particulate matterROS reactive oxygen speciesSLE systemic lupus erythematosusSNP single nucleotide polymorphismSOD superoxide dismutaseURT untranslated region
22 Oxidative Medicine and Cellular Longevity
Disclosure
Authorrsquos contribution to the Work was done as part of theAuthorrsquos official duties as a NIH employee and is a Work ofthe United States Government Therefore copyright may notbe established in the United States
Conflict of Interests
The author declares that there is no conflict of interests
Acknowledgments
The research related to this paper was supported by theIntramural Research Program of the National Institutes ofHealth of the National Institute of Environmental HealthSciences (NIEHS) Drs Steven Kleeberger and StephanieLondon at the NIEHS provided excellent critical review ofthis paper The author thanks Mrs Jacqui Marzec for herhelpful comments and English editing
References
[1] J Y Chan M C Cheung P Moi K Chan and Y W KanldquoChromosomal localization of the humanNF-E2 family of bZIPtranscription factors by fluorescence in situ hybridizationrdquoHuman Genetics vol 95 no 3 pp 265ndash269 1995
[2] K Itoh K Igarashi N Hayashi M Nishizawa and MYamamoto ldquoCloning and characterization of a novel erythroidcell-derived CNC family transcription factor heterodimerizingwith the small Maf family proteinsrdquo Molecular and CellularBiology vol 15 no 8 pp 4184ndash4193 1995
[3] W O Osburn and T W Kensler ldquoNRF2 signaling an adaptiveresponse pathway for protection against environmental toxicinsultsrdquoMutation Research vol 659 no 1-2 pp 31ndash39 2008
[4] D Papp K Lenti D Modos D Fazekas Z Dul D Tureiet al ldquoThe NRF2-related interactome and regulome containmultifunctional proteins and fine-tuned autoregulatory loopsrdquoFEBS Letters vol 586 no 13 pp 1795ndash1802 2012
[5] X Wang D J Tomso B N Chorley et al ldquoIdentificationof polymorphic antioxidant response elements in the humangenomerdquo Human Molecular Genetics vol 16 no 10 pp 1188ndash1200 2007
[6] D Malhotra E Portales-Casamar A Singh et al ldquoGlobalmapping of binding sites for NRF2 identifies novel targets incell survival response through ChIP-Seq profiling and networkanalysisrdquo Nucleic Acids Research vol 38 no 17 pp 5718ndash57342010
[7] K Chan R Lu J C Chang and Y W Kan ldquoNRF2 a memberof the NFE2 family of transcription factors is not essential formurine erythropoiesis growth and developmentrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 93 no 24 pp 13943ndash13948 1996
[8] K Itoh T Chiba S Takahashi et al ldquoAn NRF2small Mafheterodimer mediates the induction of phase II detoxifyingenzyme genes through antioxidant response elementsrdquo Bio-chemical and Biophysical Research Communications vol 236no 2 pp 313ndash322 1997
[9] FMartin J M vanDeursen R A Shivdasani CW Jackson AG Troutman and P A Ney ldquoErythroid maturation and globin
gene expression inmicewith combined deficiency ofNF-E2 andNrf-2rdquo Blood vol 91 no 9 pp 3459ndash3466 1998
[10] K Itoh N Wakabayashi Y Katoh et al ldquoKEAP1 repressesnuclear activation of antioxidant responsive elements by NRF2through binding to the amino-terminal Neh2 domainrdquo Genesand Development vol 13 no 1 pp 76ndash86 1999
[11] A Kobayashi M I Kang H Okawa et al ldquoOxidative stresssensor KEAP1 functions as an adaptor for Cul3-based E3 ligaseto regulate proteasomal degradation of NRF2rdquo Molecular andCellular Biology vol 24 no 16 pp 7130ndash7139 2004
[12] K Taguchi H Motohashi and M Yamamoto ldquoMolecularmechanisms of the KEAP1-NRF2 pathway in stress responseand cancer evolutionrdquo Genes to Cells vol 16 no 2 pp 123ndash1402011
[13] K Mukaigasa L T Nguyen L Li H Nakajima M Yamamotoand M Kobayashi ldquoGenetic evidence of an evolutionarilyconserved role for NRF2 in the protection against oxidativestressrdquoMolecular and Cellular Biology vol 32 no 21 pp 4455ndash4461 2012
[14] JMaher andMYamamoto ldquoThe rise of antioxidant signalingmdashthe evolution and hormetic actions of NRF2rdquo Toxicology andApplied Pharmacology vol 244 no 1 pp 4ndash15 2010
[15] S C Lo X Li M T Henzl L J Beamer and M HanninkldquoStructure of the KEAP1NRF2 interface provides mechanisticinsight intoNRF2 signalingrdquoThe EMBO Journal vol 25 no 15pp 3605ndash3617 2006
[16] V A McKusick ldquoMendelian inheritance in man and its onlineversion OMIMrdquo American Journal of Human Genetics vol 80no 4 pp 588ndash604 2007
[17] B Yalcin D J Adams J Flint and T M Keane ldquoNext-generation sequencing of experimental mouse strainsrdquo Mam-malian Genome vol 23 no 9-10 pp 490ndash498 2012
[18] D R Bentley S Balasubramanian H P Swerdlow et alldquoAccurate whole human genome sequencing using reversibleterminator chemistryrdquo Nature vol 456 no 7218 pp 53ndash592008
[19] P S G Chain D V Grafham R S Fulton et al ldquoGenomeproject standards in a new era of sequencingrdquo Science vol 326no 5950 pp 236ndash237 2009
[20] RHWaterstonK Lindblad-Toh E Birney J Rogers J F AbrilP Agarwal et al ldquoInitial sequencing and comparative analysis ofthe mouse genomerdquo Nature vol 420 pp 520ndash562 2002
[21] T M Keane L Goodstadt P Danecek M A White K WongB Yalcin et al ldquoMouse genomic variation and its effect onphenotypes and gene regulationrdquoNature vol 477 pp 289ndash2942011
[22] T Yamamoto K Yoh A Kobayashi et al ldquoIdentification ofpolymorphisms in the promoter region of the human NRF2generdquo Biochemical and Biophysical Research Communicationsvol 321 no 1 pp 72ndash79 2004
[23] J M Marzec J D Christie S P Reddy et al ldquoFunctionalpolymorphisms in the transcription factor NRF2 in humansincrease the risk of acute lung injuryrdquo The FASEB Journal vol21 no 9 pp 2237ndash2246 2007
[24] H Fukushima-Uesaka Y Saito K Maekawa et al ldquoGeneticvariations and haplotype structures of transcriptional factorNRF2 and its cytosolic reservoir protein KEAP1 in JapaneserdquoDrug Metabolism and Pharmacokinetics vol 22 no 3 pp 212ndash219 2007
[25] E J Cordova R Velazquez-Cruz F Centeno V Baca and LOrozco ldquoThe NRF2 gene variant -653GA is associated with
Oxidative Medicine and Cellular Longevity 23
nephritis in childhood-onset systemic lupus erythematosusrdquoLupus vol 19 no 10 pp 1237ndash1242 2010
[26] M von Otter S Landgren S Nilsson et al ldquoAssociation ofNRF2-encoding NFE2L2 haplotypes with Parkinsonrsquos diseaserdquoBMCMedical Genetics vol 11 no 1 article 36 2010
[27] JMHartikainenM TengstromVMKosmaV L Kinnula AMannermaa andY Soini ldquoGenetic polymorphisms and proteinexpression of NRF2 and sulfiredoxin predict survival outcomesin breast cancerrdquo Cancer Research vol 72 pp 5537ndash5546 2012
[28] T Arisawa T Tahara T Shibata et al ldquoThe relationshipbetween Helicobacter pylori infection and promoter polymor-phism of the NRF2 gene in chronic gastritisrdquo InternationalJournal of Molecular Medicine vol 19 no 1 pp 143ndash148 2007
[29] T Arisawa T Tahara T Shibata et al ldquoThe influence ofpromoter polymorphism of nuclear factor-erythroid 2-relatedfactor 2 gene on the aberrant DNA methylation in gastricepitheliumrdquo Oncology Reports vol 19 no 1 pp 211ndash216 2008
[30] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene pro-moter polymorphism and gastric carcinogenesisrdquo Hepato-Gastroenterology vol 55 no 82-83 pp 750ndash754 2008
[31] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene promoterpolymorphism is associated with ulcerative colitis in a Japanesepopulationrdquo Hepato-Gastroenterology vol 55 no 82-83 pp394ndash397 2008
[32] C C Hua L C Chang J C Tseng C M Chu Y C Liu andW B Shieh ldquoFunctional haplotypes in the promoter region oftranscription factor NRF2 in chronic obstructive pulmonarydiseaserdquo Disease Markers vol 28 no 3 pp 185ndash193 2010
[33] S O Shaheen R B Newson S M Ring M J Rose-ZerilliJ W Holloway and A J Henderson ldquoPrenatal and infantacetaminophen exposure antioxidant gene polymorphismsand childhood asthmardquo The Journal of Allergy and ClinicalImmunology vol 126 no 6 pp 1141e7ndash1148e7 2010
[34] H Masuko T Sakamoto Y Kaneko et al ldquoLower FEV1 innon-COPD nonasthmatic subjects association with smokingannual decline in FEV1 total IgE levels and TSLP genotypesrdquoInternational Journal of Chronic Obstructive Pulmonary Diseasevol 6 pp 181ndash189 2011
[35] C P Guan M N Zhou A E Xu et al ldquoThe susceptibilityto vitiligo is associated with NF-E2-related factor 2 (NRF2)gene polymorphisms a study on Chinese Han populationrdquoExperimental Dermatology vol 17 no 12 pp 1059ndash1062 2008
[36] J Bouligand O Cabaret M Canonico et al ldquoEffect of NFE2L2genetic polymorphism on the association between oral estrogentherapy and the risk of venous thromboembolism in post-menopausal womenrdquo Clinical Pharmacology amp Therapeuticsvol 89 no 1 pp 60ndash64 2011
[37] D S OrsquoMahony B J Glavan T D Holden C Fong R A BlackG Rona et al ldquoInflammation and immune-related candidategene associations with acute lung injury susceptibility andseverity a validation studyrdquo PLoS ONE vol 7 no 12 Article IDe51104 2012
[38] I Ungvari E Hadadi V Virag et al ldquoRelationship between airpollutionNFE2L2 gene polymorphisms and childhood asthmain aHungarian populationrdquo Journal of CommunityGenetics vol3 no 1 pp 25ndash33 2012
[39] M SiedlinskiD S Postma JMA Boer et al ldquoLevel and courseof FEV1 in relation to polymorphisms inNFE2L2 andKEAP1 inthe general populationrdquo Respiratory Research vol 10 article 732009
[40] C C Hong C B Ambrosone J Ahn et al ldquoGenetic variabilityin iron-related oxidative stress pathways (NRF2 NQ01 NOS3
and HO-1) iron intake and risk of postmenopausal breastcancerrdquo Cancer Epidemiology Biomarkers and Prevention vol16 no 9 pp 1784ndash1794 2007
[41] C Canova C Dunster F J Kelly C Minelli P L Shah CCaneja et al ldquoPM10-induced hospital admissions for asthmaand chronic obstructive pulmonary disease the modifyingeffect of individual characteristicsrdquo Epidemiology vol 23 no 4pp 607ndash615 2012
[42] A J Henderson R B Newson M Rose-Zerilli S M RingJ W Holloway and S O Shaheen ldquoMaternal NRF2 andgluthathione-S-transferase polymorphisms donotmodify asso-ciations of prenatal tobacco smoke exposure with asthma andlung function in school-aged childrenrdquo Thorax vol 65 no 10pp 897ndash902 2010
[43] C Xing A L Sestak J A Kelly et al ldquoLocalization andreplication of the systemic lupus erythematosus linkage signalat 4p16 interaction with 2p11 12q24 and 19q13 in EuropeanAmericansrdquo Human Genetics vol 120 no 5 pp 623ndash631 2007
[44] T F Wong K Yoshinaga Y Monma K Ito H NiikuraS Nagase et al ldquoAssociation of KEAP1 and NRF2 geneticmutations and polymorphisms with endometrioid endometrialadenocarcinoma survivalrdquo International Journal of Gynecologi-cal Cancer vol 21 no 8 pp 1428ndash1435 2011
[45] M von Otter S Landgren S Nilsson et al ldquoNRF2-encodingNFE2L2 haplotypes influence disease progression but not riskin Alzheimerrsquos disease and age-related cataractrdquoMechanisms ofAgeing and Development vol 131 no 2 pp 105ndash110 2010
[46] S Tsang Z Sun B Luke et al ldquoA comprehensive SNP-basedgenetic analysis of inbred mouse strainsrdquoMammalian Genomevol 16 no 7 pp 476ndash480 2005
[47] C M Wade E J Kulbokas III A W Kirby et al ldquoThe mosaicstructure of variation in the laboratory mouse genomerdquoNaturevol 420 no 6915 pp 574ndash578 2002
[48] T Wiltshire M T Pletcher S Batalov et al ldquoGenome-widesingle-nucleotide polymorphism analysis defines haplotypepatterns in mouserdquo Proceedings of the National Academy ofSciences of the United States of America vol 100 no 6 pp 3380ndash3385 2003
[49] K A Frazer E Eskin H M Kang et al ldquoA sequence-basedvariation map of 827 million SNPs in inbred mouse strainsrdquoNature vol 448 no 7157 pp 1050ndash1053 2007
[50] T P Maddatu S C Grubb C J Bult andM A Bogue ldquoMousephenome database (MPD)rdquo Nucleic Acids Research vol 40 ppD887ndashD894 2007
[51] A Kirby H M Kang C M Wade et al ldquoFine mapping in 94inbred mouse strains using a high-density haplotype resourcerdquoGenetics vol 185 no 3 pp 1081ndash1095 2010
[52] H Y Cho A E Jedlicka S P M Reddy L Y Zhang T WKensler and S R Kleeberger ldquoLinkage analysis of susceptibilityto hyperoxia NRF2 is a candidate generdquo American Journal ofRespiratory Cell and Molecular Biology vol 26 no 1 pp 42ndash512002
[53] H Y Cho A E Jedlicka S P M Reddy et al ldquoRole of NRF2in protection against hyperoxic lung injury in micerdquo AmericanJournal of Respiratory Cell and Molecular Biology vol 26 no 2pp 175ndash182 2002
[54] T Shibata T Ohta K I Tong et al ldquoCancer related mutationsin NRF2 impair its recognition by KEAP1-Cul3 E3 ligase andpromote malignancyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 36 pp13568ndash13573 2008
24 Oxidative Medicine and Cellular Longevity
[55] A Singh V Misra R K Thimmulappa et al ldquoDysfunctionalKEAP1-NRF2 interaction in non-small-cell lung cancerrdquo PLoSMedicine vol 3 no 10 article e420 2006
[56] T Ohta K Iijima M Miyamoto et al ldquoLoss of KEAP1 functionactivates NRF2 and provides advantages for lung cancer cellgrowthrdquo Cancer Research vol 68 no 5 pp 1303ndash1309 2008
[57] B Padmanabhan K I Tong T Ohta et al ldquoStructural basisfor defects of KEAP1 activity provoked by its point mutationsin lung cancerrdquoMolecular Cell vol 21 no 5 pp 689ndash700 2006
[58] T Shibata A Kokubu M Gotoh et al ldquoGenetic alteration ofKEAP1 confers constitutive NRF2 activation and resistance tochemotherapy in gallbladder cancerrdquoGastroenterology vol 135no 4 pp 1358e4ndash1368e4 2008
[59] N J Yoo H R Kim Y R Kim C H An and S H LeeldquoSomatic mutations of the KEAP1 gene in common solidcancersrdquo Histopathology vol 60 no 6 pp 943ndash952 2012
[60] Y Hu Y Ju D Lin Z Wang Y Huang S Zhang et alldquoMutation of the NRF2 gene in non-small cell lung cancerrdquoMolecular Biology Reports vol 39 no 4 pp 4743ndash4747 2012
[61] Y R Kim J EOhM S Kim et al ldquoOncogenicNRF2mutationsin squamous cell carcinomas of oesophagus and skinrdquo TheJournal of Pathology vol 220 no 4 pp 446ndash451 2010
[62] T Shibata A Kokubu S Saito et al ldquoNRF2 mutation confersmalignant potential and resistance to chemoradiation therapyin advanced esophageal squamous cancerrdquo Neoplasia vol 13pp 864ndash873 2011
[63] H Sasaki M Shitara K Yokota Y Hikosaka S MoriyamaM Yano et al ldquoIncreased NRF2 gene (NFE2L2) copy numbercorrelates with mutations in lung squamous cell carcinomasrdquoMolecular Medicine Reports vol 6 no 2 pp 391ndash394 2012
[64] H Sasaki M Shitara K Yokota Y Hikosaka S Moriyama MYano et al ldquoRagD gene expression andNRF2mutations in lungsquamous cell carcinomasrdquo Oncology Letters vol 4 pp 1167ndash1170 2012
[65] A Singh M Bodas N Wakabayashi F Bunz and S BiswalldquoGain of NRF2 function in non-small-cell lung cancer cellsconfers radioresistancerdquo Antioxidants and Redox Signaling vol13 no 11 pp 1627ndash1637 2010
[66] T Yamadori Y Ishii SHommaYMorishimaKKurishimaKItoh et al ldquoMolecular mechanisms for the regulation of NRF2-mediated cell proliferation in non-small-cell lung cancersrdquoOncogene vol 31 pp 4768ndash4777 2012
[67] A K Bauer H Y Cho L Miller-Degraff C Walker K HelmsJ Fostel et al ldquoTargeted deletion of NRF2 reduces urethane-induced lung tumor development in micerdquo PLoS ONE vol 6no 10 Article ID e26590 2011
[68] H Y Cho and S R Kleeberger ldquoNRF2 protects against airwaydisordersrdquo Toxicology and Applied Pharmacology vol 244 no1 pp 43ndash56 2010
[69] R Wang J An F Ji H Jiao H Sun and D Zhou ldquoHyperme-thylation of the KEAP1 gene in human lung cancer cell linesand lung cancer tissuesrdquo Biochemical and Biophysical ResearchCommunications vol 373 no 1 pp 151ndash154 2008
[70] P Zhang A Singh S Yegnasubramanian et al ldquoLoss ofkelch-like ECH-associated protein 1 function in prostate cancercells causes chemoresistance and radioresistance and promotestumor growthrdquoMolecular Cancer Therapeutics vol 9 no 2 pp336ndash346 2010
[71] N Hanada T Takahata Q Zhou et al ldquoMethylation of theKEAP1 gene promoter region in human colorectal cancerrdquoBMCCancer vol 12 article 66 2012
[72] L A Muscarella R Barbano V DrsquoAngelo et al ldquoRegulationof KEAP1 expression by promoter methylation in malignantgliomas and association with patientrsquos outcomerdquo Epigeneticsvol 6 no 3 pp 317ndash325 2011
[73] S Yu T O Khor K L Cheung et al ldquoNRF2 expressionis regulated by epigenetic mechanisms in prostate cancer ofTRAMP micerdquo PLoS ONE vol 5 no 1 Article ID e8579 2010
[74] T O Khor Y Huang T Y Wu L Shu J Lee and A N KongldquoPharmacodynamics of curcumin as DNA hypomethylationagent in restoring the expression of NRF2 via promoter CpGsdemethylationrdquo Biochemical Pharmacology vol 82 no 9 pp1073ndash1078 2011
[75] I M Fingerman L McDaniel X Zhang et al ldquoNCBI epige-nomics a new public resource for exploring epigenomic datasetsrdquo Nucleic Acids Research vol 39 supplemen 1 pp D908ndashD912 2011
[76] I M Fingerman X Zhang W Ratzat N Husain R F Cohenand G D Schuler ldquoNCBI epigenomics whatrsquos new for 2013rdquoNucleic Acids Research vol 41 no 1 pp D221ndashD225 2013
[77] J H Lee T O Khor L Shu Z Y Su F Fuentes andA N Kong ldquoDietary phytochemicals and cancer preventionNRF2 signaling epigenetics and cell death mechanisms inblocking cancer initiation and progressionrdquo Pharmacology ampTherapeutics vol 137 no 2 pp 153ndash171 2012
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Disease Markers
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OncologyJournal of
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Oxidative Medicine and Cellular Longevity
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18 Oxidative Medicine and Cellular Longevity
Table5Con
tinued
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs247275247
75704961
GA
(CT)
51015840Flankingminus320
NC
GG
GG
GG
GG
GG
GG
AG
GG
Grs247519047
75704922
Gdel(Cdel)
51015840Flankingminus281
NC
Grs27978309
(rs51915758)
75704887
GA
C(C
TG
)51015840Flankingminus246
NC
GA
AG
AA
GG
AA
GA
GA
CC
G
rs218139102
75704879
CA(G
T)
51015840Flankingminus238
NC
CC
CC
CC
CC
CC
CC
CC
AC
Crs251990355
75704870
TC(A
G)
51015840Flankingminus229
NC
TT
TT
TT
TT
TT
TT
TT
TC
Trs238746955
75704794
AG
(TC)
51015840Flankingminus153
NC
AA
AA
AA
AA
AA
AA
AA
GA
Ars221664
405
75704786
AG
(TC)
51015840Flankingminus145
NC
AA
AA
AA
AA
AA
AA
AA
AG
Ars217054035
75704766
ndash75704767
GAAadd(TTC
add)
51015840Flankingminus125minus126
NC
GA
rs248182931
75704759ndash75704760
Gadd(C
add)
51015840Flankingminus118
minus119
NC
AGrs217197904
75704744
GA
(CT)
51015840Flankingminus103
(+)S
p1[23]
GA
AG
AA
GG
AA
GA
AA
GG
Ars2644
9364975704704
Gdel(Cdel)
51015840Flankingminus63
NC
Grs2404
81112
757046
82CT(G
A)
51015840Flankingminus41
NC
CC
CC
CC
CC
CC
CC
CC
CT
Crs27978310
75704617
AG
(TC)
UTR
-525
NC
AA
AA
AA
AA
AA
AA
AA
GG
Ars27978311
75704610
CG(G
C)
UTR
-532
NC
GG
GG
GG
GG
GG
GG
GG
GG
Crs27978312
757046
05GA
(CT)
UTR
-537
NC
GA
AG
AA
GG
AA
GA
GA
GG
Grs214784220
757044
99AG
(TC)
UTR
-5143
NC
AA
AA
AA
AA
AA
AA
AA
GG
Ars244146318
757044
98GT
(CA
)UTR
-5144
NC
GG
GG
GG
GG
GG
GG
GG
GT
Grs27978313
757044
97GC
(CG
)UTR
-5145
NC
GC
CG
CC
GC
CC
GC
CC
CC
Crs215431944
757044
93CT(G
A)
UTR
-5149
NC
CC
CC
CC
CC
CC
CC
CC
TC
Crs257015697
757044
49GA
(CT)
UTR
-5193
NC
GG
GG
GG
GG
GG
GG
GG
GA
Grs252234782
757044
19GT
(CA
)UTR
-5223
NC
Grs1346
0861
75679262
AG
(TC)
Exon
244
6Cn
sF71L
AG
GA
GG
AG
GG
AG
AG
GG
Grs1346
0859
75679202
GA
(CT)lowast
Exon
2506
CsH91=
GA
AG
AA
GG
AA
GA
GA
GG
Grs27978436
75679187
GA
(CT)lowast
Exon
2521
CsT9
6=G
GG
GG
GG
AG
GG
GG
GA
GG
rs226131070
75679178
GA
(CT)
Exon
2530
CsS99=
GG
GG
GG
GG
GG
GG
GG
GA
Grs227683834
75678576
AT
(TA
)Ex
on3
547
CnsV
105D
AA
AA
AA
AA
AA
AA
AA
AT
Ars257321187
75678506
TC(A
G)
Exon
3617
CsP128=
TT
TT
TT
TC
TT
TT
TT
TT
Trs27978444
75678500
TA(A
T)
Exon
3623
CsV130=
TT
TT
TT
TA
TT
TT
TT
AT
Trs227743136
75677686
GA
(CT)
Exon
4662
CsH143=
GG
GG
GG
GG
GG
GG
GG
GA
Grs215327202
75677664
CG(G
C)
Exon
4684
CnsA
151P
CC
CC
CC
CG
CC
CC
CC
CC
Crs247539755
75677663
GA
(CT)
Exon
4685
CnsA
151V
GG
GG
GG
GA
GG
GG
GG
GG
Grs233164
668
75677640
TC(A
G)
Exon
4708
CnsN
159D
TT
TT
TT
TT
TT
TT
TT
TC
T
rs2340
95816
75677162ndash75677160
GCT
del
(AGCdel)
Exon
5826minus
828
Cds-in
delQ
198
GCT
Oxidative Medicine and Cellular Longevity 19
Table5Con
tinued
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs27978452
75677145
CT(G
A)
Exon
5843
CnsA
204T
CC
CC
CC
CC
CC
CC
CC
CC
T
rs225047937
75677086ndash75677078
GAG
ATCG
ATdel
(ATG
CATC
TCdel)
Exon
5902minus
910
Cds-in
del
(S224I225S226)
GAG
ATC
GAT
rs241469868
75676998
TA(G
T)
Exon
5990
CnT2
53S
TT
TT
TT
TT
TT
TT
TT
TA
Trs221602571
75676923
AC
(TG
)Ex
on5
1065
CnsS
278A
AA
AA
AA
AA
AA
AA
AA
AC
Ars252576472
75676876
GA
(CT)
Exon
51112
CsS293=
GG
GG
GG
GG
GG
GG
GG
GA
Grs250802933
75676841
TC(A
G)
Exon
5114
7Cn
sD305G
TT
TT
TT
TT
TT
TT
TT
TC
Trs225425698
75676792
CT(G
A)
Exon
5119
6Cs
P321=
CC
CC
CC
CC
CC
CC
CC
CT
Crs13459064
75676732
CT(G
A)
Exon
51256
CsT3
41=
CT
TC
TT
CT
TT
CT
CT
TC
Crs27978453
75676717
CT(G
A)
Exon
51271
CsA346=
CC
CC
CC
CC
CC
CC
CC
TC
Crs214335034
75676636
AG
(TC)
Exon
51352
CsD373=
AA
AA
AA
AA
AA
AA
AA
AG
Ars24958364
4756766
03AC
(TG
)Ex
on5
1385
CsP3
84=
AA
AA
AA
AA
AA
AA
AA
AC
Ars27978454
75676592
GA
(CT)
Exon
51396
CnsP
388L
GG
GG
GG
GG
GG
GG
GG
AG
Grs215384328
75676589
GC
(CG
)Ex
on5
1399
CnsA
389G
GG
GG
GG
GG
GG
GG
GG
GC
Grs251728286
75676571
GA
(CT)
Exon
51417
CnsT
395I
Grs247602334
75676567
TC(A
G)
Exon
51421
CsV396=
TT
TT
TT
TT
TT
TT
TT
TC
Trs234216231
75676530
TC(A
G)
Exon
51458
CnsM
409V
TT
TT
TT
TT
TT
TT
TT
TC
Trs212904337
75676526
CT(G
A)
Exon
51462
CnsR
410H
CC
CC
CC
CC
CC
CC
CC
CT
Crs252650779
75676522
TC(A
G)
Exon
51466
CsE4
11=
TT
TT
TT
TT
TT
TT
TT
TC
Trs231273560
75676516
TC(A
G)
Exon
51472
CsQ413=
TT
TT
TT
TT
TT
TT
TT
TC
Trs257886949
756764
13GT
(CA
)Ex
on5
1575
CsR4
48=
GG
GG
GG
GG
GG
GG
GG
GT
Grs241537608
756764
04GC
(CG
)Ex
on5
1584
CnsL
451V
GG
GG
GG
GG
GG
GG
GG
GC
Grs227619071
75676312
TC(A
G)
Exon
51676
CsQ481=
TT
TT
TT
TT
TT
TT
TT
TC
Trs258913831
75676290
AG
(TC)
Exon
51698
CsL4
89=
AA
AA
AA
AA
AA
AA
AA
AG
Ars225593319
75676126
AT
(TA
)Ex
on5
1862
CnsH
543Q
Ars4223233
75676105
GA
(CT)
Exon
51883
CsS550=
GG
GG
GG
GA
GG
GG
GG
GG
Grs4223232
75676032
GT
(CA
)Ex
on5
1956
CnsL
575M
Grs4223231
75675816
CG
UTR
-32172
NC
GG
GG
GG
GG
GG
GG
GG
GC
Grs1346
860
75675682
AT
UTR
-32306
NC
ASequ
ence
varia
tions
inmou
seNr
f2wereob
tained
from
Mou
sePh
enom
eDatabase(http
ph
enom
ejaxorgSNP)
andNCB
ISNPdatabase
(http
wwwncbinlm
nihgovSNP)N
CBIreference
sequ
ence
isMus
Musculusstra
inC5
7BL6J
chromosom
e2GRC
m38p1(NC
0000
687
GI37209910875704641ndash756755192
9123
bp)To
tal968
genetic
mutations
arerepo
rted
inNr
f2gene
and5k
bup
stream
(ge75704
642minus
1)and2k
bdo
wnstre
am(le75675512)sequences
asof
Janu
ary2003A
llSN
Psandallelesa
representedas
geno
miccontig
(reversedsequ
ence
indicated)E
xon175704
641ndash75704364(278
bpT
TS=75704
408)
exon
275679431ndash756791
65(267
bp)exon
375678579ndash75678490(90b
p)exon475677713ndash756775
46(168
bp)andexon
57567718
4ndash75678849
(1666
bp)Proteinam
inoacid
(aa)
resid
uesinNP00
61552
(597
aa)Cs
cod
ing-syno
nymou
sCn
scoding
-non
syno
nymou
sAminoacid
A-alanineD-aspartic
acidE
-glutamicacidF-phenylalanineG
-glycineH
-histidineI-iso
leucineL-leu
cineand
M-m
ethion
ineN-
asparagineP-prolin
eQ-glutamineR-arginineS-serineT-threon
ineandV-valin
elowastErrorsin
databasesfi
xedNC
notcon
firmed
20 Oxidative Medicine and Cellular Longevity
Table 6 NRF2 somatic mutations revealed in various human cancers
Domain Amino acid residues DNA mutation Cancer types (cases) ReferencesLocus Wild type Mutant
DLG motif
24 W (Trp) C (Cys) c72GgtCGgtT NSCLC neck ESC [54 62]K (Lys) c72TgtC ESC [62]
26 Q (Gln) E (Glu) c76CgtG NSCLC ESC [54 62]
27 D (Asp) G (Gly)lowast c80GgtA NSCLC [60]Y (Tyr) c79GgtT ESC [61]
28 I (Ile) T (Thr) c83CgtT NSCLC [54]
29 D (Asp) G (Gly) c86AgtG Head and neck ESC [54 62]H (His) c85GgtC NSCLC laynx [60 61]
30 L (Leu) F (Phe) c88CgtT NSCLC ESC [54 62]
31 G (Gly) A (Ala) c92GgtC NSCLC ESC skin [54 60ndash62]
32 V (Val) T (Thr) c95TgtG NSCLC [54]del c93 95delAGT ESC [61]
33ndash36 S-R-E-V S-R-E-V-S-R-E-Vlowast c97 108dupAGTCGAGCCGTA ESC [61]
34 R (Arg) Q (Gln) c101GgtA NCSCL [54 60 61]P (Pro) c101GgtC NSCLC [63]
ETGF motif
75 Q (Gln) H (His) c225AgtC Head and neck ESC [54 62]
77 D (Asp)
V (Val) c230AgtT NSCLC ESC [54 60 62]G (Gly) c230AgtG ESC [62]A (Ala) c230AgtC NSCLC [61]N (Asn) c229GgtA Larynx [61]
78 E (Glu) K (Lys) c232GgtA NSCLC ESC [54 62]
79 E (Glu)
K (Lys) c235GgtA NSCLC ESC [54 61 62]Q (Gln) c235GgtC NSCLC ESC [54 60ndash62]G (Gly) c236AgtG Larynx [61]E-E c234 236dupAGA ESC [61]
80 T (Thr) K (Lys) c239CgtACgtG NSCLC ESC [54 61 62]
80 T (Thr)P (Phe) c238AgtC ESC [62]I (Ile) c239CgtT Head and neck [54]A (Ala) c238AgtG NSCLC [63]
81 G (Gly) V (Val) c242GgtT ESC [61]D (Asp) c242GgtA NSCLC ESC [60ndash62]
82 E (Glu)
D (Asp) c246AgtT ESC oral cancer cell line [54 62]G (Gly) c245AgtG NSCLC [54]Q (Gln)lowast c244GgtC NSCLC ESC [60 61]V (Val)lowast c245AgtT ESC [61]
83 F (Phe) L (Leu)lowast c247TgtC NSCLC [60]NSCLC non-small cell lung cancer ESC esophageal squamous cancer and lowasterrors in reference fixed Number of cases 82 NSCLC and 10 ESC in [62] 125NSCLC 70 ESC 23 larynx and 17 skin in [61] 103 NSCLC and 12 head and neck in [54] 90 NSCLC in [63] 103 NSCLC in [60]
be involved in squamous lung cancer cell proliferation [64]suggesting that the mutation is functional and overcomesKEAP1 inhibition Singh et al [65] determined in vitrothat RNAi-mediated depletion of NRF2 in lung cancer cellsenhanced ROS production and susceptibility to cell deathby ionizing radiation These studies support the concept thatelevated NRF2 and ARE responsiveness provides cancer cellswith proliferative advantage for malignant transformation
and undue protection from anti-cancer therapy Onco-genic epidermal growth factor receptor (EGFR) signaling isrecently found to be critical in NRF2-mediated proliferationof NSCLC cells [66]
Collectively ldquogain of functionrdquo mutations in NRF2 thatreduce KEAP1 recognition are suggested to be predictivemarkers for poor responsiveness to chemotherapy and radi-ation therapy Although NRF2-mediated cellular defense
Oxidative Medicine and Cellular Longevity 21
processes are essential in the initiation stage enhancedNRF2-ARE activity in advanced stages of cancer developmentmay create a favorable intracellular environment for tumorcell growth and survival [67 68] In this context NRF2may be a potential molecular target for the treatment ofradio-resistance cancers especially those that have ldquoloss offunctionrdquo mutations in EGFR KRAS or KEAP1 as well asldquogain of functionrdquo mutations in NRF2
5 Epigenetic Alterations of NF-E2-RelatedFactor 2
Epigenetic modifications are alterations of molecules inter-acting with genes without changes to the primary DNAsequence They include post-translational modification ofhistones DNA methylation events chromatin conforma-tional changes and alterations to noncoding regulatoryRNAs Epigenetic alterations are stable and often inheritablebut are reversible and may affect expression of the geneDysregulation or defects in epigenetic processes particularlyhypermethylation of tumor suppressor gene promoters (egCpG islands) or histone modifications are thought to beassociated with carcinogenesis Investigators have reportedhypermethylation in CpG islands of KEAP1 which wereassociated with reduced KEAP1 expression in human cancersfrom lung prostate colon and so forth [69ndash72] Similar tosomatic mutations epigenetic changes on KEAP1 impairedthe function of its encoded protein leading to constitutiveNRF2 activation
Supporting the role of ldquopathogenic mutationsrdquo in NRF2expression of Nrf2 and downstream Nqo1 was suppressedin prostate tumors of mice (transgenic adenocarcinomaof mouse prostate TRAMP) Among 15 promoter CpGislands located between minus942 and minus654 (cminus1175 cminus1132and cminus1059 cminus887 gap in cminus1131 cminus1060 see Figure 3)hypermethylation of the first 5 CpG islands (minus942 minus899 andcminus1175 cminus1132) was significantly associated with tumorige-nesis [73] Moreover treatment with inhibitors for DNAmethyltransferase and histone deacetylase restored Nrf2expression in these tumor cells [73] A dietary phytochemicalcurcumin known as a DNA hypomethylation agent restoredepigenetically silent Nrf2 expression through CpG demethy-lation in carcinogen-induced mouse tumor cells [74]
The whole genome epigenetic datasets for 5 species arepublicly accessible at NCBI Epigenomics [75 76]The humanNRF2 epigenome of primary cells (breast penis) and H1stem cell line as well as mouse Nrf2 CpG island methylationdata for sperm blood and cerebellum are currently avail-able (httpwwwncbinlmnihgovepigenomics) Althoughno direct evidence of disease-associated epigenetic modu-lation has been identified in human NRF2 various phyto-chemical NRF2 agonists such as sulforaphane and curcuminhave shown their roles in DNA methylation and histonemodification (see reviews by Lee and colleagues eg [77])Taken together epigenetic modifications of theNRF2KEAP1axle are predicted to cause dysregulation of ARE-mediatedcellular defense leading to deleterious health effects and
phytochemical antioxidants as epigenetic modulators forNRF2 are suggested to be useful in cancer prevention
6 Conclusions
NRF2 is evolutionally conserved with high-sequence homol-ogy in many species However it is a highly mutable geneand numerous genetic variants have been discovered inhuman ethnic groups Importantly certain SNPs or haplo-types have been identified in various diseases as ldquoat-riskrdquoalleles and are related to functional alterations In additionto genetic variations multiple somatic mutations identifiedin the KEAP1 recognition domain of NRF2 in cancer cellshave been found to be oncogenic due to dysregulation ofNRF2 homeostasis by its excess ldquogain of functionrdquo Epigeneticalteration of theNRF2 is under investigation and is predictedto have pathogenic influences as learned from mouse andphytochemical agonist studies Continuous updates of Nrf2allelic variants in inbred mouse strains will provide a usefultool for effective experimental designs formodels of oxidativedisorders to provide insight into the disease mechanisms andintervention strategies
Abbreviations
AD Alzheimerrsquos diseaseALI acute lung injuryARE antioxidant response elementbZIP basic leucine zippercds coding DNA sequencesCI confidence intervalCOPD chronic obstructive pulmonary diseaseESC esophageal squamous cancerFEV1 forced expiratory volume in one second
Gbp giga base pairsGST glutathione S-transferaseGWAS genome-wide association studyHGVS Human Genome Variation SocietyHO-1 heme oxygenase-1119867 119901119910119897119900119903119894 119867119890119897119894119888119900119887119886119888119905119890119903 119901119910119897119900119903119894KEAP1 Kelch-like ECH activating protein 1MPD Mouse Phenome DatabaseMRP multidrug resistance proteinNCBI National Center for Biotechnology InformationNeh NRF2-ECH homologyNfe2l2 nuclear factor (erythroid-derived 2)-like 2Nrf2 NF-E2-related factor 2NSCLC nonsmall cell lung cancerNQO1 NAD(P)Hquinone oxidoreductase 1OR odds ratioPD Parkinsonrsquos diseasePM particulate matterROS reactive oxygen speciesSLE systemic lupus erythematosusSNP single nucleotide polymorphismSOD superoxide dismutaseURT untranslated region
22 Oxidative Medicine and Cellular Longevity
Disclosure
Authorrsquos contribution to the Work was done as part of theAuthorrsquos official duties as a NIH employee and is a Work ofthe United States Government Therefore copyright may notbe established in the United States
Conflict of Interests
The author declares that there is no conflict of interests
Acknowledgments
The research related to this paper was supported by theIntramural Research Program of the National Institutes ofHealth of the National Institute of Environmental HealthSciences (NIEHS) Drs Steven Kleeberger and StephanieLondon at the NIEHS provided excellent critical review ofthis paper The author thanks Mrs Jacqui Marzec for herhelpful comments and English editing
References
[1] J Y Chan M C Cheung P Moi K Chan and Y W KanldquoChromosomal localization of the humanNF-E2 family of bZIPtranscription factors by fluorescence in situ hybridizationrdquoHuman Genetics vol 95 no 3 pp 265ndash269 1995
[2] K Itoh K Igarashi N Hayashi M Nishizawa and MYamamoto ldquoCloning and characterization of a novel erythroidcell-derived CNC family transcription factor heterodimerizingwith the small Maf family proteinsrdquo Molecular and CellularBiology vol 15 no 8 pp 4184ndash4193 1995
[3] W O Osburn and T W Kensler ldquoNRF2 signaling an adaptiveresponse pathway for protection against environmental toxicinsultsrdquoMutation Research vol 659 no 1-2 pp 31ndash39 2008
[4] D Papp K Lenti D Modos D Fazekas Z Dul D Tureiet al ldquoThe NRF2-related interactome and regulome containmultifunctional proteins and fine-tuned autoregulatory loopsrdquoFEBS Letters vol 586 no 13 pp 1795ndash1802 2012
[5] X Wang D J Tomso B N Chorley et al ldquoIdentificationof polymorphic antioxidant response elements in the humangenomerdquo Human Molecular Genetics vol 16 no 10 pp 1188ndash1200 2007
[6] D Malhotra E Portales-Casamar A Singh et al ldquoGlobalmapping of binding sites for NRF2 identifies novel targets incell survival response through ChIP-Seq profiling and networkanalysisrdquo Nucleic Acids Research vol 38 no 17 pp 5718ndash57342010
[7] K Chan R Lu J C Chang and Y W Kan ldquoNRF2 a memberof the NFE2 family of transcription factors is not essential formurine erythropoiesis growth and developmentrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 93 no 24 pp 13943ndash13948 1996
[8] K Itoh T Chiba S Takahashi et al ldquoAn NRF2small Mafheterodimer mediates the induction of phase II detoxifyingenzyme genes through antioxidant response elementsrdquo Bio-chemical and Biophysical Research Communications vol 236no 2 pp 313ndash322 1997
[9] FMartin J M vanDeursen R A Shivdasani CW Jackson AG Troutman and P A Ney ldquoErythroid maturation and globin
gene expression inmicewith combined deficiency ofNF-E2 andNrf-2rdquo Blood vol 91 no 9 pp 3459ndash3466 1998
[10] K Itoh N Wakabayashi Y Katoh et al ldquoKEAP1 repressesnuclear activation of antioxidant responsive elements by NRF2through binding to the amino-terminal Neh2 domainrdquo Genesand Development vol 13 no 1 pp 76ndash86 1999
[11] A Kobayashi M I Kang H Okawa et al ldquoOxidative stresssensor KEAP1 functions as an adaptor for Cul3-based E3 ligaseto regulate proteasomal degradation of NRF2rdquo Molecular andCellular Biology vol 24 no 16 pp 7130ndash7139 2004
[12] K Taguchi H Motohashi and M Yamamoto ldquoMolecularmechanisms of the KEAP1-NRF2 pathway in stress responseand cancer evolutionrdquo Genes to Cells vol 16 no 2 pp 123ndash1402011
[13] K Mukaigasa L T Nguyen L Li H Nakajima M Yamamotoand M Kobayashi ldquoGenetic evidence of an evolutionarilyconserved role for NRF2 in the protection against oxidativestressrdquoMolecular and Cellular Biology vol 32 no 21 pp 4455ndash4461 2012
[14] JMaher andMYamamoto ldquoThe rise of antioxidant signalingmdashthe evolution and hormetic actions of NRF2rdquo Toxicology andApplied Pharmacology vol 244 no 1 pp 4ndash15 2010
[15] S C Lo X Li M T Henzl L J Beamer and M HanninkldquoStructure of the KEAP1NRF2 interface provides mechanisticinsight intoNRF2 signalingrdquoThe EMBO Journal vol 25 no 15pp 3605ndash3617 2006
[16] V A McKusick ldquoMendelian inheritance in man and its onlineversion OMIMrdquo American Journal of Human Genetics vol 80no 4 pp 588ndash604 2007
[17] B Yalcin D J Adams J Flint and T M Keane ldquoNext-generation sequencing of experimental mouse strainsrdquo Mam-malian Genome vol 23 no 9-10 pp 490ndash498 2012
[18] D R Bentley S Balasubramanian H P Swerdlow et alldquoAccurate whole human genome sequencing using reversibleterminator chemistryrdquo Nature vol 456 no 7218 pp 53ndash592008
[19] P S G Chain D V Grafham R S Fulton et al ldquoGenomeproject standards in a new era of sequencingrdquo Science vol 326no 5950 pp 236ndash237 2009
[20] RHWaterstonK Lindblad-Toh E Birney J Rogers J F AbrilP Agarwal et al ldquoInitial sequencing and comparative analysis ofthe mouse genomerdquo Nature vol 420 pp 520ndash562 2002
[21] T M Keane L Goodstadt P Danecek M A White K WongB Yalcin et al ldquoMouse genomic variation and its effect onphenotypes and gene regulationrdquoNature vol 477 pp 289ndash2942011
[22] T Yamamoto K Yoh A Kobayashi et al ldquoIdentification ofpolymorphisms in the promoter region of the human NRF2generdquo Biochemical and Biophysical Research Communicationsvol 321 no 1 pp 72ndash79 2004
[23] J M Marzec J D Christie S P Reddy et al ldquoFunctionalpolymorphisms in the transcription factor NRF2 in humansincrease the risk of acute lung injuryrdquo The FASEB Journal vol21 no 9 pp 2237ndash2246 2007
[24] H Fukushima-Uesaka Y Saito K Maekawa et al ldquoGeneticvariations and haplotype structures of transcriptional factorNRF2 and its cytosolic reservoir protein KEAP1 in JapaneserdquoDrug Metabolism and Pharmacokinetics vol 22 no 3 pp 212ndash219 2007
[25] E J Cordova R Velazquez-Cruz F Centeno V Baca and LOrozco ldquoThe NRF2 gene variant -653GA is associated with
Oxidative Medicine and Cellular Longevity 23
nephritis in childhood-onset systemic lupus erythematosusrdquoLupus vol 19 no 10 pp 1237ndash1242 2010
[26] M von Otter S Landgren S Nilsson et al ldquoAssociation ofNRF2-encoding NFE2L2 haplotypes with Parkinsonrsquos diseaserdquoBMCMedical Genetics vol 11 no 1 article 36 2010
[27] JMHartikainenM TengstromVMKosmaV L Kinnula AMannermaa andY Soini ldquoGenetic polymorphisms and proteinexpression of NRF2 and sulfiredoxin predict survival outcomesin breast cancerrdquo Cancer Research vol 72 pp 5537ndash5546 2012
[28] T Arisawa T Tahara T Shibata et al ldquoThe relationshipbetween Helicobacter pylori infection and promoter polymor-phism of the NRF2 gene in chronic gastritisrdquo InternationalJournal of Molecular Medicine vol 19 no 1 pp 143ndash148 2007
[29] T Arisawa T Tahara T Shibata et al ldquoThe influence ofpromoter polymorphism of nuclear factor-erythroid 2-relatedfactor 2 gene on the aberrant DNA methylation in gastricepitheliumrdquo Oncology Reports vol 19 no 1 pp 211ndash216 2008
[30] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene pro-moter polymorphism and gastric carcinogenesisrdquo Hepato-Gastroenterology vol 55 no 82-83 pp 750ndash754 2008
[31] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene promoterpolymorphism is associated with ulcerative colitis in a Japanesepopulationrdquo Hepato-Gastroenterology vol 55 no 82-83 pp394ndash397 2008
[32] C C Hua L C Chang J C Tseng C M Chu Y C Liu andW B Shieh ldquoFunctional haplotypes in the promoter region oftranscription factor NRF2 in chronic obstructive pulmonarydiseaserdquo Disease Markers vol 28 no 3 pp 185ndash193 2010
[33] S O Shaheen R B Newson S M Ring M J Rose-ZerilliJ W Holloway and A J Henderson ldquoPrenatal and infantacetaminophen exposure antioxidant gene polymorphismsand childhood asthmardquo The Journal of Allergy and ClinicalImmunology vol 126 no 6 pp 1141e7ndash1148e7 2010
[34] H Masuko T Sakamoto Y Kaneko et al ldquoLower FEV1 innon-COPD nonasthmatic subjects association with smokingannual decline in FEV1 total IgE levels and TSLP genotypesrdquoInternational Journal of Chronic Obstructive Pulmonary Diseasevol 6 pp 181ndash189 2011
[35] C P Guan M N Zhou A E Xu et al ldquoThe susceptibilityto vitiligo is associated with NF-E2-related factor 2 (NRF2)gene polymorphisms a study on Chinese Han populationrdquoExperimental Dermatology vol 17 no 12 pp 1059ndash1062 2008
[36] J Bouligand O Cabaret M Canonico et al ldquoEffect of NFE2L2genetic polymorphism on the association between oral estrogentherapy and the risk of venous thromboembolism in post-menopausal womenrdquo Clinical Pharmacology amp Therapeuticsvol 89 no 1 pp 60ndash64 2011
[37] D S OrsquoMahony B J Glavan T D Holden C Fong R A BlackG Rona et al ldquoInflammation and immune-related candidategene associations with acute lung injury susceptibility andseverity a validation studyrdquo PLoS ONE vol 7 no 12 Article IDe51104 2012
[38] I Ungvari E Hadadi V Virag et al ldquoRelationship between airpollutionNFE2L2 gene polymorphisms and childhood asthmain aHungarian populationrdquo Journal of CommunityGenetics vol3 no 1 pp 25ndash33 2012
[39] M SiedlinskiD S Postma JMA Boer et al ldquoLevel and courseof FEV1 in relation to polymorphisms inNFE2L2 andKEAP1 inthe general populationrdquo Respiratory Research vol 10 article 732009
[40] C C Hong C B Ambrosone J Ahn et al ldquoGenetic variabilityin iron-related oxidative stress pathways (NRF2 NQ01 NOS3
and HO-1) iron intake and risk of postmenopausal breastcancerrdquo Cancer Epidemiology Biomarkers and Prevention vol16 no 9 pp 1784ndash1794 2007
[41] C Canova C Dunster F J Kelly C Minelli P L Shah CCaneja et al ldquoPM10-induced hospital admissions for asthmaand chronic obstructive pulmonary disease the modifyingeffect of individual characteristicsrdquo Epidemiology vol 23 no 4pp 607ndash615 2012
[42] A J Henderson R B Newson M Rose-Zerilli S M RingJ W Holloway and S O Shaheen ldquoMaternal NRF2 andgluthathione-S-transferase polymorphisms donotmodify asso-ciations of prenatal tobacco smoke exposure with asthma andlung function in school-aged childrenrdquo Thorax vol 65 no 10pp 897ndash902 2010
[43] C Xing A L Sestak J A Kelly et al ldquoLocalization andreplication of the systemic lupus erythematosus linkage signalat 4p16 interaction with 2p11 12q24 and 19q13 in EuropeanAmericansrdquo Human Genetics vol 120 no 5 pp 623ndash631 2007
[44] T F Wong K Yoshinaga Y Monma K Ito H NiikuraS Nagase et al ldquoAssociation of KEAP1 and NRF2 geneticmutations and polymorphisms with endometrioid endometrialadenocarcinoma survivalrdquo International Journal of Gynecologi-cal Cancer vol 21 no 8 pp 1428ndash1435 2011
[45] M von Otter S Landgren S Nilsson et al ldquoNRF2-encodingNFE2L2 haplotypes influence disease progression but not riskin Alzheimerrsquos disease and age-related cataractrdquoMechanisms ofAgeing and Development vol 131 no 2 pp 105ndash110 2010
[46] S Tsang Z Sun B Luke et al ldquoA comprehensive SNP-basedgenetic analysis of inbred mouse strainsrdquoMammalian Genomevol 16 no 7 pp 476ndash480 2005
[47] C M Wade E J Kulbokas III A W Kirby et al ldquoThe mosaicstructure of variation in the laboratory mouse genomerdquoNaturevol 420 no 6915 pp 574ndash578 2002
[48] T Wiltshire M T Pletcher S Batalov et al ldquoGenome-widesingle-nucleotide polymorphism analysis defines haplotypepatterns in mouserdquo Proceedings of the National Academy ofSciences of the United States of America vol 100 no 6 pp 3380ndash3385 2003
[49] K A Frazer E Eskin H M Kang et al ldquoA sequence-basedvariation map of 827 million SNPs in inbred mouse strainsrdquoNature vol 448 no 7157 pp 1050ndash1053 2007
[50] T P Maddatu S C Grubb C J Bult andM A Bogue ldquoMousephenome database (MPD)rdquo Nucleic Acids Research vol 40 ppD887ndashD894 2007
[51] A Kirby H M Kang C M Wade et al ldquoFine mapping in 94inbred mouse strains using a high-density haplotype resourcerdquoGenetics vol 185 no 3 pp 1081ndash1095 2010
[52] H Y Cho A E Jedlicka S P M Reddy L Y Zhang T WKensler and S R Kleeberger ldquoLinkage analysis of susceptibilityto hyperoxia NRF2 is a candidate generdquo American Journal ofRespiratory Cell and Molecular Biology vol 26 no 1 pp 42ndash512002
[53] H Y Cho A E Jedlicka S P M Reddy et al ldquoRole of NRF2in protection against hyperoxic lung injury in micerdquo AmericanJournal of Respiratory Cell and Molecular Biology vol 26 no 2pp 175ndash182 2002
[54] T Shibata T Ohta K I Tong et al ldquoCancer related mutationsin NRF2 impair its recognition by KEAP1-Cul3 E3 ligase andpromote malignancyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 36 pp13568ndash13573 2008
24 Oxidative Medicine and Cellular Longevity
[55] A Singh V Misra R K Thimmulappa et al ldquoDysfunctionalKEAP1-NRF2 interaction in non-small-cell lung cancerrdquo PLoSMedicine vol 3 no 10 article e420 2006
[56] T Ohta K Iijima M Miyamoto et al ldquoLoss of KEAP1 functionactivates NRF2 and provides advantages for lung cancer cellgrowthrdquo Cancer Research vol 68 no 5 pp 1303ndash1309 2008
[57] B Padmanabhan K I Tong T Ohta et al ldquoStructural basisfor defects of KEAP1 activity provoked by its point mutationsin lung cancerrdquoMolecular Cell vol 21 no 5 pp 689ndash700 2006
[58] T Shibata A Kokubu M Gotoh et al ldquoGenetic alteration ofKEAP1 confers constitutive NRF2 activation and resistance tochemotherapy in gallbladder cancerrdquoGastroenterology vol 135no 4 pp 1358e4ndash1368e4 2008
[59] N J Yoo H R Kim Y R Kim C H An and S H LeeldquoSomatic mutations of the KEAP1 gene in common solidcancersrdquo Histopathology vol 60 no 6 pp 943ndash952 2012
[60] Y Hu Y Ju D Lin Z Wang Y Huang S Zhang et alldquoMutation of the NRF2 gene in non-small cell lung cancerrdquoMolecular Biology Reports vol 39 no 4 pp 4743ndash4747 2012
[61] Y R Kim J EOhM S Kim et al ldquoOncogenicNRF2mutationsin squamous cell carcinomas of oesophagus and skinrdquo TheJournal of Pathology vol 220 no 4 pp 446ndash451 2010
[62] T Shibata A Kokubu S Saito et al ldquoNRF2 mutation confersmalignant potential and resistance to chemoradiation therapyin advanced esophageal squamous cancerrdquo Neoplasia vol 13pp 864ndash873 2011
[63] H Sasaki M Shitara K Yokota Y Hikosaka S MoriyamaM Yano et al ldquoIncreased NRF2 gene (NFE2L2) copy numbercorrelates with mutations in lung squamous cell carcinomasrdquoMolecular Medicine Reports vol 6 no 2 pp 391ndash394 2012
[64] H Sasaki M Shitara K Yokota Y Hikosaka S Moriyama MYano et al ldquoRagD gene expression andNRF2mutations in lungsquamous cell carcinomasrdquo Oncology Letters vol 4 pp 1167ndash1170 2012
[65] A Singh M Bodas N Wakabayashi F Bunz and S BiswalldquoGain of NRF2 function in non-small-cell lung cancer cellsconfers radioresistancerdquo Antioxidants and Redox Signaling vol13 no 11 pp 1627ndash1637 2010
[66] T Yamadori Y Ishii SHommaYMorishimaKKurishimaKItoh et al ldquoMolecular mechanisms for the regulation of NRF2-mediated cell proliferation in non-small-cell lung cancersrdquoOncogene vol 31 pp 4768ndash4777 2012
[67] A K Bauer H Y Cho L Miller-Degraff C Walker K HelmsJ Fostel et al ldquoTargeted deletion of NRF2 reduces urethane-induced lung tumor development in micerdquo PLoS ONE vol 6no 10 Article ID e26590 2011
[68] H Y Cho and S R Kleeberger ldquoNRF2 protects against airwaydisordersrdquo Toxicology and Applied Pharmacology vol 244 no1 pp 43ndash56 2010
[69] R Wang J An F Ji H Jiao H Sun and D Zhou ldquoHyperme-thylation of the KEAP1 gene in human lung cancer cell linesand lung cancer tissuesrdquo Biochemical and Biophysical ResearchCommunications vol 373 no 1 pp 151ndash154 2008
[70] P Zhang A Singh S Yegnasubramanian et al ldquoLoss ofkelch-like ECH-associated protein 1 function in prostate cancercells causes chemoresistance and radioresistance and promotestumor growthrdquoMolecular Cancer Therapeutics vol 9 no 2 pp336ndash346 2010
[71] N Hanada T Takahata Q Zhou et al ldquoMethylation of theKEAP1 gene promoter region in human colorectal cancerrdquoBMCCancer vol 12 article 66 2012
[72] L A Muscarella R Barbano V DrsquoAngelo et al ldquoRegulationof KEAP1 expression by promoter methylation in malignantgliomas and association with patientrsquos outcomerdquo Epigeneticsvol 6 no 3 pp 317ndash325 2011
[73] S Yu T O Khor K L Cheung et al ldquoNRF2 expressionis regulated by epigenetic mechanisms in prostate cancer ofTRAMP micerdquo PLoS ONE vol 5 no 1 Article ID e8579 2010
[74] T O Khor Y Huang T Y Wu L Shu J Lee and A N KongldquoPharmacodynamics of curcumin as DNA hypomethylationagent in restoring the expression of NRF2 via promoter CpGsdemethylationrdquo Biochemical Pharmacology vol 82 no 9 pp1073ndash1078 2011
[75] I M Fingerman L McDaniel X Zhang et al ldquoNCBI epige-nomics a new public resource for exploring epigenomic datasetsrdquo Nucleic Acids Research vol 39 supplemen 1 pp D908ndashD912 2011
[76] I M Fingerman X Zhang W Ratzat N Husain R F Cohenand G D Schuler ldquoNCBI epigenomics whatrsquos new for 2013rdquoNucleic Acids Research vol 41 no 1 pp D221ndashD225 2013
[77] J H Lee T O Khor L Shu Z Y Su F Fuentes andA N Kong ldquoDietary phytochemicals and cancer preventionNRF2 signaling epigenetics and cell death mechanisms inblocking cancer initiation and progressionrdquo Pharmacology ampTherapeutics vol 137 no 2 pp 153ndash171 2012
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
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Computational and Mathematical Methods in Medicine
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Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Oxidative Medicine and Cellular Longevity 19
Table5Con
tinued
SNPID
Chr2
locatio
n(bp)
SNPallele
Region
Locatio
nfro
mTS
Sreversed
SNP
allele
Con
sequ
ences
Strains
1 B62
34
56
78
910
1112
1314
1516
17
rs27978452
75677145
CT(G
A)
Exon
5843
CnsA
204T
CC
CC
CC
CC
CC
CC
CC
CC
T
rs225047937
75677086ndash75677078
GAG
ATCG
ATdel
(ATG
CATC
TCdel)
Exon
5902minus
910
Cds-in
del
(S224I225S226)
GAG
ATC
GAT
rs241469868
75676998
TA(G
T)
Exon
5990
CnT2
53S
TT
TT
TT
TT
TT
TT
TT
TA
Trs221602571
75676923
AC
(TG
)Ex
on5
1065
CnsS
278A
AA
AA
AA
AA
AA
AA
AA
AC
Ars252576472
75676876
GA
(CT)
Exon
51112
CsS293=
GG
GG
GG
GG
GG
GG
GG
GA
Grs250802933
75676841
TC(A
G)
Exon
5114
7Cn
sD305G
TT
TT
TT
TT
TT
TT
TT
TC
Trs225425698
75676792
CT(G
A)
Exon
5119
6Cs
P321=
CC
CC
CC
CC
CC
CC
CC
CT
Crs13459064
75676732
CT(G
A)
Exon
51256
CsT3
41=
CT
TC
TT
CT
TT
CT
CT
TC
Crs27978453
75676717
CT(G
A)
Exon
51271
CsA346=
CC
CC
CC
CC
CC
CC
CC
TC
Crs214335034
75676636
AG
(TC)
Exon
51352
CsD373=
AA
AA
AA
AA
AA
AA
AA
AG
Ars24958364
4756766
03AC
(TG
)Ex
on5
1385
CsP3
84=
AA
AA
AA
AA
AA
AA
AA
AC
Ars27978454
75676592
GA
(CT)
Exon
51396
CnsP
388L
GG
GG
GG
GG
GG
GG
GG
AG
Grs215384328
75676589
GC
(CG
)Ex
on5
1399
CnsA
389G
GG
GG
GG
GG
GG
GG
GG
GC
Grs251728286
75676571
GA
(CT)
Exon
51417
CnsT
395I
Grs247602334
75676567
TC(A
G)
Exon
51421
CsV396=
TT
TT
TT
TT
TT
TT
TT
TC
Trs234216231
75676530
TC(A
G)
Exon
51458
CnsM
409V
TT
TT
TT
TT
TT
TT
TT
TC
Trs212904337
75676526
CT(G
A)
Exon
51462
CnsR
410H
CC
CC
CC
CC
CC
CC
CC
CT
Crs252650779
75676522
TC(A
G)
Exon
51466
CsE4
11=
TT
TT
TT
TT
TT
TT
TT
TC
Trs231273560
75676516
TC(A
G)
Exon
51472
CsQ413=
TT
TT
TT
TT
TT
TT
TT
TC
Trs257886949
756764
13GT
(CA
)Ex
on5
1575
CsR4
48=
GG
GG
GG
GG
GG
GG
GG
GT
Grs241537608
756764
04GC
(CG
)Ex
on5
1584
CnsL
451V
GG
GG
GG
GG
GG
GG
GG
GC
Grs227619071
75676312
TC(A
G)
Exon
51676
CsQ481=
TT
TT
TT
TT
TT
TT
TT
TC
Trs258913831
75676290
AG
(TC)
Exon
51698
CsL4
89=
AA
AA
AA
AA
AA
AA
AA
AG
Ars225593319
75676126
AT
(TA
)Ex
on5
1862
CnsH
543Q
Ars4223233
75676105
GA
(CT)
Exon
51883
CsS550=
GG
GG
GG
GA
GG
GG
GG
GG
Grs4223232
75676032
GT
(CA
)Ex
on5
1956
CnsL
575M
Grs4223231
75675816
CG
UTR
-32172
NC
GG
GG
GG
GG
GG
GG
GG
GC
Grs1346
860
75675682
AT
UTR
-32306
NC
ASequ
ence
varia
tions
inmou
seNr
f2wereob
tained
from
Mou
sePh
enom
eDatabase(http
ph
enom
ejaxorgSNP)
andNCB
ISNPdatabase
(http
wwwncbinlm
nihgovSNP)N
CBIreference
sequ
ence
isMus
Musculusstra
inC5
7BL6J
chromosom
e2GRC
m38p1(NC
0000
687
GI37209910875704641ndash756755192
9123
bp)To
tal968
genetic
mutations
arerepo
rted
inNr
f2gene
and5k
bup
stream
(ge75704
642minus
1)and2k
bdo
wnstre
am(le75675512)sequences
asof
Janu
ary2003A
llSN
Psandallelesa
representedas
geno
miccontig
(reversedsequ
ence
indicated)E
xon175704
641ndash75704364(278
bpT
TS=75704
408)
exon
275679431ndash756791
65(267
bp)exon
375678579ndash75678490(90b
p)exon475677713ndash756775
46(168
bp)andexon
57567718
4ndash75678849
(1666
bp)Proteinam
inoacid
(aa)
resid
uesinNP00
61552
(597
aa)Cs
cod
ing-syno
nymou
sCn
scoding
-non
syno
nymou
sAminoacid
A-alanineD-aspartic
acidE
-glutamicacidF-phenylalanineG
-glycineH
-histidineI-iso
leucineL-leu
cineand
M-m
ethion
ineN-
asparagineP-prolin
eQ-glutamineR-arginineS-serineT-threon
ineandV-valin
elowastErrorsin
databasesfi
xedNC
notcon
firmed
20 Oxidative Medicine and Cellular Longevity
Table 6 NRF2 somatic mutations revealed in various human cancers
Domain Amino acid residues DNA mutation Cancer types (cases) ReferencesLocus Wild type Mutant
DLG motif
24 W (Trp) C (Cys) c72GgtCGgtT NSCLC neck ESC [54 62]K (Lys) c72TgtC ESC [62]
26 Q (Gln) E (Glu) c76CgtG NSCLC ESC [54 62]
27 D (Asp) G (Gly)lowast c80GgtA NSCLC [60]Y (Tyr) c79GgtT ESC [61]
28 I (Ile) T (Thr) c83CgtT NSCLC [54]
29 D (Asp) G (Gly) c86AgtG Head and neck ESC [54 62]H (His) c85GgtC NSCLC laynx [60 61]
30 L (Leu) F (Phe) c88CgtT NSCLC ESC [54 62]
31 G (Gly) A (Ala) c92GgtC NSCLC ESC skin [54 60ndash62]
32 V (Val) T (Thr) c95TgtG NSCLC [54]del c93 95delAGT ESC [61]
33ndash36 S-R-E-V S-R-E-V-S-R-E-Vlowast c97 108dupAGTCGAGCCGTA ESC [61]
34 R (Arg) Q (Gln) c101GgtA NCSCL [54 60 61]P (Pro) c101GgtC NSCLC [63]
ETGF motif
75 Q (Gln) H (His) c225AgtC Head and neck ESC [54 62]
77 D (Asp)
V (Val) c230AgtT NSCLC ESC [54 60 62]G (Gly) c230AgtG ESC [62]A (Ala) c230AgtC NSCLC [61]N (Asn) c229GgtA Larynx [61]
78 E (Glu) K (Lys) c232GgtA NSCLC ESC [54 62]
79 E (Glu)
K (Lys) c235GgtA NSCLC ESC [54 61 62]Q (Gln) c235GgtC NSCLC ESC [54 60ndash62]G (Gly) c236AgtG Larynx [61]E-E c234 236dupAGA ESC [61]
80 T (Thr) K (Lys) c239CgtACgtG NSCLC ESC [54 61 62]
80 T (Thr)P (Phe) c238AgtC ESC [62]I (Ile) c239CgtT Head and neck [54]A (Ala) c238AgtG NSCLC [63]
81 G (Gly) V (Val) c242GgtT ESC [61]D (Asp) c242GgtA NSCLC ESC [60ndash62]
82 E (Glu)
D (Asp) c246AgtT ESC oral cancer cell line [54 62]G (Gly) c245AgtG NSCLC [54]Q (Gln)lowast c244GgtC NSCLC ESC [60 61]V (Val)lowast c245AgtT ESC [61]
83 F (Phe) L (Leu)lowast c247TgtC NSCLC [60]NSCLC non-small cell lung cancer ESC esophageal squamous cancer and lowasterrors in reference fixed Number of cases 82 NSCLC and 10 ESC in [62] 125NSCLC 70 ESC 23 larynx and 17 skin in [61] 103 NSCLC and 12 head and neck in [54] 90 NSCLC in [63] 103 NSCLC in [60]
be involved in squamous lung cancer cell proliferation [64]suggesting that the mutation is functional and overcomesKEAP1 inhibition Singh et al [65] determined in vitrothat RNAi-mediated depletion of NRF2 in lung cancer cellsenhanced ROS production and susceptibility to cell deathby ionizing radiation These studies support the concept thatelevated NRF2 and ARE responsiveness provides cancer cellswith proliferative advantage for malignant transformation
and undue protection from anti-cancer therapy Onco-genic epidermal growth factor receptor (EGFR) signaling isrecently found to be critical in NRF2-mediated proliferationof NSCLC cells [66]
Collectively ldquogain of functionrdquo mutations in NRF2 thatreduce KEAP1 recognition are suggested to be predictivemarkers for poor responsiveness to chemotherapy and radi-ation therapy Although NRF2-mediated cellular defense
Oxidative Medicine and Cellular Longevity 21
processes are essential in the initiation stage enhancedNRF2-ARE activity in advanced stages of cancer developmentmay create a favorable intracellular environment for tumorcell growth and survival [67 68] In this context NRF2may be a potential molecular target for the treatment ofradio-resistance cancers especially those that have ldquoloss offunctionrdquo mutations in EGFR KRAS or KEAP1 as well asldquogain of functionrdquo mutations in NRF2
5 Epigenetic Alterations of NF-E2-RelatedFactor 2
Epigenetic modifications are alterations of molecules inter-acting with genes without changes to the primary DNAsequence They include post-translational modification ofhistones DNA methylation events chromatin conforma-tional changes and alterations to noncoding regulatoryRNAs Epigenetic alterations are stable and often inheritablebut are reversible and may affect expression of the geneDysregulation or defects in epigenetic processes particularlyhypermethylation of tumor suppressor gene promoters (egCpG islands) or histone modifications are thought to beassociated with carcinogenesis Investigators have reportedhypermethylation in CpG islands of KEAP1 which wereassociated with reduced KEAP1 expression in human cancersfrom lung prostate colon and so forth [69ndash72] Similar tosomatic mutations epigenetic changes on KEAP1 impairedthe function of its encoded protein leading to constitutiveNRF2 activation
Supporting the role of ldquopathogenic mutationsrdquo in NRF2expression of Nrf2 and downstream Nqo1 was suppressedin prostate tumors of mice (transgenic adenocarcinomaof mouse prostate TRAMP) Among 15 promoter CpGislands located between minus942 and minus654 (cminus1175 cminus1132and cminus1059 cminus887 gap in cminus1131 cminus1060 see Figure 3)hypermethylation of the first 5 CpG islands (minus942 minus899 andcminus1175 cminus1132) was significantly associated with tumorige-nesis [73] Moreover treatment with inhibitors for DNAmethyltransferase and histone deacetylase restored Nrf2expression in these tumor cells [73] A dietary phytochemicalcurcumin known as a DNA hypomethylation agent restoredepigenetically silent Nrf2 expression through CpG demethy-lation in carcinogen-induced mouse tumor cells [74]
The whole genome epigenetic datasets for 5 species arepublicly accessible at NCBI Epigenomics [75 76]The humanNRF2 epigenome of primary cells (breast penis) and H1stem cell line as well as mouse Nrf2 CpG island methylationdata for sperm blood and cerebellum are currently avail-able (httpwwwncbinlmnihgovepigenomics) Althoughno direct evidence of disease-associated epigenetic modu-lation has been identified in human NRF2 various phyto-chemical NRF2 agonists such as sulforaphane and curcuminhave shown their roles in DNA methylation and histonemodification (see reviews by Lee and colleagues eg [77])Taken together epigenetic modifications of theNRF2KEAP1axle are predicted to cause dysregulation of ARE-mediatedcellular defense leading to deleterious health effects and
phytochemical antioxidants as epigenetic modulators forNRF2 are suggested to be useful in cancer prevention
6 Conclusions
NRF2 is evolutionally conserved with high-sequence homol-ogy in many species However it is a highly mutable geneand numerous genetic variants have been discovered inhuman ethnic groups Importantly certain SNPs or haplo-types have been identified in various diseases as ldquoat-riskrdquoalleles and are related to functional alterations In additionto genetic variations multiple somatic mutations identifiedin the KEAP1 recognition domain of NRF2 in cancer cellshave been found to be oncogenic due to dysregulation ofNRF2 homeostasis by its excess ldquogain of functionrdquo Epigeneticalteration of theNRF2 is under investigation and is predictedto have pathogenic influences as learned from mouse andphytochemical agonist studies Continuous updates of Nrf2allelic variants in inbred mouse strains will provide a usefultool for effective experimental designs formodels of oxidativedisorders to provide insight into the disease mechanisms andintervention strategies
Abbreviations
AD Alzheimerrsquos diseaseALI acute lung injuryARE antioxidant response elementbZIP basic leucine zippercds coding DNA sequencesCI confidence intervalCOPD chronic obstructive pulmonary diseaseESC esophageal squamous cancerFEV1 forced expiratory volume in one second
Gbp giga base pairsGST glutathione S-transferaseGWAS genome-wide association studyHGVS Human Genome Variation SocietyHO-1 heme oxygenase-1119867 119901119910119897119900119903119894 119867119890119897119894119888119900119887119886119888119905119890119903 119901119910119897119900119903119894KEAP1 Kelch-like ECH activating protein 1MPD Mouse Phenome DatabaseMRP multidrug resistance proteinNCBI National Center for Biotechnology InformationNeh NRF2-ECH homologyNfe2l2 nuclear factor (erythroid-derived 2)-like 2Nrf2 NF-E2-related factor 2NSCLC nonsmall cell lung cancerNQO1 NAD(P)Hquinone oxidoreductase 1OR odds ratioPD Parkinsonrsquos diseasePM particulate matterROS reactive oxygen speciesSLE systemic lupus erythematosusSNP single nucleotide polymorphismSOD superoxide dismutaseURT untranslated region
22 Oxidative Medicine and Cellular Longevity
Disclosure
Authorrsquos contribution to the Work was done as part of theAuthorrsquos official duties as a NIH employee and is a Work ofthe United States Government Therefore copyright may notbe established in the United States
Conflict of Interests
The author declares that there is no conflict of interests
Acknowledgments
The research related to this paper was supported by theIntramural Research Program of the National Institutes ofHealth of the National Institute of Environmental HealthSciences (NIEHS) Drs Steven Kleeberger and StephanieLondon at the NIEHS provided excellent critical review ofthis paper The author thanks Mrs Jacqui Marzec for herhelpful comments and English editing
References
[1] J Y Chan M C Cheung P Moi K Chan and Y W KanldquoChromosomal localization of the humanNF-E2 family of bZIPtranscription factors by fluorescence in situ hybridizationrdquoHuman Genetics vol 95 no 3 pp 265ndash269 1995
[2] K Itoh K Igarashi N Hayashi M Nishizawa and MYamamoto ldquoCloning and characterization of a novel erythroidcell-derived CNC family transcription factor heterodimerizingwith the small Maf family proteinsrdquo Molecular and CellularBiology vol 15 no 8 pp 4184ndash4193 1995
[3] W O Osburn and T W Kensler ldquoNRF2 signaling an adaptiveresponse pathway for protection against environmental toxicinsultsrdquoMutation Research vol 659 no 1-2 pp 31ndash39 2008
[4] D Papp K Lenti D Modos D Fazekas Z Dul D Tureiet al ldquoThe NRF2-related interactome and regulome containmultifunctional proteins and fine-tuned autoregulatory loopsrdquoFEBS Letters vol 586 no 13 pp 1795ndash1802 2012
[5] X Wang D J Tomso B N Chorley et al ldquoIdentificationof polymorphic antioxidant response elements in the humangenomerdquo Human Molecular Genetics vol 16 no 10 pp 1188ndash1200 2007
[6] D Malhotra E Portales-Casamar A Singh et al ldquoGlobalmapping of binding sites for NRF2 identifies novel targets incell survival response through ChIP-Seq profiling and networkanalysisrdquo Nucleic Acids Research vol 38 no 17 pp 5718ndash57342010
[7] K Chan R Lu J C Chang and Y W Kan ldquoNRF2 a memberof the NFE2 family of transcription factors is not essential formurine erythropoiesis growth and developmentrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 93 no 24 pp 13943ndash13948 1996
[8] K Itoh T Chiba S Takahashi et al ldquoAn NRF2small Mafheterodimer mediates the induction of phase II detoxifyingenzyme genes through antioxidant response elementsrdquo Bio-chemical and Biophysical Research Communications vol 236no 2 pp 313ndash322 1997
[9] FMartin J M vanDeursen R A Shivdasani CW Jackson AG Troutman and P A Ney ldquoErythroid maturation and globin
gene expression inmicewith combined deficiency ofNF-E2 andNrf-2rdquo Blood vol 91 no 9 pp 3459ndash3466 1998
[10] K Itoh N Wakabayashi Y Katoh et al ldquoKEAP1 repressesnuclear activation of antioxidant responsive elements by NRF2through binding to the amino-terminal Neh2 domainrdquo Genesand Development vol 13 no 1 pp 76ndash86 1999
[11] A Kobayashi M I Kang H Okawa et al ldquoOxidative stresssensor KEAP1 functions as an adaptor for Cul3-based E3 ligaseto regulate proteasomal degradation of NRF2rdquo Molecular andCellular Biology vol 24 no 16 pp 7130ndash7139 2004
[12] K Taguchi H Motohashi and M Yamamoto ldquoMolecularmechanisms of the KEAP1-NRF2 pathway in stress responseand cancer evolutionrdquo Genes to Cells vol 16 no 2 pp 123ndash1402011
[13] K Mukaigasa L T Nguyen L Li H Nakajima M Yamamotoand M Kobayashi ldquoGenetic evidence of an evolutionarilyconserved role for NRF2 in the protection against oxidativestressrdquoMolecular and Cellular Biology vol 32 no 21 pp 4455ndash4461 2012
[14] JMaher andMYamamoto ldquoThe rise of antioxidant signalingmdashthe evolution and hormetic actions of NRF2rdquo Toxicology andApplied Pharmacology vol 244 no 1 pp 4ndash15 2010
[15] S C Lo X Li M T Henzl L J Beamer and M HanninkldquoStructure of the KEAP1NRF2 interface provides mechanisticinsight intoNRF2 signalingrdquoThe EMBO Journal vol 25 no 15pp 3605ndash3617 2006
[16] V A McKusick ldquoMendelian inheritance in man and its onlineversion OMIMrdquo American Journal of Human Genetics vol 80no 4 pp 588ndash604 2007
[17] B Yalcin D J Adams J Flint and T M Keane ldquoNext-generation sequencing of experimental mouse strainsrdquo Mam-malian Genome vol 23 no 9-10 pp 490ndash498 2012
[18] D R Bentley S Balasubramanian H P Swerdlow et alldquoAccurate whole human genome sequencing using reversibleterminator chemistryrdquo Nature vol 456 no 7218 pp 53ndash592008
[19] P S G Chain D V Grafham R S Fulton et al ldquoGenomeproject standards in a new era of sequencingrdquo Science vol 326no 5950 pp 236ndash237 2009
[20] RHWaterstonK Lindblad-Toh E Birney J Rogers J F AbrilP Agarwal et al ldquoInitial sequencing and comparative analysis ofthe mouse genomerdquo Nature vol 420 pp 520ndash562 2002
[21] T M Keane L Goodstadt P Danecek M A White K WongB Yalcin et al ldquoMouse genomic variation and its effect onphenotypes and gene regulationrdquoNature vol 477 pp 289ndash2942011
[22] T Yamamoto K Yoh A Kobayashi et al ldquoIdentification ofpolymorphisms in the promoter region of the human NRF2generdquo Biochemical and Biophysical Research Communicationsvol 321 no 1 pp 72ndash79 2004
[23] J M Marzec J D Christie S P Reddy et al ldquoFunctionalpolymorphisms in the transcription factor NRF2 in humansincrease the risk of acute lung injuryrdquo The FASEB Journal vol21 no 9 pp 2237ndash2246 2007
[24] H Fukushima-Uesaka Y Saito K Maekawa et al ldquoGeneticvariations and haplotype structures of transcriptional factorNRF2 and its cytosolic reservoir protein KEAP1 in JapaneserdquoDrug Metabolism and Pharmacokinetics vol 22 no 3 pp 212ndash219 2007
[25] E J Cordova R Velazquez-Cruz F Centeno V Baca and LOrozco ldquoThe NRF2 gene variant -653GA is associated with
Oxidative Medicine and Cellular Longevity 23
nephritis in childhood-onset systemic lupus erythematosusrdquoLupus vol 19 no 10 pp 1237ndash1242 2010
[26] M von Otter S Landgren S Nilsson et al ldquoAssociation ofNRF2-encoding NFE2L2 haplotypes with Parkinsonrsquos diseaserdquoBMCMedical Genetics vol 11 no 1 article 36 2010
[27] JMHartikainenM TengstromVMKosmaV L Kinnula AMannermaa andY Soini ldquoGenetic polymorphisms and proteinexpression of NRF2 and sulfiredoxin predict survival outcomesin breast cancerrdquo Cancer Research vol 72 pp 5537ndash5546 2012
[28] T Arisawa T Tahara T Shibata et al ldquoThe relationshipbetween Helicobacter pylori infection and promoter polymor-phism of the NRF2 gene in chronic gastritisrdquo InternationalJournal of Molecular Medicine vol 19 no 1 pp 143ndash148 2007
[29] T Arisawa T Tahara T Shibata et al ldquoThe influence ofpromoter polymorphism of nuclear factor-erythroid 2-relatedfactor 2 gene on the aberrant DNA methylation in gastricepitheliumrdquo Oncology Reports vol 19 no 1 pp 211ndash216 2008
[30] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene pro-moter polymorphism and gastric carcinogenesisrdquo Hepato-Gastroenterology vol 55 no 82-83 pp 750ndash754 2008
[31] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene promoterpolymorphism is associated with ulcerative colitis in a Japanesepopulationrdquo Hepato-Gastroenterology vol 55 no 82-83 pp394ndash397 2008
[32] C C Hua L C Chang J C Tseng C M Chu Y C Liu andW B Shieh ldquoFunctional haplotypes in the promoter region oftranscription factor NRF2 in chronic obstructive pulmonarydiseaserdquo Disease Markers vol 28 no 3 pp 185ndash193 2010
[33] S O Shaheen R B Newson S M Ring M J Rose-ZerilliJ W Holloway and A J Henderson ldquoPrenatal and infantacetaminophen exposure antioxidant gene polymorphismsand childhood asthmardquo The Journal of Allergy and ClinicalImmunology vol 126 no 6 pp 1141e7ndash1148e7 2010
[34] H Masuko T Sakamoto Y Kaneko et al ldquoLower FEV1 innon-COPD nonasthmatic subjects association with smokingannual decline in FEV1 total IgE levels and TSLP genotypesrdquoInternational Journal of Chronic Obstructive Pulmonary Diseasevol 6 pp 181ndash189 2011
[35] C P Guan M N Zhou A E Xu et al ldquoThe susceptibilityto vitiligo is associated with NF-E2-related factor 2 (NRF2)gene polymorphisms a study on Chinese Han populationrdquoExperimental Dermatology vol 17 no 12 pp 1059ndash1062 2008
[36] J Bouligand O Cabaret M Canonico et al ldquoEffect of NFE2L2genetic polymorphism on the association between oral estrogentherapy and the risk of venous thromboembolism in post-menopausal womenrdquo Clinical Pharmacology amp Therapeuticsvol 89 no 1 pp 60ndash64 2011
[37] D S OrsquoMahony B J Glavan T D Holden C Fong R A BlackG Rona et al ldquoInflammation and immune-related candidategene associations with acute lung injury susceptibility andseverity a validation studyrdquo PLoS ONE vol 7 no 12 Article IDe51104 2012
[38] I Ungvari E Hadadi V Virag et al ldquoRelationship between airpollutionNFE2L2 gene polymorphisms and childhood asthmain aHungarian populationrdquo Journal of CommunityGenetics vol3 no 1 pp 25ndash33 2012
[39] M SiedlinskiD S Postma JMA Boer et al ldquoLevel and courseof FEV1 in relation to polymorphisms inNFE2L2 andKEAP1 inthe general populationrdquo Respiratory Research vol 10 article 732009
[40] C C Hong C B Ambrosone J Ahn et al ldquoGenetic variabilityin iron-related oxidative stress pathways (NRF2 NQ01 NOS3
and HO-1) iron intake and risk of postmenopausal breastcancerrdquo Cancer Epidemiology Biomarkers and Prevention vol16 no 9 pp 1784ndash1794 2007
[41] C Canova C Dunster F J Kelly C Minelli P L Shah CCaneja et al ldquoPM10-induced hospital admissions for asthmaand chronic obstructive pulmonary disease the modifyingeffect of individual characteristicsrdquo Epidemiology vol 23 no 4pp 607ndash615 2012
[42] A J Henderson R B Newson M Rose-Zerilli S M RingJ W Holloway and S O Shaheen ldquoMaternal NRF2 andgluthathione-S-transferase polymorphisms donotmodify asso-ciations of prenatal tobacco smoke exposure with asthma andlung function in school-aged childrenrdquo Thorax vol 65 no 10pp 897ndash902 2010
[43] C Xing A L Sestak J A Kelly et al ldquoLocalization andreplication of the systemic lupus erythematosus linkage signalat 4p16 interaction with 2p11 12q24 and 19q13 in EuropeanAmericansrdquo Human Genetics vol 120 no 5 pp 623ndash631 2007
[44] T F Wong K Yoshinaga Y Monma K Ito H NiikuraS Nagase et al ldquoAssociation of KEAP1 and NRF2 geneticmutations and polymorphisms with endometrioid endometrialadenocarcinoma survivalrdquo International Journal of Gynecologi-cal Cancer vol 21 no 8 pp 1428ndash1435 2011
[45] M von Otter S Landgren S Nilsson et al ldquoNRF2-encodingNFE2L2 haplotypes influence disease progression but not riskin Alzheimerrsquos disease and age-related cataractrdquoMechanisms ofAgeing and Development vol 131 no 2 pp 105ndash110 2010
[46] S Tsang Z Sun B Luke et al ldquoA comprehensive SNP-basedgenetic analysis of inbred mouse strainsrdquoMammalian Genomevol 16 no 7 pp 476ndash480 2005
[47] C M Wade E J Kulbokas III A W Kirby et al ldquoThe mosaicstructure of variation in the laboratory mouse genomerdquoNaturevol 420 no 6915 pp 574ndash578 2002
[48] T Wiltshire M T Pletcher S Batalov et al ldquoGenome-widesingle-nucleotide polymorphism analysis defines haplotypepatterns in mouserdquo Proceedings of the National Academy ofSciences of the United States of America vol 100 no 6 pp 3380ndash3385 2003
[49] K A Frazer E Eskin H M Kang et al ldquoA sequence-basedvariation map of 827 million SNPs in inbred mouse strainsrdquoNature vol 448 no 7157 pp 1050ndash1053 2007
[50] T P Maddatu S C Grubb C J Bult andM A Bogue ldquoMousephenome database (MPD)rdquo Nucleic Acids Research vol 40 ppD887ndashD894 2007
[51] A Kirby H M Kang C M Wade et al ldquoFine mapping in 94inbred mouse strains using a high-density haplotype resourcerdquoGenetics vol 185 no 3 pp 1081ndash1095 2010
[52] H Y Cho A E Jedlicka S P M Reddy L Y Zhang T WKensler and S R Kleeberger ldquoLinkage analysis of susceptibilityto hyperoxia NRF2 is a candidate generdquo American Journal ofRespiratory Cell and Molecular Biology vol 26 no 1 pp 42ndash512002
[53] H Y Cho A E Jedlicka S P M Reddy et al ldquoRole of NRF2in protection against hyperoxic lung injury in micerdquo AmericanJournal of Respiratory Cell and Molecular Biology vol 26 no 2pp 175ndash182 2002
[54] T Shibata T Ohta K I Tong et al ldquoCancer related mutationsin NRF2 impair its recognition by KEAP1-Cul3 E3 ligase andpromote malignancyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 36 pp13568ndash13573 2008
24 Oxidative Medicine and Cellular Longevity
[55] A Singh V Misra R K Thimmulappa et al ldquoDysfunctionalKEAP1-NRF2 interaction in non-small-cell lung cancerrdquo PLoSMedicine vol 3 no 10 article e420 2006
[56] T Ohta K Iijima M Miyamoto et al ldquoLoss of KEAP1 functionactivates NRF2 and provides advantages for lung cancer cellgrowthrdquo Cancer Research vol 68 no 5 pp 1303ndash1309 2008
[57] B Padmanabhan K I Tong T Ohta et al ldquoStructural basisfor defects of KEAP1 activity provoked by its point mutationsin lung cancerrdquoMolecular Cell vol 21 no 5 pp 689ndash700 2006
[58] T Shibata A Kokubu M Gotoh et al ldquoGenetic alteration ofKEAP1 confers constitutive NRF2 activation and resistance tochemotherapy in gallbladder cancerrdquoGastroenterology vol 135no 4 pp 1358e4ndash1368e4 2008
[59] N J Yoo H R Kim Y R Kim C H An and S H LeeldquoSomatic mutations of the KEAP1 gene in common solidcancersrdquo Histopathology vol 60 no 6 pp 943ndash952 2012
[60] Y Hu Y Ju D Lin Z Wang Y Huang S Zhang et alldquoMutation of the NRF2 gene in non-small cell lung cancerrdquoMolecular Biology Reports vol 39 no 4 pp 4743ndash4747 2012
[61] Y R Kim J EOhM S Kim et al ldquoOncogenicNRF2mutationsin squamous cell carcinomas of oesophagus and skinrdquo TheJournal of Pathology vol 220 no 4 pp 446ndash451 2010
[62] T Shibata A Kokubu S Saito et al ldquoNRF2 mutation confersmalignant potential and resistance to chemoradiation therapyin advanced esophageal squamous cancerrdquo Neoplasia vol 13pp 864ndash873 2011
[63] H Sasaki M Shitara K Yokota Y Hikosaka S MoriyamaM Yano et al ldquoIncreased NRF2 gene (NFE2L2) copy numbercorrelates with mutations in lung squamous cell carcinomasrdquoMolecular Medicine Reports vol 6 no 2 pp 391ndash394 2012
[64] H Sasaki M Shitara K Yokota Y Hikosaka S Moriyama MYano et al ldquoRagD gene expression andNRF2mutations in lungsquamous cell carcinomasrdquo Oncology Letters vol 4 pp 1167ndash1170 2012
[65] A Singh M Bodas N Wakabayashi F Bunz and S BiswalldquoGain of NRF2 function in non-small-cell lung cancer cellsconfers radioresistancerdquo Antioxidants and Redox Signaling vol13 no 11 pp 1627ndash1637 2010
[66] T Yamadori Y Ishii SHommaYMorishimaKKurishimaKItoh et al ldquoMolecular mechanisms for the regulation of NRF2-mediated cell proliferation in non-small-cell lung cancersrdquoOncogene vol 31 pp 4768ndash4777 2012
[67] A K Bauer H Y Cho L Miller-Degraff C Walker K HelmsJ Fostel et al ldquoTargeted deletion of NRF2 reduces urethane-induced lung tumor development in micerdquo PLoS ONE vol 6no 10 Article ID e26590 2011
[68] H Y Cho and S R Kleeberger ldquoNRF2 protects against airwaydisordersrdquo Toxicology and Applied Pharmacology vol 244 no1 pp 43ndash56 2010
[69] R Wang J An F Ji H Jiao H Sun and D Zhou ldquoHyperme-thylation of the KEAP1 gene in human lung cancer cell linesand lung cancer tissuesrdquo Biochemical and Biophysical ResearchCommunications vol 373 no 1 pp 151ndash154 2008
[70] P Zhang A Singh S Yegnasubramanian et al ldquoLoss ofkelch-like ECH-associated protein 1 function in prostate cancercells causes chemoresistance and radioresistance and promotestumor growthrdquoMolecular Cancer Therapeutics vol 9 no 2 pp336ndash346 2010
[71] N Hanada T Takahata Q Zhou et al ldquoMethylation of theKEAP1 gene promoter region in human colorectal cancerrdquoBMCCancer vol 12 article 66 2012
[72] L A Muscarella R Barbano V DrsquoAngelo et al ldquoRegulationof KEAP1 expression by promoter methylation in malignantgliomas and association with patientrsquos outcomerdquo Epigeneticsvol 6 no 3 pp 317ndash325 2011
[73] S Yu T O Khor K L Cheung et al ldquoNRF2 expressionis regulated by epigenetic mechanisms in prostate cancer ofTRAMP micerdquo PLoS ONE vol 5 no 1 Article ID e8579 2010
[74] T O Khor Y Huang T Y Wu L Shu J Lee and A N KongldquoPharmacodynamics of curcumin as DNA hypomethylationagent in restoring the expression of NRF2 via promoter CpGsdemethylationrdquo Biochemical Pharmacology vol 82 no 9 pp1073ndash1078 2011
[75] I M Fingerman L McDaniel X Zhang et al ldquoNCBI epige-nomics a new public resource for exploring epigenomic datasetsrdquo Nucleic Acids Research vol 39 supplemen 1 pp D908ndashD912 2011
[76] I M Fingerman X Zhang W Ratzat N Husain R F Cohenand G D Schuler ldquoNCBI epigenomics whatrsquos new for 2013rdquoNucleic Acids Research vol 41 no 1 pp D221ndashD225 2013
[77] J H Lee T O Khor L Shu Z Y Su F Fuentes andA N Kong ldquoDietary phytochemicals and cancer preventionNRF2 signaling epigenetics and cell death mechanisms inblocking cancer initiation and progressionrdquo Pharmacology ampTherapeutics vol 137 no 2 pp 153ndash171 2012
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Disease Markers
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OncologyJournal of
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Oxidative Medicine and Cellular Longevity
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Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
20 Oxidative Medicine and Cellular Longevity
Table 6 NRF2 somatic mutations revealed in various human cancers
Domain Amino acid residues DNA mutation Cancer types (cases) ReferencesLocus Wild type Mutant
DLG motif
24 W (Trp) C (Cys) c72GgtCGgtT NSCLC neck ESC [54 62]K (Lys) c72TgtC ESC [62]
26 Q (Gln) E (Glu) c76CgtG NSCLC ESC [54 62]
27 D (Asp) G (Gly)lowast c80GgtA NSCLC [60]Y (Tyr) c79GgtT ESC [61]
28 I (Ile) T (Thr) c83CgtT NSCLC [54]
29 D (Asp) G (Gly) c86AgtG Head and neck ESC [54 62]H (His) c85GgtC NSCLC laynx [60 61]
30 L (Leu) F (Phe) c88CgtT NSCLC ESC [54 62]
31 G (Gly) A (Ala) c92GgtC NSCLC ESC skin [54 60ndash62]
32 V (Val) T (Thr) c95TgtG NSCLC [54]del c93 95delAGT ESC [61]
33ndash36 S-R-E-V S-R-E-V-S-R-E-Vlowast c97 108dupAGTCGAGCCGTA ESC [61]
34 R (Arg) Q (Gln) c101GgtA NCSCL [54 60 61]P (Pro) c101GgtC NSCLC [63]
ETGF motif
75 Q (Gln) H (His) c225AgtC Head and neck ESC [54 62]
77 D (Asp)
V (Val) c230AgtT NSCLC ESC [54 60 62]G (Gly) c230AgtG ESC [62]A (Ala) c230AgtC NSCLC [61]N (Asn) c229GgtA Larynx [61]
78 E (Glu) K (Lys) c232GgtA NSCLC ESC [54 62]
79 E (Glu)
K (Lys) c235GgtA NSCLC ESC [54 61 62]Q (Gln) c235GgtC NSCLC ESC [54 60ndash62]G (Gly) c236AgtG Larynx [61]E-E c234 236dupAGA ESC [61]
80 T (Thr) K (Lys) c239CgtACgtG NSCLC ESC [54 61 62]
80 T (Thr)P (Phe) c238AgtC ESC [62]I (Ile) c239CgtT Head and neck [54]A (Ala) c238AgtG NSCLC [63]
81 G (Gly) V (Val) c242GgtT ESC [61]D (Asp) c242GgtA NSCLC ESC [60ndash62]
82 E (Glu)
D (Asp) c246AgtT ESC oral cancer cell line [54 62]G (Gly) c245AgtG NSCLC [54]Q (Gln)lowast c244GgtC NSCLC ESC [60 61]V (Val)lowast c245AgtT ESC [61]
83 F (Phe) L (Leu)lowast c247TgtC NSCLC [60]NSCLC non-small cell lung cancer ESC esophageal squamous cancer and lowasterrors in reference fixed Number of cases 82 NSCLC and 10 ESC in [62] 125NSCLC 70 ESC 23 larynx and 17 skin in [61] 103 NSCLC and 12 head and neck in [54] 90 NSCLC in [63] 103 NSCLC in [60]
be involved in squamous lung cancer cell proliferation [64]suggesting that the mutation is functional and overcomesKEAP1 inhibition Singh et al [65] determined in vitrothat RNAi-mediated depletion of NRF2 in lung cancer cellsenhanced ROS production and susceptibility to cell deathby ionizing radiation These studies support the concept thatelevated NRF2 and ARE responsiveness provides cancer cellswith proliferative advantage for malignant transformation
and undue protection from anti-cancer therapy Onco-genic epidermal growth factor receptor (EGFR) signaling isrecently found to be critical in NRF2-mediated proliferationof NSCLC cells [66]
Collectively ldquogain of functionrdquo mutations in NRF2 thatreduce KEAP1 recognition are suggested to be predictivemarkers for poor responsiveness to chemotherapy and radi-ation therapy Although NRF2-mediated cellular defense
Oxidative Medicine and Cellular Longevity 21
processes are essential in the initiation stage enhancedNRF2-ARE activity in advanced stages of cancer developmentmay create a favorable intracellular environment for tumorcell growth and survival [67 68] In this context NRF2may be a potential molecular target for the treatment ofradio-resistance cancers especially those that have ldquoloss offunctionrdquo mutations in EGFR KRAS or KEAP1 as well asldquogain of functionrdquo mutations in NRF2
5 Epigenetic Alterations of NF-E2-RelatedFactor 2
Epigenetic modifications are alterations of molecules inter-acting with genes without changes to the primary DNAsequence They include post-translational modification ofhistones DNA methylation events chromatin conforma-tional changes and alterations to noncoding regulatoryRNAs Epigenetic alterations are stable and often inheritablebut are reversible and may affect expression of the geneDysregulation or defects in epigenetic processes particularlyhypermethylation of tumor suppressor gene promoters (egCpG islands) or histone modifications are thought to beassociated with carcinogenesis Investigators have reportedhypermethylation in CpG islands of KEAP1 which wereassociated with reduced KEAP1 expression in human cancersfrom lung prostate colon and so forth [69ndash72] Similar tosomatic mutations epigenetic changes on KEAP1 impairedthe function of its encoded protein leading to constitutiveNRF2 activation
Supporting the role of ldquopathogenic mutationsrdquo in NRF2expression of Nrf2 and downstream Nqo1 was suppressedin prostate tumors of mice (transgenic adenocarcinomaof mouse prostate TRAMP) Among 15 promoter CpGislands located between minus942 and minus654 (cminus1175 cminus1132and cminus1059 cminus887 gap in cminus1131 cminus1060 see Figure 3)hypermethylation of the first 5 CpG islands (minus942 minus899 andcminus1175 cminus1132) was significantly associated with tumorige-nesis [73] Moreover treatment with inhibitors for DNAmethyltransferase and histone deacetylase restored Nrf2expression in these tumor cells [73] A dietary phytochemicalcurcumin known as a DNA hypomethylation agent restoredepigenetically silent Nrf2 expression through CpG demethy-lation in carcinogen-induced mouse tumor cells [74]
The whole genome epigenetic datasets for 5 species arepublicly accessible at NCBI Epigenomics [75 76]The humanNRF2 epigenome of primary cells (breast penis) and H1stem cell line as well as mouse Nrf2 CpG island methylationdata for sperm blood and cerebellum are currently avail-able (httpwwwncbinlmnihgovepigenomics) Althoughno direct evidence of disease-associated epigenetic modu-lation has been identified in human NRF2 various phyto-chemical NRF2 agonists such as sulforaphane and curcuminhave shown their roles in DNA methylation and histonemodification (see reviews by Lee and colleagues eg [77])Taken together epigenetic modifications of theNRF2KEAP1axle are predicted to cause dysregulation of ARE-mediatedcellular defense leading to deleterious health effects and
phytochemical antioxidants as epigenetic modulators forNRF2 are suggested to be useful in cancer prevention
6 Conclusions
NRF2 is evolutionally conserved with high-sequence homol-ogy in many species However it is a highly mutable geneand numerous genetic variants have been discovered inhuman ethnic groups Importantly certain SNPs or haplo-types have been identified in various diseases as ldquoat-riskrdquoalleles and are related to functional alterations In additionto genetic variations multiple somatic mutations identifiedin the KEAP1 recognition domain of NRF2 in cancer cellshave been found to be oncogenic due to dysregulation ofNRF2 homeostasis by its excess ldquogain of functionrdquo Epigeneticalteration of theNRF2 is under investigation and is predictedto have pathogenic influences as learned from mouse andphytochemical agonist studies Continuous updates of Nrf2allelic variants in inbred mouse strains will provide a usefultool for effective experimental designs formodels of oxidativedisorders to provide insight into the disease mechanisms andintervention strategies
Abbreviations
AD Alzheimerrsquos diseaseALI acute lung injuryARE antioxidant response elementbZIP basic leucine zippercds coding DNA sequencesCI confidence intervalCOPD chronic obstructive pulmonary diseaseESC esophageal squamous cancerFEV1 forced expiratory volume in one second
Gbp giga base pairsGST glutathione S-transferaseGWAS genome-wide association studyHGVS Human Genome Variation SocietyHO-1 heme oxygenase-1119867 119901119910119897119900119903119894 119867119890119897119894119888119900119887119886119888119905119890119903 119901119910119897119900119903119894KEAP1 Kelch-like ECH activating protein 1MPD Mouse Phenome DatabaseMRP multidrug resistance proteinNCBI National Center for Biotechnology InformationNeh NRF2-ECH homologyNfe2l2 nuclear factor (erythroid-derived 2)-like 2Nrf2 NF-E2-related factor 2NSCLC nonsmall cell lung cancerNQO1 NAD(P)Hquinone oxidoreductase 1OR odds ratioPD Parkinsonrsquos diseasePM particulate matterROS reactive oxygen speciesSLE systemic lupus erythematosusSNP single nucleotide polymorphismSOD superoxide dismutaseURT untranslated region
22 Oxidative Medicine and Cellular Longevity
Disclosure
Authorrsquos contribution to the Work was done as part of theAuthorrsquos official duties as a NIH employee and is a Work ofthe United States Government Therefore copyright may notbe established in the United States
Conflict of Interests
The author declares that there is no conflict of interests
Acknowledgments
The research related to this paper was supported by theIntramural Research Program of the National Institutes ofHealth of the National Institute of Environmental HealthSciences (NIEHS) Drs Steven Kleeberger and StephanieLondon at the NIEHS provided excellent critical review ofthis paper The author thanks Mrs Jacqui Marzec for herhelpful comments and English editing
References
[1] J Y Chan M C Cheung P Moi K Chan and Y W KanldquoChromosomal localization of the humanNF-E2 family of bZIPtranscription factors by fluorescence in situ hybridizationrdquoHuman Genetics vol 95 no 3 pp 265ndash269 1995
[2] K Itoh K Igarashi N Hayashi M Nishizawa and MYamamoto ldquoCloning and characterization of a novel erythroidcell-derived CNC family transcription factor heterodimerizingwith the small Maf family proteinsrdquo Molecular and CellularBiology vol 15 no 8 pp 4184ndash4193 1995
[3] W O Osburn and T W Kensler ldquoNRF2 signaling an adaptiveresponse pathway for protection against environmental toxicinsultsrdquoMutation Research vol 659 no 1-2 pp 31ndash39 2008
[4] D Papp K Lenti D Modos D Fazekas Z Dul D Tureiet al ldquoThe NRF2-related interactome and regulome containmultifunctional proteins and fine-tuned autoregulatory loopsrdquoFEBS Letters vol 586 no 13 pp 1795ndash1802 2012
[5] X Wang D J Tomso B N Chorley et al ldquoIdentificationof polymorphic antioxidant response elements in the humangenomerdquo Human Molecular Genetics vol 16 no 10 pp 1188ndash1200 2007
[6] D Malhotra E Portales-Casamar A Singh et al ldquoGlobalmapping of binding sites for NRF2 identifies novel targets incell survival response through ChIP-Seq profiling and networkanalysisrdquo Nucleic Acids Research vol 38 no 17 pp 5718ndash57342010
[7] K Chan R Lu J C Chang and Y W Kan ldquoNRF2 a memberof the NFE2 family of transcription factors is not essential formurine erythropoiesis growth and developmentrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 93 no 24 pp 13943ndash13948 1996
[8] K Itoh T Chiba S Takahashi et al ldquoAn NRF2small Mafheterodimer mediates the induction of phase II detoxifyingenzyme genes through antioxidant response elementsrdquo Bio-chemical and Biophysical Research Communications vol 236no 2 pp 313ndash322 1997
[9] FMartin J M vanDeursen R A Shivdasani CW Jackson AG Troutman and P A Ney ldquoErythroid maturation and globin
gene expression inmicewith combined deficiency ofNF-E2 andNrf-2rdquo Blood vol 91 no 9 pp 3459ndash3466 1998
[10] K Itoh N Wakabayashi Y Katoh et al ldquoKEAP1 repressesnuclear activation of antioxidant responsive elements by NRF2through binding to the amino-terminal Neh2 domainrdquo Genesand Development vol 13 no 1 pp 76ndash86 1999
[11] A Kobayashi M I Kang H Okawa et al ldquoOxidative stresssensor KEAP1 functions as an adaptor for Cul3-based E3 ligaseto regulate proteasomal degradation of NRF2rdquo Molecular andCellular Biology vol 24 no 16 pp 7130ndash7139 2004
[12] K Taguchi H Motohashi and M Yamamoto ldquoMolecularmechanisms of the KEAP1-NRF2 pathway in stress responseand cancer evolutionrdquo Genes to Cells vol 16 no 2 pp 123ndash1402011
[13] K Mukaigasa L T Nguyen L Li H Nakajima M Yamamotoand M Kobayashi ldquoGenetic evidence of an evolutionarilyconserved role for NRF2 in the protection against oxidativestressrdquoMolecular and Cellular Biology vol 32 no 21 pp 4455ndash4461 2012
[14] JMaher andMYamamoto ldquoThe rise of antioxidant signalingmdashthe evolution and hormetic actions of NRF2rdquo Toxicology andApplied Pharmacology vol 244 no 1 pp 4ndash15 2010
[15] S C Lo X Li M T Henzl L J Beamer and M HanninkldquoStructure of the KEAP1NRF2 interface provides mechanisticinsight intoNRF2 signalingrdquoThe EMBO Journal vol 25 no 15pp 3605ndash3617 2006
[16] V A McKusick ldquoMendelian inheritance in man and its onlineversion OMIMrdquo American Journal of Human Genetics vol 80no 4 pp 588ndash604 2007
[17] B Yalcin D J Adams J Flint and T M Keane ldquoNext-generation sequencing of experimental mouse strainsrdquo Mam-malian Genome vol 23 no 9-10 pp 490ndash498 2012
[18] D R Bentley S Balasubramanian H P Swerdlow et alldquoAccurate whole human genome sequencing using reversibleterminator chemistryrdquo Nature vol 456 no 7218 pp 53ndash592008
[19] P S G Chain D V Grafham R S Fulton et al ldquoGenomeproject standards in a new era of sequencingrdquo Science vol 326no 5950 pp 236ndash237 2009
[20] RHWaterstonK Lindblad-Toh E Birney J Rogers J F AbrilP Agarwal et al ldquoInitial sequencing and comparative analysis ofthe mouse genomerdquo Nature vol 420 pp 520ndash562 2002
[21] T M Keane L Goodstadt P Danecek M A White K WongB Yalcin et al ldquoMouse genomic variation and its effect onphenotypes and gene regulationrdquoNature vol 477 pp 289ndash2942011
[22] T Yamamoto K Yoh A Kobayashi et al ldquoIdentification ofpolymorphisms in the promoter region of the human NRF2generdquo Biochemical and Biophysical Research Communicationsvol 321 no 1 pp 72ndash79 2004
[23] J M Marzec J D Christie S P Reddy et al ldquoFunctionalpolymorphisms in the transcription factor NRF2 in humansincrease the risk of acute lung injuryrdquo The FASEB Journal vol21 no 9 pp 2237ndash2246 2007
[24] H Fukushima-Uesaka Y Saito K Maekawa et al ldquoGeneticvariations and haplotype structures of transcriptional factorNRF2 and its cytosolic reservoir protein KEAP1 in JapaneserdquoDrug Metabolism and Pharmacokinetics vol 22 no 3 pp 212ndash219 2007
[25] E J Cordova R Velazquez-Cruz F Centeno V Baca and LOrozco ldquoThe NRF2 gene variant -653GA is associated with
Oxidative Medicine and Cellular Longevity 23
nephritis in childhood-onset systemic lupus erythematosusrdquoLupus vol 19 no 10 pp 1237ndash1242 2010
[26] M von Otter S Landgren S Nilsson et al ldquoAssociation ofNRF2-encoding NFE2L2 haplotypes with Parkinsonrsquos diseaserdquoBMCMedical Genetics vol 11 no 1 article 36 2010
[27] JMHartikainenM TengstromVMKosmaV L Kinnula AMannermaa andY Soini ldquoGenetic polymorphisms and proteinexpression of NRF2 and sulfiredoxin predict survival outcomesin breast cancerrdquo Cancer Research vol 72 pp 5537ndash5546 2012
[28] T Arisawa T Tahara T Shibata et al ldquoThe relationshipbetween Helicobacter pylori infection and promoter polymor-phism of the NRF2 gene in chronic gastritisrdquo InternationalJournal of Molecular Medicine vol 19 no 1 pp 143ndash148 2007
[29] T Arisawa T Tahara T Shibata et al ldquoThe influence ofpromoter polymorphism of nuclear factor-erythroid 2-relatedfactor 2 gene on the aberrant DNA methylation in gastricepitheliumrdquo Oncology Reports vol 19 no 1 pp 211ndash216 2008
[30] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene pro-moter polymorphism and gastric carcinogenesisrdquo Hepato-Gastroenterology vol 55 no 82-83 pp 750ndash754 2008
[31] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene promoterpolymorphism is associated with ulcerative colitis in a Japanesepopulationrdquo Hepato-Gastroenterology vol 55 no 82-83 pp394ndash397 2008
[32] C C Hua L C Chang J C Tseng C M Chu Y C Liu andW B Shieh ldquoFunctional haplotypes in the promoter region oftranscription factor NRF2 in chronic obstructive pulmonarydiseaserdquo Disease Markers vol 28 no 3 pp 185ndash193 2010
[33] S O Shaheen R B Newson S M Ring M J Rose-ZerilliJ W Holloway and A J Henderson ldquoPrenatal and infantacetaminophen exposure antioxidant gene polymorphismsand childhood asthmardquo The Journal of Allergy and ClinicalImmunology vol 126 no 6 pp 1141e7ndash1148e7 2010
[34] H Masuko T Sakamoto Y Kaneko et al ldquoLower FEV1 innon-COPD nonasthmatic subjects association with smokingannual decline in FEV1 total IgE levels and TSLP genotypesrdquoInternational Journal of Chronic Obstructive Pulmonary Diseasevol 6 pp 181ndash189 2011
[35] C P Guan M N Zhou A E Xu et al ldquoThe susceptibilityto vitiligo is associated with NF-E2-related factor 2 (NRF2)gene polymorphisms a study on Chinese Han populationrdquoExperimental Dermatology vol 17 no 12 pp 1059ndash1062 2008
[36] J Bouligand O Cabaret M Canonico et al ldquoEffect of NFE2L2genetic polymorphism on the association between oral estrogentherapy and the risk of venous thromboembolism in post-menopausal womenrdquo Clinical Pharmacology amp Therapeuticsvol 89 no 1 pp 60ndash64 2011
[37] D S OrsquoMahony B J Glavan T D Holden C Fong R A BlackG Rona et al ldquoInflammation and immune-related candidategene associations with acute lung injury susceptibility andseverity a validation studyrdquo PLoS ONE vol 7 no 12 Article IDe51104 2012
[38] I Ungvari E Hadadi V Virag et al ldquoRelationship between airpollutionNFE2L2 gene polymorphisms and childhood asthmain aHungarian populationrdquo Journal of CommunityGenetics vol3 no 1 pp 25ndash33 2012
[39] M SiedlinskiD S Postma JMA Boer et al ldquoLevel and courseof FEV1 in relation to polymorphisms inNFE2L2 andKEAP1 inthe general populationrdquo Respiratory Research vol 10 article 732009
[40] C C Hong C B Ambrosone J Ahn et al ldquoGenetic variabilityin iron-related oxidative stress pathways (NRF2 NQ01 NOS3
and HO-1) iron intake and risk of postmenopausal breastcancerrdquo Cancer Epidemiology Biomarkers and Prevention vol16 no 9 pp 1784ndash1794 2007
[41] C Canova C Dunster F J Kelly C Minelli P L Shah CCaneja et al ldquoPM10-induced hospital admissions for asthmaand chronic obstructive pulmonary disease the modifyingeffect of individual characteristicsrdquo Epidemiology vol 23 no 4pp 607ndash615 2012
[42] A J Henderson R B Newson M Rose-Zerilli S M RingJ W Holloway and S O Shaheen ldquoMaternal NRF2 andgluthathione-S-transferase polymorphisms donotmodify asso-ciations of prenatal tobacco smoke exposure with asthma andlung function in school-aged childrenrdquo Thorax vol 65 no 10pp 897ndash902 2010
[43] C Xing A L Sestak J A Kelly et al ldquoLocalization andreplication of the systemic lupus erythematosus linkage signalat 4p16 interaction with 2p11 12q24 and 19q13 in EuropeanAmericansrdquo Human Genetics vol 120 no 5 pp 623ndash631 2007
[44] T F Wong K Yoshinaga Y Monma K Ito H NiikuraS Nagase et al ldquoAssociation of KEAP1 and NRF2 geneticmutations and polymorphisms with endometrioid endometrialadenocarcinoma survivalrdquo International Journal of Gynecologi-cal Cancer vol 21 no 8 pp 1428ndash1435 2011
[45] M von Otter S Landgren S Nilsson et al ldquoNRF2-encodingNFE2L2 haplotypes influence disease progression but not riskin Alzheimerrsquos disease and age-related cataractrdquoMechanisms ofAgeing and Development vol 131 no 2 pp 105ndash110 2010
[46] S Tsang Z Sun B Luke et al ldquoA comprehensive SNP-basedgenetic analysis of inbred mouse strainsrdquoMammalian Genomevol 16 no 7 pp 476ndash480 2005
[47] C M Wade E J Kulbokas III A W Kirby et al ldquoThe mosaicstructure of variation in the laboratory mouse genomerdquoNaturevol 420 no 6915 pp 574ndash578 2002
[48] T Wiltshire M T Pletcher S Batalov et al ldquoGenome-widesingle-nucleotide polymorphism analysis defines haplotypepatterns in mouserdquo Proceedings of the National Academy ofSciences of the United States of America vol 100 no 6 pp 3380ndash3385 2003
[49] K A Frazer E Eskin H M Kang et al ldquoA sequence-basedvariation map of 827 million SNPs in inbred mouse strainsrdquoNature vol 448 no 7157 pp 1050ndash1053 2007
[50] T P Maddatu S C Grubb C J Bult andM A Bogue ldquoMousephenome database (MPD)rdquo Nucleic Acids Research vol 40 ppD887ndashD894 2007
[51] A Kirby H M Kang C M Wade et al ldquoFine mapping in 94inbred mouse strains using a high-density haplotype resourcerdquoGenetics vol 185 no 3 pp 1081ndash1095 2010
[52] H Y Cho A E Jedlicka S P M Reddy L Y Zhang T WKensler and S R Kleeberger ldquoLinkage analysis of susceptibilityto hyperoxia NRF2 is a candidate generdquo American Journal ofRespiratory Cell and Molecular Biology vol 26 no 1 pp 42ndash512002
[53] H Y Cho A E Jedlicka S P M Reddy et al ldquoRole of NRF2in protection against hyperoxic lung injury in micerdquo AmericanJournal of Respiratory Cell and Molecular Biology vol 26 no 2pp 175ndash182 2002
[54] T Shibata T Ohta K I Tong et al ldquoCancer related mutationsin NRF2 impair its recognition by KEAP1-Cul3 E3 ligase andpromote malignancyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 36 pp13568ndash13573 2008
24 Oxidative Medicine and Cellular Longevity
[55] A Singh V Misra R K Thimmulappa et al ldquoDysfunctionalKEAP1-NRF2 interaction in non-small-cell lung cancerrdquo PLoSMedicine vol 3 no 10 article e420 2006
[56] T Ohta K Iijima M Miyamoto et al ldquoLoss of KEAP1 functionactivates NRF2 and provides advantages for lung cancer cellgrowthrdquo Cancer Research vol 68 no 5 pp 1303ndash1309 2008
[57] B Padmanabhan K I Tong T Ohta et al ldquoStructural basisfor defects of KEAP1 activity provoked by its point mutationsin lung cancerrdquoMolecular Cell vol 21 no 5 pp 689ndash700 2006
[58] T Shibata A Kokubu M Gotoh et al ldquoGenetic alteration ofKEAP1 confers constitutive NRF2 activation and resistance tochemotherapy in gallbladder cancerrdquoGastroenterology vol 135no 4 pp 1358e4ndash1368e4 2008
[59] N J Yoo H R Kim Y R Kim C H An and S H LeeldquoSomatic mutations of the KEAP1 gene in common solidcancersrdquo Histopathology vol 60 no 6 pp 943ndash952 2012
[60] Y Hu Y Ju D Lin Z Wang Y Huang S Zhang et alldquoMutation of the NRF2 gene in non-small cell lung cancerrdquoMolecular Biology Reports vol 39 no 4 pp 4743ndash4747 2012
[61] Y R Kim J EOhM S Kim et al ldquoOncogenicNRF2mutationsin squamous cell carcinomas of oesophagus and skinrdquo TheJournal of Pathology vol 220 no 4 pp 446ndash451 2010
[62] T Shibata A Kokubu S Saito et al ldquoNRF2 mutation confersmalignant potential and resistance to chemoradiation therapyin advanced esophageal squamous cancerrdquo Neoplasia vol 13pp 864ndash873 2011
[63] H Sasaki M Shitara K Yokota Y Hikosaka S MoriyamaM Yano et al ldquoIncreased NRF2 gene (NFE2L2) copy numbercorrelates with mutations in lung squamous cell carcinomasrdquoMolecular Medicine Reports vol 6 no 2 pp 391ndash394 2012
[64] H Sasaki M Shitara K Yokota Y Hikosaka S Moriyama MYano et al ldquoRagD gene expression andNRF2mutations in lungsquamous cell carcinomasrdquo Oncology Letters vol 4 pp 1167ndash1170 2012
[65] A Singh M Bodas N Wakabayashi F Bunz and S BiswalldquoGain of NRF2 function in non-small-cell lung cancer cellsconfers radioresistancerdquo Antioxidants and Redox Signaling vol13 no 11 pp 1627ndash1637 2010
[66] T Yamadori Y Ishii SHommaYMorishimaKKurishimaKItoh et al ldquoMolecular mechanisms for the regulation of NRF2-mediated cell proliferation in non-small-cell lung cancersrdquoOncogene vol 31 pp 4768ndash4777 2012
[67] A K Bauer H Y Cho L Miller-Degraff C Walker K HelmsJ Fostel et al ldquoTargeted deletion of NRF2 reduces urethane-induced lung tumor development in micerdquo PLoS ONE vol 6no 10 Article ID e26590 2011
[68] H Y Cho and S R Kleeberger ldquoNRF2 protects against airwaydisordersrdquo Toxicology and Applied Pharmacology vol 244 no1 pp 43ndash56 2010
[69] R Wang J An F Ji H Jiao H Sun and D Zhou ldquoHyperme-thylation of the KEAP1 gene in human lung cancer cell linesand lung cancer tissuesrdquo Biochemical and Biophysical ResearchCommunications vol 373 no 1 pp 151ndash154 2008
[70] P Zhang A Singh S Yegnasubramanian et al ldquoLoss ofkelch-like ECH-associated protein 1 function in prostate cancercells causes chemoresistance and radioresistance and promotestumor growthrdquoMolecular Cancer Therapeutics vol 9 no 2 pp336ndash346 2010
[71] N Hanada T Takahata Q Zhou et al ldquoMethylation of theKEAP1 gene promoter region in human colorectal cancerrdquoBMCCancer vol 12 article 66 2012
[72] L A Muscarella R Barbano V DrsquoAngelo et al ldquoRegulationof KEAP1 expression by promoter methylation in malignantgliomas and association with patientrsquos outcomerdquo Epigeneticsvol 6 no 3 pp 317ndash325 2011
[73] S Yu T O Khor K L Cheung et al ldquoNRF2 expressionis regulated by epigenetic mechanisms in prostate cancer ofTRAMP micerdquo PLoS ONE vol 5 no 1 Article ID e8579 2010
[74] T O Khor Y Huang T Y Wu L Shu J Lee and A N KongldquoPharmacodynamics of curcumin as DNA hypomethylationagent in restoring the expression of NRF2 via promoter CpGsdemethylationrdquo Biochemical Pharmacology vol 82 no 9 pp1073ndash1078 2011
[75] I M Fingerman L McDaniel X Zhang et al ldquoNCBI epige-nomics a new public resource for exploring epigenomic datasetsrdquo Nucleic Acids Research vol 39 supplemen 1 pp D908ndashD912 2011
[76] I M Fingerman X Zhang W Ratzat N Husain R F Cohenand G D Schuler ldquoNCBI epigenomics whatrsquos new for 2013rdquoNucleic Acids Research vol 41 no 1 pp D221ndashD225 2013
[77] J H Lee T O Khor L Shu Z Y Su F Fuentes andA N Kong ldquoDietary phytochemicals and cancer preventionNRF2 signaling epigenetics and cell death mechanisms inblocking cancer initiation and progressionrdquo Pharmacology ampTherapeutics vol 137 no 2 pp 153ndash171 2012
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
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Diabetes ResearchJournal of
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Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Oxidative Medicine and Cellular Longevity 21
processes are essential in the initiation stage enhancedNRF2-ARE activity in advanced stages of cancer developmentmay create a favorable intracellular environment for tumorcell growth and survival [67 68] In this context NRF2may be a potential molecular target for the treatment ofradio-resistance cancers especially those that have ldquoloss offunctionrdquo mutations in EGFR KRAS or KEAP1 as well asldquogain of functionrdquo mutations in NRF2
5 Epigenetic Alterations of NF-E2-RelatedFactor 2
Epigenetic modifications are alterations of molecules inter-acting with genes without changes to the primary DNAsequence They include post-translational modification ofhistones DNA methylation events chromatin conforma-tional changes and alterations to noncoding regulatoryRNAs Epigenetic alterations are stable and often inheritablebut are reversible and may affect expression of the geneDysregulation or defects in epigenetic processes particularlyhypermethylation of tumor suppressor gene promoters (egCpG islands) or histone modifications are thought to beassociated with carcinogenesis Investigators have reportedhypermethylation in CpG islands of KEAP1 which wereassociated with reduced KEAP1 expression in human cancersfrom lung prostate colon and so forth [69ndash72] Similar tosomatic mutations epigenetic changes on KEAP1 impairedthe function of its encoded protein leading to constitutiveNRF2 activation
Supporting the role of ldquopathogenic mutationsrdquo in NRF2expression of Nrf2 and downstream Nqo1 was suppressedin prostate tumors of mice (transgenic adenocarcinomaof mouse prostate TRAMP) Among 15 promoter CpGislands located between minus942 and minus654 (cminus1175 cminus1132and cminus1059 cminus887 gap in cminus1131 cminus1060 see Figure 3)hypermethylation of the first 5 CpG islands (minus942 minus899 andcminus1175 cminus1132) was significantly associated with tumorige-nesis [73] Moreover treatment with inhibitors for DNAmethyltransferase and histone deacetylase restored Nrf2expression in these tumor cells [73] A dietary phytochemicalcurcumin known as a DNA hypomethylation agent restoredepigenetically silent Nrf2 expression through CpG demethy-lation in carcinogen-induced mouse tumor cells [74]
The whole genome epigenetic datasets for 5 species arepublicly accessible at NCBI Epigenomics [75 76]The humanNRF2 epigenome of primary cells (breast penis) and H1stem cell line as well as mouse Nrf2 CpG island methylationdata for sperm blood and cerebellum are currently avail-able (httpwwwncbinlmnihgovepigenomics) Althoughno direct evidence of disease-associated epigenetic modu-lation has been identified in human NRF2 various phyto-chemical NRF2 agonists such as sulforaphane and curcuminhave shown their roles in DNA methylation and histonemodification (see reviews by Lee and colleagues eg [77])Taken together epigenetic modifications of theNRF2KEAP1axle are predicted to cause dysregulation of ARE-mediatedcellular defense leading to deleterious health effects and
phytochemical antioxidants as epigenetic modulators forNRF2 are suggested to be useful in cancer prevention
6 Conclusions
NRF2 is evolutionally conserved with high-sequence homol-ogy in many species However it is a highly mutable geneand numerous genetic variants have been discovered inhuman ethnic groups Importantly certain SNPs or haplo-types have been identified in various diseases as ldquoat-riskrdquoalleles and are related to functional alterations In additionto genetic variations multiple somatic mutations identifiedin the KEAP1 recognition domain of NRF2 in cancer cellshave been found to be oncogenic due to dysregulation ofNRF2 homeostasis by its excess ldquogain of functionrdquo Epigeneticalteration of theNRF2 is under investigation and is predictedto have pathogenic influences as learned from mouse andphytochemical agonist studies Continuous updates of Nrf2allelic variants in inbred mouse strains will provide a usefultool for effective experimental designs formodels of oxidativedisorders to provide insight into the disease mechanisms andintervention strategies
Abbreviations
AD Alzheimerrsquos diseaseALI acute lung injuryARE antioxidant response elementbZIP basic leucine zippercds coding DNA sequencesCI confidence intervalCOPD chronic obstructive pulmonary diseaseESC esophageal squamous cancerFEV1 forced expiratory volume in one second
Gbp giga base pairsGST glutathione S-transferaseGWAS genome-wide association studyHGVS Human Genome Variation SocietyHO-1 heme oxygenase-1119867 119901119910119897119900119903119894 119867119890119897119894119888119900119887119886119888119905119890119903 119901119910119897119900119903119894KEAP1 Kelch-like ECH activating protein 1MPD Mouse Phenome DatabaseMRP multidrug resistance proteinNCBI National Center for Biotechnology InformationNeh NRF2-ECH homologyNfe2l2 nuclear factor (erythroid-derived 2)-like 2Nrf2 NF-E2-related factor 2NSCLC nonsmall cell lung cancerNQO1 NAD(P)Hquinone oxidoreductase 1OR odds ratioPD Parkinsonrsquos diseasePM particulate matterROS reactive oxygen speciesSLE systemic lupus erythematosusSNP single nucleotide polymorphismSOD superoxide dismutaseURT untranslated region
22 Oxidative Medicine and Cellular Longevity
Disclosure
Authorrsquos contribution to the Work was done as part of theAuthorrsquos official duties as a NIH employee and is a Work ofthe United States Government Therefore copyright may notbe established in the United States
Conflict of Interests
The author declares that there is no conflict of interests
Acknowledgments
The research related to this paper was supported by theIntramural Research Program of the National Institutes ofHealth of the National Institute of Environmental HealthSciences (NIEHS) Drs Steven Kleeberger and StephanieLondon at the NIEHS provided excellent critical review ofthis paper The author thanks Mrs Jacqui Marzec for herhelpful comments and English editing
References
[1] J Y Chan M C Cheung P Moi K Chan and Y W KanldquoChromosomal localization of the humanNF-E2 family of bZIPtranscription factors by fluorescence in situ hybridizationrdquoHuman Genetics vol 95 no 3 pp 265ndash269 1995
[2] K Itoh K Igarashi N Hayashi M Nishizawa and MYamamoto ldquoCloning and characterization of a novel erythroidcell-derived CNC family transcription factor heterodimerizingwith the small Maf family proteinsrdquo Molecular and CellularBiology vol 15 no 8 pp 4184ndash4193 1995
[3] W O Osburn and T W Kensler ldquoNRF2 signaling an adaptiveresponse pathway for protection against environmental toxicinsultsrdquoMutation Research vol 659 no 1-2 pp 31ndash39 2008
[4] D Papp K Lenti D Modos D Fazekas Z Dul D Tureiet al ldquoThe NRF2-related interactome and regulome containmultifunctional proteins and fine-tuned autoregulatory loopsrdquoFEBS Letters vol 586 no 13 pp 1795ndash1802 2012
[5] X Wang D J Tomso B N Chorley et al ldquoIdentificationof polymorphic antioxidant response elements in the humangenomerdquo Human Molecular Genetics vol 16 no 10 pp 1188ndash1200 2007
[6] D Malhotra E Portales-Casamar A Singh et al ldquoGlobalmapping of binding sites for NRF2 identifies novel targets incell survival response through ChIP-Seq profiling and networkanalysisrdquo Nucleic Acids Research vol 38 no 17 pp 5718ndash57342010
[7] K Chan R Lu J C Chang and Y W Kan ldquoNRF2 a memberof the NFE2 family of transcription factors is not essential formurine erythropoiesis growth and developmentrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 93 no 24 pp 13943ndash13948 1996
[8] K Itoh T Chiba S Takahashi et al ldquoAn NRF2small Mafheterodimer mediates the induction of phase II detoxifyingenzyme genes through antioxidant response elementsrdquo Bio-chemical and Biophysical Research Communications vol 236no 2 pp 313ndash322 1997
[9] FMartin J M vanDeursen R A Shivdasani CW Jackson AG Troutman and P A Ney ldquoErythroid maturation and globin
gene expression inmicewith combined deficiency ofNF-E2 andNrf-2rdquo Blood vol 91 no 9 pp 3459ndash3466 1998
[10] K Itoh N Wakabayashi Y Katoh et al ldquoKEAP1 repressesnuclear activation of antioxidant responsive elements by NRF2through binding to the amino-terminal Neh2 domainrdquo Genesand Development vol 13 no 1 pp 76ndash86 1999
[11] A Kobayashi M I Kang H Okawa et al ldquoOxidative stresssensor KEAP1 functions as an adaptor for Cul3-based E3 ligaseto regulate proteasomal degradation of NRF2rdquo Molecular andCellular Biology vol 24 no 16 pp 7130ndash7139 2004
[12] K Taguchi H Motohashi and M Yamamoto ldquoMolecularmechanisms of the KEAP1-NRF2 pathway in stress responseand cancer evolutionrdquo Genes to Cells vol 16 no 2 pp 123ndash1402011
[13] K Mukaigasa L T Nguyen L Li H Nakajima M Yamamotoand M Kobayashi ldquoGenetic evidence of an evolutionarilyconserved role for NRF2 in the protection against oxidativestressrdquoMolecular and Cellular Biology vol 32 no 21 pp 4455ndash4461 2012
[14] JMaher andMYamamoto ldquoThe rise of antioxidant signalingmdashthe evolution and hormetic actions of NRF2rdquo Toxicology andApplied Pharmacology vol 244 no 1 pp 4ndash15 2010
[15] S C Lo X Li M T Henzl L J Beamer and M HanninkldquoStructure of the KEAP1NRF2 interface provides mechanisticinsight intoNRF2 signalingrdquoThe EMBO Journal vol 25 no 15pp 3605ndash3617 2006
[16] V A McKusick ldquoMendelian inheritance in man and its onlineversion OMIMrdquo American Journal of Human Genetics vol 80no 4 pp 588ndash604 2007
[17] B Yalcin D J Adams J Flint and T M Keane ldquoNext-generation sequencing of experimental mouse strainsrdquo Mam-malian Genome vol 23 no 9-10 pp 490ndash498 2012
[18] D R Bentley S Balasubramanian H P Swerdlow et alldquoAccurate whole human genome sequencing using reversibleterminator chemistryrdquo Nature vol 456 no 7218 pp 53ndash592008
[19] P S G Chain D V Grafham R S Fulton et al ldquoGenomeproject standards in a new era of sequencingrdquo Science vol 326no 5950 pp 236ndash237 2009
[20] RHWaterstonK Lindblad-Toh E Birney J Rogers J F AbrilP Agarwal et al ldquoInitial sequencing and comparative analysis ofthe mouse genomerdquo Nature vol 420 pp 520ndash562 2002
[21] T M Keane L Goodstadt P Danecek M A White K WongB Yalcin et al ldquoMouse genomic variation and its effect onphenotypes and gene regulationrdquoNature vol 477 pp 289ndash2942011
[22] T Yamamoto K Yoh A Kobayashi et al ldquoIdentification ofpolymorphisms in the promoter region of the human NRF2generdquo Biochemical and Biophysical Research Communicationsvol 321 no 1 pp 72ndash79 2004
[23] J M Marzec J D Christie S P Reddy et al ldquoFunctionalpolymorphisms in the transcription factor NRF2 in humansincrease the risk of acute lung injuryrdquo The FASEB Journal vol21 no 9 pp 2237ndash2246 2007
[24] H Fukushima-Uesaka Y Saito K Maekawa et al ldquoGeneticvariations and haplotype structures of transcriptional factorNRF2 and its cytosolic reservoir protein KEAP1 in JapaneserdquoDrug Metabolism and Pharmacokinetics vol 22 no 3 pp 212ndash219 2007
[25] E J Cordova R Velazquez-Cruz F Centeno V Baca and LOrozco ldquoThe NRF2 gene variant -653GA is associated with
Oxidative Medicine and Cellular Longevity 23
nephritis in childhood-onset systemic lupus erythematosusrdquoLupus vol 19 no 10 pp 1237ndash1242 2010
[26] M von Otter S Landgren S Nilsson et al ldquoAssociation ofNRF2-encoding NFE2L2 haplotypes with Parkinsonrsquos diseaserdquoBMCMedical Genetics vol 11 no 1 article 36 2010
[27] JMHartikainenM TengstromVMKosmaV L Kinnula AMannermaa andY Soini ldquoGenetic polymorphisms and proteinexpression of NRF2 and sulfiredoxin predict survival outcomesin breast cancerrdquo Cancer Research vol 72 pp 5537ndash5546 2012
[28] T Arisawa T Tahara T Shibata et al ldquoThe relationshipbetween Helicobacter pylori infection and promoter polymor-phism of the NRF2 gene in chronic gastritisrdquo InternationalJournal of Molecular Medicine vol 19 no 1 pp 143ndash148 2007
[29] T Arisawa T Tahara T Shibata et al ldquoThe influence ofpromoter polymorphism of nuclear factor-erythroid 2-relatedfactor 2 gene on the aberrant DNA methylation in gastricepitheliumrdquo Oncology Reports vol 19 no 1 pp 211ndash216 2008
[30] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene pro-moter polymorphism and gastric carcinogenesisrdquo Hepato-Gastroenterology vol 55 no 82-83 pp 750ndash754 2008
[31] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene promoterpolymorphism is associated with ulcerative colitis in a Japanesepopulationrdquo Hepato-Gastroenterology vol 55 no 82-83 pp394ndash397 2008
[32] C C Hua L C Chang J C Tseng C M Chu Y C Liu andW B Shieh ldquoFunctional haplotypes in the promoter region oftranscription factor NRF2 in chronic obstructive pulmonarydiseaserdquo Disease Markers vol 28 no 3 pp 185ndash193 2010
[33] S O Shaheen R B Newson S M Ring M J Rose-ZerilliJ W Holloway and A J Henderson ldquoPrenatal and infantacetaminophen exposure antioxidant gene polymorphismsand childhood asthmardquo The Journal of Allergy and ClinicalImmunology vol 126 no 6 pp 1141e7ndash1148e7 2010
[34] H Masuko T Sakamoto Y Kaneko et al ldquoLower FEV1 innon-COPD nonasthmatic subjects association with smokingannual decline in FEV1 total IgE levels and TSLP genotypesrdquoInternational Journal of Chronic Obstructive Pulmonary Diseasevol 6 pp 181ndash189 2011
[35] C P Guan M N Zhou A E Xu et al ldquoThe susceptibilityto vitiligo is associated with NF-E2-related factor 2 (NRF2)gene polymorphisms a study on Chinese Han populationrdquoExperimental Dermatology vol 17 no 12 pp 1059ndash1062 2008
[36] J Bouligand O Cabaret M Canonico et al ldquoEffect of NFE2L2genetic polymorphism on the association between oral estrogentherapy and the risk of venous thromboembolism in post-menopausal womenrdquo Clinical Pharmacology amp Therapeuticsvol 89 no 1 pp 60ndash64 2011
[37] D S OrsquoMahony B J Glavan T D Holden C Fong R A BlackG Rona et al ldquoInflammation and immune-related candidategene associations with acute lung injury susceptibility andseverity a validation studyrdquo PLoS ONE vol 7 no 12 Article IDe51104 2012
[38] I Ungvari E Hadadi V Virag et al ldquoRelationship between airpollutionNFE2L2 gene polymorphisms and childhood asthmain aHungarian populationrdquo Journal of CommunityGenetics vol3 no 1 pp 25ndash33 2012
[39] M SiedlinskiD S Postma JMA Boer et al ldquoLevel and courseof FEV1 in relation to polymorphisms inNFE2L2 andKEAP1 inthe general populationrdquo Respiratory Research vol 10 article 732009
[40] C C Hong C B Ambrosone J Ahn et al ldquoGenetic variabilityin iron-related oxidative stress pathways (NRF2 NQ01 NOS3
and HO-1) iron intake and risk of postmenopausal breastcancerrdquo Cancer Epidemiology Biomarkers and Prevention vol16 no 9 pp 1784ndash1794 2007
[41] C Canova C Dunster F J Kelly C Minelli P L Shah CCaneja et al ldquoPM10-induced hospital admissions for asthmaand chronic obstructive pulmonary disease the modifyingeffect of individual characteristicsrdquo Epidemiology vol 23 no 4pp 607ndash615 2012
[42] A J Henderson R B Newson M Rose-Zerilli S M RingJ W Holloway and S O Shaheen ldquoMaternal NRF2 andgluthathione-S-transferase polymorphisms donotmodify asso-ciations of prenatal tobacco smoke exposure with asthma andlung function in school-aged childrenrdquo Thorax vol 65 no 10pp 897ndash902 2010
[43] C Xing A L Sestak J A Kelly et al ldquoLocalization andreplication of the systemic lupus erythematosus linkage signalat 4p16 interaction with 2p11 12q24 and 19q13 in EuropeanAmericansrdquo Human Genetics vol 120 no 5 pp 623ndash631 2007
[44] T F Wong K Yoshinaga Y Monma K Ito H NiikuraS Nagase et al ldquoAssociation of KEAP1 and NRF2 geneticmutations and polymorphisms with endometrioid endometrialadenocarcinoma survivalrdquo International Journal of Gynecologi-cal Cancer vol 21 no 8 pp 1428ndash1435 2011
[45] M von Otter S Landgren S Nilsson et al ldquoNRF2-encodingNFE2L2 haplotypes influence disease progression but not riskin Alzheimerrsquos disease and age-related cataractrdquoMechanisms ofAgeing and Development vol 131 no 2 pp 105ndash110 2010
[46] S Tsang Z Sun B Luke et al ldquoA comprehensive SNP-basedgenetic analysis of inbred mouse strainsrdquoMammalian Genomevol 16 no 7 pp 476ndash480 2005
[47] C M Wade E J Kulbokas III A W Kirby et al ldquoThe mosaicstructure of variation in the laboratory mouse genomerdquoNaturevol 420 no 6915 pp 574ndash578 2002
[48] T Wiltshire M T Pletcher S Batalov et al ldquoGenome-widesingle-nucleotide polymorphism analysis defines haplotypepatterns in mouserdquo Proceedings of the National Academy ofSciences of the United States of America vol 100 no 6 pp 3380ndash3385 2003
[49] K A Frazer E Eskin H M Kang et al ldquoA sequence-basedvariation map of 827 million SNPs in inbred mouse strainsrdquoNature vol 448 no 7157 pp 1050ndash1053 2007
[50] T P Maddatu S C Grubb C J Bult andM A Bogue ldquoMousephenome database (MPD)rdquo Nucleic Acids Research vol 40 ppD887ndashD894 2007
[51] A Kirby H M Kang C M Wade et al ldquoFine mapping in 94inbred mouse strains using a high-density haplotype resourcerdquoGenetics vol 185 no 3 pp 1081ndash1095 2010
[52] H Y Cho A E Jedlicka S P M Reddy L Y Zhang T WKensler and S R Kleeberger ldquoLinkage analysis of susceptibilityto hyperoxia NRF2 is a candidate generdquo American Journal ofRespiratory Cell and Molecular Biology vol 26 no 1 pp 42ndash512002
[53] H Y Cho A E Jedlicka S P M Reddy et al ldquoRole of NRF2in protection against hyperoxic lung injury in micerdquo AmericanJournal of Respiratory Cell and Molecular Biology vol 26 no 2pp 175ndash182 2002
[54] T Shibata T Ohta K I Tong et al ldquoCancer related mutationsin NRF2 impair its recognition by KEAP1-Cul3 E3 ligase andpromote malignancyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 36 pp13568ndash13573 2008
24 Oxidative Medicine and Cellular Longevity
[55] A Singh V Misra R K Thimmulappa et al ldquoDysfunctionalKEAP1-NRF2 interaction in non-small-cell lung cancerrdquo PLoSMedicine vol 3 no 10 article e420 2006
[56] T Ohta K Iijima M Miyamoto et al ldquoLoss of KEAP1 functionactivates NRF2 and provides advantages for lung cancer cellgrowthrdquo Cancer Research vol 68 no 5 pp 1303ndash1309 2008
[57] B Padmanabhan K I Tong T Ohta et al ldquoStructural basisfor defects of KEAP1 activity provoked by its point mutationsin lung cancerrdquoMolecular Cell vol 21 no 5 pp 689ndash700 2006
[58] T Shibata A Kokubu M Gotoh et al ldquoGenetic alteration ofKEAP1 confers constitutive NRF2 activation and resistance tochemotherapy in gallbladder cancerrdquoGastroenterology vol 135no 4 pp 1358e4ndash1368e4 2008
[59] N J Yoo H R Kim Y R Kim C H An and S H LeeldquoSomatic mutations of the KEAP1 gene in common solidcancersrdquo Histopathology vol 60 no 6 pp 943ndash952 2012
[60] Y Hu Y Ju D Lin Z Wang Y Huang S Zhang et alldquoMutation of the NRF2 gene in non-small cell lung cancerrdquoMolecular Biology Reports vol 39 no 4 pp 4743ndash4747 2012
[61] Y R Kim J EOhM S Kim et al ldquoOncogenicNRF2mutationsin squamous cell carcinomas of oesophagus and skinrdquo TheJournal of Pathology vol 220 no 4 pp 446ndash451 2010
[62] T Shibata A Kokubu S Saito et al ldquoNRF2 mutation confersmalignant potential and resistance to chemoradiation therapyin advanced esophageal squamous cancerrdquo Neoplasia vol 13pp 864ndash873 2011
[63] H Sasaki M Shitara K Yokota Y Hikosaka S MoriyamaM Yano et al ldquoIncreased NRF2 gene (NFE2L2) copy numbercorrelates with mutations in lung squamous cell carcinomasrdquoMolecular Medicine Reports vol 6 no 2 pp 391ndash394 2012
[64] H Sasaki M Shitara K Yokota Y Hikosaka S Moriyama MYano et al ldquoRagD gene expression andNRF2mutations in lungsquamous cell carcinomasrdquo Oncology Letters vol 4 pp 1167ndash1170 2012
[65] A Singh M Bodas N Wakabayashi F Bunz and S BiswalldquoGain of NRF2 function in non-small-cell lung cancer cellsconfers radioresistancerdquo Antioxidants and Redox Signaling vol13 no 11 pp 1627ndash1637 2010
[66] T Yamadori Y Ishii SHommaYMorishimaKKurishimaKItoh et al ldquoMolecular mechanisms for the regulation of NRF2-mediated cell proliferation in non-small-cell lung cancersrdquoOncogene vol 31 pp 4768ndash4777 2012
[67] A K Bauer H Y Cho L Miller-Degraff C Walker K HelmsJ Fostel et al ldquoTargeted deletion of NRF2 reduces urethane-induced lung tumor development in micerdquo PLoS ONE vol 6no 10 Article ID e26590 2011
[68] H Y Cho and S R Kleeberger ldquoNRF2 protects against airwaydisordersrdquo Toxicology and Applied Pharmacology vol 244 no1 pp 43ndash56 2010
[69] R Wang J An F Ji H Jiao H Sun and D Zhou ldquoHyperme-thylation of the KEAP1 gene in human lung cancer cell linesand lung cancer tissuesrdquo Biochemical and Biophysical ResearchCommunications vol 373 no 1 pp 151ndash154 2008
[70] P Zhang A Singh S Yegnasubramanian et al ldquoLoss ofkelch-like ECH-associated protein 1 function in prostate cancercells causes chemoresistance and radioresistance and promotestumor growthrdquoMolecular Cancer Therapeutics vol 9 no 2 pp336ndash346 2010
[71] N Hanada T Takahata Q Zhou et al ldquoMethylation of theKEAP1 gene promoter region in human colorectal cancerrdquoBMCCancer vol 12 article 66 2012
[72] L A Muscarella R Barbano V DrsquoAngelo et al ldquoRegulationof KEAP1 expression by promoter methylation in malignantgliomas and association with patientrsquos outcomerdquo Epigeneticsvol 6 no 3 pp 317ndash325 2011
[73] S Yu T O Khor K L Cheung et al ldquoNRF2 expressionis regulated by epigenetic mechanisms in prostate cancer ofTRAMP micerdquo PLoS ONE vol 5 no 1 Article ID e8579 2010
[74] T O Khor Y Huang T Y Wu L Shu J Lee and A N KongldquoPharmacodynamics of curcumin as DNA hypomethylationagent in restoring the expression of NRF2 via promoter CpGsdemethylationrdquo Biochemical Pharmacology vol 82 no 9 pp1073ndash1078 2011
[75] I M Fingerman L McDaniel X Zhang et al ldquoNCBI epige-nomics a new public resource for exploring epigenomic datasetsrdquo Nucleic Acids Research vol 39 supplemen 1 pp D908ndashD912 2011
[76] I M Fingerman X Zhang W Ratzat N Husain R F Cohenand G D Schuler ldquoNCBI epigenomics whatrsquos new for 2013rdquoNucleic Acids Research vol 41 no 1 pp D221ndashD225 2013
[77] J H Lee T O Khor L Shu Z Y Su F Fuentes andA N Kong ldquoDietary phytochemicals and cancer preventionNRF2 signaling epigenetics and cell death mechanisms inblocking cancer initiation and progressionrdquo Pharmacology ampTherapeutics vol 137 no 2 pp 153ndash171 2012
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
22 Oxidative Medicine and Cellular Longevity
Disclosure
Authorrsquos contribution to the Work was done as part of theAuthorrsquos official duties as a NIH employee and is a Work ofthe United States Government Therefore copyright may notbe established in the United States
Conflict of Interests
The author declares that there is no conflict of interests
Acknowledgments
The research related to this paper was supported by theIntramural Research Program of the National Institutes ofHealth of the National Institute of Environmental HealthSciences (NIEHS) Drs Steven Kleeberger and StephanieLondon at the NIEHS provided excellent critical review ofthis paper The author thanks Mrs Jacqui Marzec for herhelpful comments and English editing
References
[1] J Y Chan M C Cheung P Moi K Chan and Y W KanldquoChromosomal localization of the humanNF-E2 family of bZIPtranscription factors by fluorescence in situ hybridizationrdquoHuman Genetics vol 95 no 3 pp 265ndash269 1995
[2] K Itoh K Igarashi N Hayashi M Nishizawa and MYamamoto ldquoCloning and characterization of a novel erythroidcell-derived CNC family transcription factor heterodimerizingwith the small Maf family proteinsrdquo Molecular and CellularBiology vol 15 no 8 pp 4184ndash4193 1995
[3] W O Osburn and T W Kensler ldquoNRF2 signaling an adaptiveresponse pathway for protection against environmental toxicinsultsrdquoMutation Research vol 659 no 1-2 pp 31ndash39 2008
[4] D Papp K Lenti D Modos D Fazekas Z Dul D Tureiet al ldquoThe NRF2-related interactome and regulome containmultifunctional proteins and fine-tuned autoregulatory loopsrdquoFEBS Letters vol 586 no 13 pp 1795ndash1802 2012
[5] X Wang D J Tomso B N Chorley et al ldquoIdentificationof polymorphic antioxidant response elements in the humangenomerdquo Human Molecular Genetics vol 16 no 10 pp 1188ndash1200 2007
[6] D Malhotra E Portales-Casamar A Singh et al ldquoGlobalmapping of binding sites for NRF2 identifies novel targets incell survival response through ChIP-Seq profiling and networkanalysisrdquo Nucleic Acids Research vol 38 no 17 pp 5718ndash57342010
[7] K Chan R Lu J C Chang and Y W Kan ldquoNRF2 a memberof the NFE2 family of transcription factors is not essential formurine erythropoiesis growth and developmentrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 93 no 24 pp 13943ndash13948 1996
[8] K Itoh T Chiba S Takahashi et al ldquoAn NRF2small Mafheterodimer mediates the induction of phase II detoxifyingenzyme genes through antioxidant response elementsrdquo Bio-chemical and Biophysical Research Communications vol 236no 2 pp 313ndash322 1997
[9] FMartin J M vanDeursen R A Shivdasani CW Jackson AG Troutman and P A Ney ldquoErythroid maturation and globin
gene expression inmicewith combined deficiency ofNF-E2 andNrf-2rdquo Blood vol 91 no 9 pp 3459ndash3466 1998
[10] K Itoh N Wakabayashi Y Katoh et al ldquoKEAP1 repressesnuclear activation of antioxidant responsive elements by NRF2through binding to the amino-terminal Neh2 domainrdquo Genesand Development vol 13 no 1 pp 76ndash86 1999
[11] A Kobayashi M I Kang H Okawa et al ldquoOxidative stresssensor KEAP1 functions as an adaptor for Cul3-based E3 ligaseto regulate proteasomal degradation of NRF2rdquo Molecular andCellular Biology vol 24 no 16 pp 7130ndash7139 2004
[12] K Taguchi H Motohashi and M Yamamoto ldquoMolecularmechanisms of the KEAP1-NRF2 pathway in stress responseand cancer evolutionrdquo Genes to Cells vol 16 no 2 pp 123ndash1402011
[13] K Mukaigasa L T Nguyen L Li H Nakajima M Yamamotoand M Kobayashi ldquoGenetic evidence of an evolutionarilyconserved role for NRF2 in the protection against oxidativestressrdquoMolecular and Cellular Biology vol 32 no 21 pp 4455ndash4461 2012
[14] JMaher andMYamamoto ldquoThe rise of antioxidant signalingmdashthe evolution and hormetic actions of NRF2rdquo Toxicology andApplied Pharmacology vol 244 no 1 pp 4ndash15 2010
[15] S C Lo X Li M T Henzl L J Beamer and M HanninkldquoStructure of the KEAP1NRF2 interface provides mechanisticinsight intoNRF2 signalingrdquoThe EMBO Journal vol 25 no 15pp 3605ndash3617 2006
[16] V A McKusick ldquoMendelian inheritance in man and its onlineversion OMIMrdquo American Journal of Human Genetics vol 80no 4 pp 588ndash604 2007
[17] B Yalcin D J Adams J Flint and T M Keane ldquoNext-generation sequencing of experimental mouse strainsrdquo Mam-malian Genome vol 23 no 9-10 pp 490ndash498 2012
[18] D R Bentley S Balasubramanian H P Swerdlow et alldquoAccurate whole human genome sequencing using reversibleterminator chemistryrdquo Nature vol 456 no 7218 pp 53ndash592008
[19] P S G Chain D V Grafham R S Fulton et al ldquoGenomeproject standards in a new era of sequencingrdquo Science vol 326no 5950 pp 236ndash237 2009
[20] RHWaterstonK Lindblad-Toh E Birney J Rogers J F AbrilP Agarwal et al ldquoInitial sequencing and comparative analysis ofthe mouse genomerdquo Nature vol 420 pp 520ndash562 2002
[21] T M Keane L Goodstadt P Danecek M A White K WongB Yalcin et al ldquoMouse genomic variation and its effect onphenotypes and gene regulationrdquoNature vol 477 pp 289ndash2942011
[22] T Yamamoto K Yoh A Kobayashi et al ldquoIdentification ofpolymorphisms in the promoter region of the human NRF2generdquo Biochemical and Biophysical Research Communicationsvol 321 no 1 pp 72ndash79 2004
[23] J M Marzec J D Christie S P Reddy et al ldquoFunctionalpolymorphisms in the transcription factor NRF2 in humansincrease the risk of acute lung injuryrdquo The FASEB Journal vol21 no 9 pp 2237ndash2246 2007
[24] H Fukushima-Uesaka Y Saito K Maekawa et al ldquoGeneticvariations and haplotype structures of transcriptional factorNRF2 and its cytosolic reservoir protein KEAP1 in JapaneserdquoDrug Metabolism and Pharmacokinetics vol 22 no 3 pp 212ndash219 2007
[25] E J Cordova R Velazquez-Cruz F Centeno V Baca and LOrozco ldquoThe NRF2 gene variant -653GA is associated with
Oxidative Medicine and Cellular Longevity 23
nephritis in childhood-onset systemic lupus erythematosusrdquoLupus vol 19 no 10 pp 1237ndash1242 2010
[26] M von Otter S Landgren S Nilsson et al ldquoAssociation ofNRF2-encoding NFE2L2 haplotypes with Parkinsonrsquos diseaserdquoBMCMedical Genetics vol 11 no 1 article 36 2010
[27] JMHartikainenM TengstromVMKosmaV L Kinnula AMannermaa andY Soini ldquoGenetic polymorphisms and proteinexpression of NRF2 and sulfiredoxin predict survival outcomesin breast cancerrdquo Cancer Research vol 72 pp 5537ndash5546 2012
[28] T Arisawa T Tahara T Shibata et al ldquoThe relationshipbetween Helicobacter pylori infection and promoter polymor-phism of the NRF2 gene in chronic gastritisrdquo InternationalJournal of Molecular Medicine vol 19 no 1 pp 143ndash148 2007
[29] T Arisawa T Tahara T Shibata et al ldquoThe influence ofpromoter polymorphism of nuclear factor-erythroid 2-relatedfactor 2 gene on the aberrant DNA methylation in gastricepitheliumrdquo Oncology Reports vol 19 no 1 pp 211ndash216 2008
[30] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene pro-moter polymorphism and gastric carcinogenesisrdquo Hepato-Gastroenterology vol 55 no 82-83 pp 750ndash754 2008
[31] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene promoterpolymorphism is associated with ulcerative colitis in a Japanesepopulationrdquo Hepato-Gastroenterology vol 55 no 82-83 pp394ndash397 2008
[32] C C Hua L C Chang J C Tseng C M Chu Y C Liu andW B Shieh ldquoFunctional haplotypes in the promoter region oftranscription factor NRF2 in chronic obstructive pulmonarydiseaserdquo Disease Markers vol 28 no 3 pp 185ndash193 2010
[33] S O Shaheen R B Newson S M Ring M J Rose-ZerilliJ W Holloway and A J Henderson ldquoPrenatal and infantacetaminophen exposure antioxidant gene polymorphismsand childhood asthmardquo The Journal of Allergy and ClinicalImmunology vol 126 no 6 pp 1141e7ndash1148e7 2010
[34] H Masuko T Sakamoto Y Kaneko et al ldquoLower FEV1 innon-COPD nonasthmatic subjects association with smokingannual decline in FEV1 total IgE levels and TSLP genotypesrdquoInternational Journal of Chronic Obstructive Pulmonary Diseasevol 6 pp 181ndash189 2011
[35] C P Guan M N Zhou A E Xu et al ldquoThe susceptibilityto vitiligo is associated with NF-E2-related factor 2 (NRF2)gene polymorphisms a study on Chinese Han populationrdquoExperimental Dermatology vol 17 no 12 pp 1059ndash1062 2008
[36] J Bouligand O Cabaret M Canonico et al ldquoEffect of NFE2L2genetic polymorphism on the association between oral estrogentherapy and the risk of venous thromboembolism in post-menopausal womenrdquo Clinical Pharmacology amp Therapeuticsvol 89 no 1 pp 60ndash64 2011
[37] D S OrsquoMahony B J Glavan T D Holden C Fong R A BlackG Rona et al ldquoInflammation and immune-related candidategene associations with acute lung injury susceptibility andseverity a validation studyrdquo PLoS ONE vol 7 no 12 Article IDe51104 2012
[38] I Ungvari E Hadadi V Virag et al ldquoRelationship between airpollutionNFE2L2 gene polymorphisms and childhood asthmain aHungarian populationrdquo Journal of CommunityGenetics vol3 no 1 pp 25ndash33 2012
[39] M SiedlinskiD S Postma JMA Boer et al ldquoLevel and courseof FEV1 in relation to polymorphisms inNFE2L2 andKEAP1 inthe general populationrdquo Respiratory Research vol 10 article 732009
[40] C C Hong C B Ambrosone J Ahn et al ldquoGenetic variabilityin iron-related oxidative stress pathways (NRF2 NQ01 NOS3
and HO-1) iron intake and risk of postmenopausal breastcancerrdquo Cancer Epidemiology Biomarkers and Prevention vol16 no 9 pp 1784ndash1794 2007
[41] C Canova C Dunster F J Kelly C Minelli P L Shah CCaneja et al ldquoPM10-induced hospital admissions for asthmaand chronic obstructive pulmonary disease the modifyingeffect of individual characteristicsrdquo Epidemiology vol 23 no 4pp 607ndash615 2012
[42] A J Henderson R B Newson M Rose-Zerilli S M RingJ W Holloway and S O Shaheen ldquoMaternal NRF2 andgluthathione-S-transferase polymorphisms donotmodify asso-ciations of prenatal tobacco smoke exposure with asthma andlung function in school-aged childrenrdquo Thorax vol 65 no 10pp 897ndash902 2010
[43] C Xing A L Sestak J A Kelly et al ldquoLocalization andreplication of the systemic lupus erythematosus linkage signalat 4p16 interaction with 2p11 12q24 and 19q13 in EuropeanAmericansrdquo Human Genetics vol 120 no 5 pp 623ndash631 2007
[44] T F Wong K Yoshinaga Y Monma K Ito H NiikuraS Nagase et al ldquoAssociation of KEAP1 and NRF2 geneticmutations and polymorphisms with endometrioid endometrialadenocarcinoma survivalrdquo International Journal of Gynecologi-cal Cancer vol 21 no 8 pp 1428ndash1435 2011
[45] M von Otter S Landgren S Nilsson et al ldquoNRF2-encodingNFE2L2 haplotypes influence disease progression but not riskin Alzheimerrsquos disease and age-related cataractrdquoMechanisms ofAgeing and Development vol 131 no 2 pp 105ndash110 2010
[46] S Tsang Z Sun B Luke et al ldquoA comprehensive SNP-basedgenetic analysis of inbred mouse strainsrdquoMammalian Genomevol 16 no 7 pp 476ndash480 2005
[47] C M Wade E J Kulbokas III A W Kirby et al ldquoThe mosaicstructure of variation in the laboratory mouse genomerdquoNaturevol 420 no 6915 pp 574ndash578 2002
[48] T Wiltshire M T Pletcher S Batalov et al ldquoGenome-widesingle-nucleotide polymorphism analysis defines haplotypepatterns in mouserdquo Proceedings of the National Academy ofSciences of the United States of America vol 100 no 6 pp 3380ndash3385 2003
[49] K A Frazer E Eskin H M Kang et al ldquoA sequence-basedvariation map of 827 million SNPs in inbred mouse strainsrdquoNature vol 448 no 7157 pp 1050ndash1053 2007
[50] T P Maddatu S C Grubb C J Bult andM A Bogue ldquoMousephenome database (MPD)rdquo Nucleic Acids Research vol 40 ppD887ndashD894 2007
[51] A Kirby H M Kang C M Wade et al ldquoFine mapping in 94inbred mouse strains using a high-density haplotype resourcerdquoGenetics vol 185 no 3 pp 1081ndash1095 2010
[52] H Y Cho A E Jedlicka S P M Reddy L Y Zhang T WKensler and S R Kleeberger ldquoLinkage analysis of susceptibilityto hyperoxia NRF2 is a candidate generdquo American Journal ofRespiratory Cell and Molecular Biology vol 26 no 1 pp 42ndash512002
[53] H Y Cho A E Jedlicka S P M Reddy et al ldquoRole of NRF2in protection against hyperoxic lung injury in micerdquo AmericanJournal of Respiratory Cell and Molecular Biology vol 26 no 2pp 175ndash182 2002
[54] T Shibata T Ohta K I Tong et al ldquoCancer related mutationsin NRF2 impair its recognition by KEAP1-Cul3 E3 ligase andpromote malignancyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 36 pp13568ndash13573 2008
24 Oxidative Medicine and Cellular Longevity
[55] A Singh V Misra R K Thimmulappa et al ldquoDysfunctionalKEAP1-NRF2 interaction in non-small-cell lung cancerrdquo PLoSMedicine vol 3 no 10 article e420 2006
[56] T Ohta K Iijima M Miyamoto et al ldquoLoss of KEAP1 functionactivates NRF2 and provides advantages for lung cancer cellgrowthrdquo Cancer Research vol 68 no 5 pp 1303ndash1309 2008
[57] B Padmanabhan K I Tong T Ohta et al ldquoStructural basisfor defects of KEAP1 activity provoked by its point mutationsin lung cancerrdquoMolecular Cell vol 21 no 5 pp 689ndash700 2006
[58] T Shibata A Kokubu M Gotoh et al ldquoGenetic alteration ofKEAP1 confers constitutive NRF2 activation and resistance tochemotherapy in gallbladder cancerrdquoGastroenterology vol 135no 4 pp 1358e4ndash1368e4 2008
[59] N J Yoo H R Kim Y R Kim C H An and S H LeeldquoSomatic mutations of the KEAP1 gene in common solidcancersrdquo Histopathology vol 60 no 6 pp 943ndash952 2012
[60] Y Hu Y Ju D Lin Z Wang Y Huang S Zhang et alldquoMutation of the NRF2 gene in non-small cell lung cancerrdquoMolecular Biology Reports vol 39 no 4 pp 4743ndash4747 2012
[61] Y R Kim J EOhM S Kim et al ldquoOncogenicNRF2mutationsin squamous cell carcinomas of oesophagus and skinrdquo TheJournal of Pathology vol 220 no 4 pp 446ndash451 2010
[62] T Shibata A Kokubu S Saito et al ldquoNRF2 mutation confersmalignant potential and resistance to chemoradiation therapyin advanced esophageal squamous cancerrdquo Neoplasia vol 13pp 864ndash873 2011
[63] H Sasaki M Shitara K Yokota Y Hikosaka S MoriyamaM Yano et al ldquoIncreased NRF2 gene (NFE2L2) copy numbercorrelates with mutations in lung squamous cell carcinomasrdquoMolecular Medicine Reports vol 6 no 2 pp 391ndash394 2012
[64] H Sasaki M Shitara K Yokota Y Hikosaka S Moriyama MYano et al ldquoRagD gene expression andNRF2mutations in lungsquamous cell carcinomasrdquo Oncology Letters vol 4 pp 1167ndash1170 2012
[65] A Singh M Bodas N Wakabayashi F Bunz and S BiswalldquoGain of NRF2 function in non-small-cell lung cancer cellsconfers radioresistancerdquo Antioxidants and Redox Signaling vol13 no 11 pp 1627ndash1637 2010
[66] T Yamadori Y Ishii SHommaYMorishimaKKurishimaKItoh et al ldquoMolecular mechanisms for the regulation of NRF2-mediated cell proliferation in non-small-cell lung cancersrdquoOncogene vol 31 pp 4768ndash4777 2012
[67] A K Bauer H Y Cho L Miller-Degraff C Walker K HelmsJ Fostel et al ldquoTargeted deletion of NRF2 reduces urethane-induced lung tumor development in micerdquo PLoS ONE vol 6no 10 Article ID e26590 2011
[68] H Y Cho and S R Kleeberger ldquoNRF2 protects against airwaydisordersrdquo Toxicology and Applied Pharmacology vol 244 no1 pp 43ndash56 2010
[69] R Wang J An F Ji H Jiao H Sun and D Zhou ldquoHyperme-thylation of the KEAP1 gene in human lung cancer cell linesand lung cancer tissuesrdquo Biochemical and Biophysical ResearchCommunications vol 373 no 1 pp 151ndash154 2008
[70] P Zhang A Singh S Yegnasubramanian et al ldquoLoss ofkelch-like ECH-associated protein 1 function in prostate cancercells causes chemoresistance and radioresistance and promotestumor growthrdquoMolecular Cancer Therapeutics vol 9 no 2 pp336ndash346 2010
[71] N Hanada T Takahata Q Zhou et al ldquoMethylation of theKEAP1 gene promoter region in human colorectal cancerrdquoBMCCancer vol 12 article 66 2012
[72] L A Muscarella R Barbano V DrsquoAngelo et al ldquoRegulationof KEAP1 expression by promoter methylation in malignantgliomas and association with patientrsquos outcomerdquo Epigeneticsvol 6 no 3 pp 317ndash325 2011
[73] S Yu T O Khor K L Cheung et al ldquoNRF2 expressionis regulated by epigenetic mechanisms in prostate cancer ofTRAMP micerdquo PLoS ONE vol 5 no 1 Article ID e8579 2010
[74] T O Khor Y Huang T Y Wu L Shu J Lee and A N KongldquoPharmacodynamics of curcumin as DNA hypomethylationagent in restoring the expression of NRF2 via promoter CpGsdemethylationrdquo Biochemical Pharmacology vol 82 no 9 pp1073ndash1078 2011
[75] I M Fingerman L McDaniel X Zhang et al ldquoNCBI epige-nomics a new public resource for exploring epigenomic datasetsrdquo Nucleic Acids Research vol 39 supplemen 1 pp D908ndashD912 2011
[76] I M Fingerman X Zhang W Ratzat N Husain R F Cohenand G D Schuler ldquoNCBI epigenomics whatrsquos new for 2013rdquoNucleic Acids Research vol 41 no 1 pp D221ndashD225 2013
[77] J H Lee T O Khor L Shu Z Y Su F Fuentes andA N Kong ldquoDietary phytochemicals and cancer preventionNRF2 signaling epigenetics and cell death mechanisms inblocking cancer initiation and progressionrdquo Pharmacology ampTherapeutics vol 137 no 2 pp 153ndash171 2012
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Oxidative Medicine and Cellular Longevity 23
nephritis in childhood-onset systemic lupus erythematosusrdquoLupus vol 19 no 10 pp 1237ndash1242 2010
[26] M von Otter S Landgren S Nilsson et al ldquoAssociation ofNRF2-encoding NFE2L2 haplotypes with Parkinsonrsquos diseaserdquoBMCMedical Genetics vol 11 no 1 article 36 2010
[27] JMHartikainenM TengstromVMKosmaV L Kinnula AMannermaa andY Soini ldquoGenetic polymorphisms and proteinexpression of NRF2 and sulfiredoxin predict survival outcomesin breast cancerrdquo Cancer Research vol 72 pp 5537ndash5546 2012
[28] T Arisawa T Tahara T Shibata et al ldquoThe relationshipbetween Helicobacter pylori infection and promoter polymor-phism of the NRF2 gene in chronic gastritisrdquo InternationalJournal of Molecular Medicine vol 19 no 1 pp 143ndash148 2007
[29] T Arisawa T Tahara T Shibata et al ldquoThe influence ofpromoter polymorphism of nuclear factor-erythroid 2-relatedfactor 2 gene on the aberrant DNA methylation in gastricepitheliumrdquo Oncology Reports vol 19 no 1 pp 211ndash216 2008
[30] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene pro-moter polymorphism and gastric carcinogenesisrdquo Hepato-Gastroenterology vol 55 no 82-83 pp 750ndash754 2008
[31] T Arisawa T Tahara T Shibata et al ldquoNRF2 gene promoterpolymorphism is associated with ulcerative colitis in a Japanesepopulationrdquo Hepato-Gastroenterology vol 55 no 82-83 pp394ndash397 2008
[32] C C Hua L C Chang J C Tseng C M Chu Y C Liu andW B Shieh ldquoFunctional haplotypes in the promoter region oftranscription factor NRF2 in chronic obstructive pulmonarydiseaserdquo Disease Markers vol 28 no 3 pp 185ndash193 2010
[33] S O Shaheen R B Newson S M Ring M J Rose-ZerilliJ W Holloway and A J Henderson ldquoPrenatal and infantacetaminophen exposure antioxidant gene polymorphismsand childhood asthmardquo The Journal of Allergy and ClinicalImmunology vol 126 no 6 pp 1141e7ndash1148e7 2010
[34] H Masuko T Sakamoto Y Kaneko et al ldquoLower FEV1 innon-COPD nonasthmatic subjects association with smokingannual decline in FEV1 total IgE levels and TSLP genotypesrdquoInternational Journal of Chronic Obstructive Pulmonary Diseasevol 6 pp 181ndash189 2011
[35] C P Guan M N Zhou A E Xu et al ldquoThe susceptibilityto vitiligo is associated with NF-E2-related factor 2 (NRF2)gene polymorphisms a study on Chinese Han populationrdquoExperimental Dermatology vol 17 no 12 pp 1059ndash1062 2008
[36] J Bouligand O Cabaret M Canonico et al ldquoEffect of NFE2L2genetic polymorphism on the association between oral estrogentherapy and the risk of venous thromboembolism in post-menopausal womenrdquo Clinical Pharmacology amp Therapeuticsvol 89 no 1 pp 60ndash64 2011
[37] D S OrsquoMahony B J Glavan T D Holden C Fong R A BlackG Rona et al ldquoInflammation and immune-related candidategene associations with acute lung injury susceptibility andseverity a validation studyrdquo PLoS ONE vol 7 no 12 Article IDe51104 2012
[38] I Ungvari E Hadadi V Virag et al ldquoRelationship between airpollutionNFE2L2 gene polymorphisms and childhood asthmain aHungarian populationrdquo Journal of CommunityGenetics vol3 no 1 pp 25ndash33 2012
[39] M SiedlinskiD S Postma JMA Boer et al ldquoLevel and courseof FEV1 in relation to polymorphisms inNFE2L2 andKEAP1 inthe general populationrdquo Respiratory Research vol 10 article 732009
[40] C C Hong C B Ambrosone J Ahn et al ldquoGenetic variabilityin iron-related oxidative stress pathways (NRF2 NQ01 NOS3
and HO-1) iron intake and risk of postmenopausal breastcancerrdquo Cancer Epidemiology Biomarkers and Prevention vol16 no 9 pp 1784ndash1794 2007
[41] C Canova C Dunster F J Kelly C Minelli P L Shah CCaneja et al ldquoPM10-induced hospital admissions for asthmaand chronic obstructive pulmonary disease the modifyingeffect of individual characteristicsrdquo Epidemiology vol 23 no 4pp 607ndash615 2012
[42] A J Henderson R B Newson M Rose-Zerilli S M RingJ W Holloway and S O Shaheen ldquoMaternal NRF2 andgluthathione-S-transferase polymorphisms donotmodify asso-ciations of prenatal tobacco smoke exposure with asthma andlung function in school-aged childrenrdquo Thorax vol 65 no 10pp 897ndash902 2010
[43] C Xing A L Sestak J A Kelly et al ldquoLocalization andreplication of the systemic lupus erythematosus linkage signalat 4p16 interaction with 2p11 12q24 and 19q13 in EuropeanAmericansrdquo Human Genetics vol 120 no 5 pp 623ndash631 2007
[44] T F Wong K Yoshinaga Y Monma K Ito H NiikuraS Nagase et al ldquoAssociation of KEAP1 and NRF2 geneticmutations and polymorphisms with endometrioid endometrialadenocarcinoma survivalrdquo International Journal of Gynecologi-cal Cancer vol 21 no 8 pp 1428ndash1435 2011
[45] M von Otter S Landgren S Nilsson et al ldquoNRF2-encodingNFE2L2 haplotypes influence disease progression but not riskin Alzheimerrsquos disease and age-related cataractrdquoMechanisms ofAgeing and Development vol 131 no 2 pp 105ndash110 2010
[46] S Tsang Z Sun B Luke et al ldquoA comprehensive SNP-basedgenetic analysis of inbred mouse strainsrdquoMammalian Genomevol 16 no 7 pp 476ndash480 2005
[47] C M Wade E J Kulbokas III A W Kirby et al ldquoThe mosaicstructure of variation in the laboratory mouse genomerdquoNaturevol 420 no 6915 pp 574ndash578 2002
[48] T Wiltshire M T Pletcher S Batalov et al ldquoGenome-widesingle-nucleotide polymorphism analysis defines haplotypepatterns in mouserdquo Proceedings of the National Academy ofSciences of the United States of America vol 100 no 6 pp 3380ndash3385 2003
[49] K A Frazer E Eskin H M Kang et al ldquoA sequence-basedvariation map of 827 million SNPs in inbred mouse strainsrdquoNature vol 448 no 7157 pp 1050ndash1053 2007
[50] T P Maddatu S C Grubb C J Bult andM A Bogue ldquoMousephenome database (MPD)rdquo Nucleic Acids Research vol 40 ppD887ndashD894 2007
[51] A Kirby H M Kang C M Wade et al ldquoFine mapping in 94inbred mouse strains using a high-density haplotype resourcerdquoGenetics vol 185 no 3 pp 1081ndash1095 2010
[52] H Y Cho A E Jedlicka S P M Reddy L Y Zhang T WKensler and S R Kleeberger ldquoLinkage analysis of susceptibilityto hyperoxia NRF2 is a candidate generdquo American Journal ofRespiratory Cell and Molecular Biology vol 26 no 1 pp 42ndash512002
[53] H Y Cho A E Jedlicka S P M Reddy et al ldquoRole of NRF2in protection against hyperoxic lung injury in micerdquo AmericanJournal of Respiratory Cell and Molecular Biology vol 26 no 2pp 175ndash182 2002
[54] T Shibata T Ohta K I Tong et al ldquoCancer related mutationsin NRF2 impair its recognition by KEAP1-Cul3 E3 ligase andpromote malignancyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 36 pp13568ndash13573 2008
24 Oxidative Medicine and Cellular Longevity
[55] A Singh V Misra R K Thimmulappa et al ldquoDysfunctionalKEAP1-NRF2 interaction in non-small-cell lung cancerrdquo PLoSMedicine vol 3 no 10 article e420 2006
[56] T Ohta K Iijima M Miyamoto et al ldquoLoss of KEAP1 functionactivates NRF2 and provides advantages for lung cancer cellgrowthrdquo Cancer Research vol 68 no 5 pp 1303ndash1309 2008
[57] B Padmanabhan K I Tong T Ohta et al ldquoStructural basisfor defects of KEAP1 activity provoked by its point mutationsin lung cancerrdquoMolecular Cell vol 21 no 5 pp 689ndash700 2006
[58] T Shibata A Kokubu M Gotoh et al ldquoGenetic alteration ofKEAP1 confers constitutive NRF2 activation and resistance tochemotherapy in gallbladder cancerrdquoGastroenterology vol 135no 4 pp 1358e4ndash1368e4 2008
[59] N J Yoo H R Kim Y R Kim C H An and S H LeeldquoSomatic mutations of the KEAP1 gene in common solidcancersrdquo Histopathology vol 60 no 6 pp 943ndash952 2012
[60] Y Hu Y Ju D Lin Z Wang Y Huang S Zhang et alldquoMutation of the NRF2 gene in non-small cell lung cancerrdquoMolecular Biology Reports vol 39 no 4 pp 4743ndash4747 2012
[61] Y R Kim J EOhM S Kim et al ldquoOncogenicNRF2mutationsin squamous cell carcinomas of oesophagus and skinrdquo TheJournal of Pathology vol 220 no 4 pp 446ndash451 2010
[62] T Shibata A Kokubu S Saito et al ldquoNRF2 mutation confersmalignant potential and resistance to chemoradiation therapyin advanced esophageal squamous cancerrdquo Neoplasia vol 13pp 864ndash873 2011
[63] H Sasaki M Shitara K Yokota Y Hikosaka S MoriyamaM Yano et al ldquoIncreased NRF2 gene (NFE2L2) copy numbercorrelates with mutations in lung squamous cell carcinomasrdquoMolecular Medicine Reports vol 6 no 2 pp 391ndash394 2012
[64] H Sasaki M Shitara K Yokota Y Hikosaka S Moriyama MYano et al ldquoRagD gene expression andNRF2mutations in lungsquamous cell carcinomasrdquo Oncology Letters vol 4 pp 1167ndash1170 2012
[65] A Singh M Bodas N Wakabayashi F Bunz and S BiswalldquoGain of NRF2 function in non-small-cell lung cancer cellsconfers radioresistancerdquo Antioxidants and Redox Signaling vol13 no 11 pp 1627ndash1637 2010
[66] T Yamadori Y Ishii SHommaYMorishimaKKurishimaKItoh et al ldquoMolecular mechanisms for the regulation of NRF2-mediated cell proliferation in non-small-cell lung cancersrdquoOncogene vol 31 pp 4768ndash4777 2012
[67] A K Bauer H Y Cho L Miller-Degraff C Walker K HelmsJ Fostel et al ldquoTargeted deletion of NRF2 reduces urethane-induced lung tumor development in micerdquo PLoS ONE vol 6no 10 Article ID e26590 2011
[68] H Y Cho and S R Kleeberger ldquoNRF2 protects against airwaydisordersrdquo Toxicology and Applied Pharmacology vol 244 no1 pp 43ndash56 2010
[69] R Wang J An F Ji H Jiao H Sun and D Zhou ldquoHyperme-thylation of the KEAP1 gene in human lung cancer cell linesand lung cancer tissuesrdquo Biochemical and Biophysical ResearchCommunications vol 373 no 1 pp 151ndash154 2008
[70] P Zhang A Singh S Yegnasubramanian et al ldquoLoss ofkelch-like ECH-associated protein 1 function in prostate cancercells causes chemoresistance and radioresistance and promotestumor growthrdquoMolecular Cancer Therapeutics vol 9 no 2 pp336ndash346 2010
[71] N Hanada T Takahata Q Zhou et al ldquoMethylation of theKEAP1 gene promoter region in human colorectal cancerrdquoBMCCancer vol 12 article 66 2012
[72] L A Muscarella R Barbano V DrsquoAngelo et al ldquoRegulationof KEAP1 expression by promoter methylation in malignantgliomas and association with patientrsquos outcomerdquo Epigeneticsvol 6 no 3 pp 317ndash325 2011
[73] S Yu T O Khor K L Cheung et al ldquoNRF2 expressionis regulated by epigenetic mechanisms in prostate cancer ofTRAMP micerdquo PLoS ONE vol 5 no 1 Article ID e8579 2010
[74] T O Khor Y Huang T Y Wu L Shu J Lee and A N KongldquoPharmacodynamics of curcumin as DNA hypomethylationagent in restoring the expression of NRF2 via promoter CpGsdemethylationrdquo Biochemical Pharmacology vol 82 no 9 pp1073ndash1078 2011
[75] I M Fingerman L McDaniel X Zhang et al ldquoNCBI epige-nomics a new public resource for exploring epigenomic datasetsrdquo Nucleic Acids Research vol 39 supplemen 1 pp D908ndashD912 2011
[76] I M Fingerman X Zhang W Ratzat N Husain R F Cohenand G D Schuler ldquoNCBI epigenomics whatrsquos new for 2013rdquoNucleic Acids Research vol 41 no 1 pp D221ndashD225 2013
[77] J H Lee T O Khor L Shu Z Y Su F Fuentes andA N Kong ldquoDietary phytochemicals and cancer preventionNRF2 signaling epigenetics and cell death mechanisms inblocking cancer initiation and progressionrdquo Pharmacology ampTherapeutics vol 137 no 2 pp 153ndash171 2012
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
24 Oxidative Medicine and Cellular Longevity
[55] A Singh V Misra R K Thimmulappa et al ldquoDysfunctionalKEAP1-NRF2 interaction in non-small-cell lung cancerrdquo PLoSMedicine vol 3 no 10 article e420 2006
[56] T Ohta K Iijima M Miyamoto et al ldquoLoss of KEAP1 functionactivates NRF2 and provides advantages for lung cancer cellgrowthrdquo Cancer Research vol 68 no 5 pp 1303ndash1309 2008
[57] B Padmanabhan K I Tong T Ohta et al ldquoStructural basisfor defects of KEAP1 activity provoked by its point mutationsin lung cancerrdquoMolecular Cell vol 21 no 5 pp 689ndash700 2006
[58] T Shibata A Kokubu M Gotoh et al ldquoGenetic alteration ofKEAP1 confers constitutive NRF2 activation and resistance tochemotherapy in gallbladder cancerrdquoGastroenterology vol 135no 4 pp 1358e4ndash1368e4 2008
[59] N J Yoo H R Kim Y R Kim C H An and S H LeeldquoSomatic mutations of the KEAP1 gene in common solidcancersrdquo Histopathology vol 60 no 6 pp 943ndash952 2012
[60] Y Hu Y Ju D Lin Z Wang Y Huang S Zhang et alldquoMutation of the NRF2 gene in non-small cell lung cancerrdquoMolecular Biology Reports vol 39 no 4 pp 4743ndash4747 2012
[61] Y R Kim J EOhM S Kim et al ldquoOncogenicNRF2mutationsin squamous cell carcinomas of oesophagus and skinrdquo TheJournal of Pathology vol 220 no 4 pp 446ndash451 2010
[62] T Shibata A Kokubu S Saito et al ldquoNRF2 mutation confersmalignant potential and resistance to chemoradiation therapyin advanced esophageal squamous cancerrdquo Neoplasia vol 13pp 864ndash873 2011
[63] H Sasaki M Shitara K Yokota Y Hikosaka S MoriyamaM Yano et al ldquoIncreased NRF2 gene (NFE2L2) copy numbercorrelates with mutations in lung squamous cell carcinomasrdquoMolecular Medicine Reports vol 6 no 2 pp 391ndash394 2012
[64] H Sasaki M Shitara K Yokota Y Hikosaka S Moriyama MYano et al ldquoRagD gene expression andNRF2mutations in lungsquamous cell carcinomasrdquo Oncology Letters vol 4 pp 1167ndash1170 2012
[65] A Singh M Bodas N Wakabayashi F Bunz and S BiswalldquoGain of NRF2 function in non-small-cell lung cancer cellsconfers radioresistancerdquo Antioxidants and Redox Signaling vol13 no 11 pp 1627ndash1637 2010
[66] T Yamadori Y Ishii SHommaYMorishimaKKurishimaKItoh et al ldquoMolecular mechanisms for the regulation of NRF2-mediated cell proliferation in non-small-cell lung cancersrdquoOncogene vol 31 pp 4768ndash4777 2012
[67] A K Bauer H Y Cho L Miller-Degraff C Walker K HelmsJ Fostel et al ldquoTargeted deletion of NRF2 reduces urethane-induced lung tumor development in micerdquo PLoS ONE vol 6no 10 Article ID e26590 2011
[68] H Y Cho and S R Kleeberger ldquoNRF2 protects against airwaydisordersrdquo Toxicology and Applied Pharmacology vol 244 no1 pp 43ndash56 2010
[69] R Wang J An F Ji H Jiao H Sun and D Zhou ldquoHyperme-thylation of the KEAP1 gene in human lung cancer cell linesand lung cancer tissuesrdquo Biochemical and Biophysical ResearchCommunications vol 373 no 1 pp 151ndash154 2008
[70] P Zhang A Singh S Yegnasubramanian et al ldquoLoss ofkelch-like ECH-associated protein 1 function in prostate cancercells causes chemoresistance and radioresistance and promotestumor growthrdquoMolecular Cancer Therapeutics vol 9 no 2 pp336ndash346 2010
[71] N Hanada T Takahata Q Zhou et al ldquoMethylation of theKEAP1 gene promoter region in human colorectal cancerrdquoBMCCancer vol 12 article 66 2012
[72] L A Muscarella R Barbano V DrsquoAngelo et al ldquoRegulationof KEAP1 expression by promoter methylation in malignantgliomas and association with patientrsquos outcomerdquo Epigeneticsvol 6 no 3 pp 317ndash325 2011
[73] S Yu T O Khor K L Cheung et al ldquoNRF2 expressionis regulated by epigenetic mechanisms in prostate cancer ofTRAMP micerdquo PLoS ONE vol 5 no 1 Article ID e8579 2010
[74] T O Khor Y Huang T Y Wu L Shu J Lee and A N KongldquoPharmacodynamics of curcumin as DNA hypomethylationagent in restoring the expression of NRF2 via promoter CpGsdemethylationrdquo Biochemical Pharmacology vol 82 no 9 pp1073ndash1078 2011
[75] I M Fingerman L McDaniel X Zhang et al ldquoNCBI epige-nomics a new public resource for exploring epigenomic datasetsrdquo Nucleic Acids Research vol 39 supplemen 1 pp D908ndashD912 2011
[76] I M Fingerman X Zhang W Ratzat N Husain R F Cohenand G D Schuler ldquoNCBI epigenomics whatrsquos new for 2013rdquoNucleic Acids Research vol 41 no 1 pp D221ndashD225 2013
[77] J H Lee T O Khor L Shu Z Y Su F Fuentes andA N Kong ldquoDietary phytochemicals and cancer preventionNRF2 signaling epigenetics and cell death mechanisms inblocking cancer initiation and progressionrdquo Pharmacology ampTherapeutics vol 137 no 2 pp 153ndash171 2012
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom