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The NIEHS Environmental Genome Project: The NIEHS Environmental Genome Project: Enabling Studies of Gene-Environment Enabling Studies of Gene-Environment
InteractionInteraction
Douglas A. Bell, Ph.D.Douglas A. Bell, Ph.D.
Environmental Genomics SectionEnvironmental Genomics Section
National Institute of Environmental Health National Institute of Environmental Health SciencesSciences
Professor, Dept of EpidemiologyProfessor, Dept of Epidemiology
UNC School of Public HealthUNC School of Public Health
NIEHS’s Environmental Genome ProjectNIEHS’s Environmental Genome Project
Resequencing of ~500 Candidate Genes Resequencing of ~500 Candidate Genes Potentially Involved in Environmental DiseasePotentially Involved in Environmental Disease
Concept and rationaleConcept and rationale Examples of gene-environment interactionExamples of gene-environment interaction Resequencing studies, accomplishments, and Resequencing studies, accomplishments, and
accessing data.accessing data.
Modulation of Response to Modulation of Response to ExposureExposure
ExposureExposure Early Early EffectsEffects
Genetic SusceptibilityGenetic Susceptibility
DiseaseDisease
Genetic Modulation of Exposure, Damage, and Biological
Response
DiseaseDisease
Genetic Variation in: Genetic Variation in:
• Metabolism, or distribution, affects dose to the tissueMetabolism, or distribution, affects dose to the tissue• Detection and repair of damageDetection and repair of damage• Differences in growth and recovery from damageDifferences in growth and recovery from damage
Exposure Exposure TargetTargettissuetissue
Biological Response
Genetic Modulation of Exposure Risk Genetic Modulation of Exposure Risk
Exposure
No No ExposureExposure
4-Fold Risk
2-Fold Risk
BackgroundRisk Level
(low)Sensitive
Genotype
Sensitive
Genotype
Resistant
Genotype
Resistant
Genotype
PAH-oxide
GST + Glutathione
Inactive
Benzo[a]pyrene Metabolism Benzo[a]pyrene Metabolism Glutathione
HO
HO
CYP450
DNA ReactiveDNA Reactive
PAH-oxide
GST + Glutathione
Inactive
DNA ReactiveDNA Reactive
Benzo[a]pyrene Metabolism Benzo[a]pyrene Metabolism Glutathione
HO
HO
CYP450
GSTM1 GSTM1 NullNull
Bladder Cancer Risk Associated with Bladder Cancer Risk Associated with Smoking and GSTM1 Null GenotypeSmoking and GSTM1 Null Genotype
*P<0.001; Bell et al, JNCI 85:1559,1993
Nonsmokers Nonsmokers
1- 50 Packyears 1- 50 Packyears SmokingSmoking
>50 Packyears>50 PackyearsSmokingSmoking
1 1.31 1.3
2.2* 4.3*2.2* 4.3*
3.5* 5.9*3.5* 5.9*
GSTM1 GSTM1 (+)(+)
GSTM1 GSTM1 nullnull
Exposure R
iskGenetic Risk
Examples of Gene-Environment Interaction Examples of Gene-Environment Interaction (gene modifies environmental effect)(gene modifies environmental effect)
Malaria and Sickle Cell gene.Malaria and Sickle Cell gene. HIV infection and CCR5 receptor variant.HIV infection and CCR5 receptor variant. LPS sensitivity and Toll Receptor (TLR4) LPS sensitivity and Toll Receptor (TLR4) Adverse drug response and CYP2D6 poor Adverse drug response and CYP2D6 poor
metabolism.metabolism. Alcohol intolerance and aldehyde dehydrogenase.Alcohol intolerance and aldehyde dehydrogenase. Smoking, GSTM1 null, NAT2 slow genotypes, and Smoking, GSTM1 null, NAT2 slow genotypes, and
bladder cancer risk .bladder cancer risk .
Variation in Risk Estimates in Human Variation in Risk Estimates in Human PopulationsPopulations
Phenotypic variation Phenotypic variation in response due to: in response due to:
PhysiologyPhysiology
MetabolismMetabolism
RepairRepair
GrowthGrowth
Timing of ExposureTiming of Exposure
RiskRisk
ExposureExposure
Example: Metabolism PolymorphismsExample: Metabolism Polymorphisms
freq
uen
cy
Activity
No Phenotypic No Phenotypic PolymorphismPolymorphism
Range of Enzyme Activity in Range of Enzyme Activity in Human PopulationsHuman Populations
Distribution of Polymorphic Enzyme Activity Distribution of Polymorphic Enzyme Activity in a Populationin a Population
freq
uen
cyfr
equ
ency
ActivityActivity ActivityActivity
HighHighLowLowHighHighLowLow
+/++/++/++/+
+/-+/-+/-+/--/--/- -/--/-
Examples: N-Acetyltransferase 2, GSTM1, CYP2D6Examples: N-Acetyltransferase 2, GSTM1, CYP2D6
How does frequency of a risk How does frequency of a risk factor impact exposure induced factor impact exposure induced (G x E) risk in the population?(G x E) risk in the population?
freq
uen
cyfr
equ
ency
ActivityActivity
5% 5% 95% 95%
Effects of Exposure in High and Low Risk Effects of Exposure in High and Low Risk Human PopulationsHuman Populations
RiskRisk
ExposureExposurefr
eq
ue
nc
yfr
eq
ue
nc
y
ActivityActivity
5% 5% 95% 95%
100
10
0
High Risk
Low RiskLow Risk
Average
How will genetic data be used in How will genetic data be used in public health risk assessment?public health risk assessment?
Given detailed information on the Given detailed information on the relationship between genotype and relationship between genotype and phenotype, more accurate risk phenotype, more accurate risk assessments may be possible. assessments may be possible.
Risk Management
MoreMore/Less /Less ControlControlHuman
Genetic Susceptibility
ExposureAssessment
Engineering design
Risk Assessment ProcessRisk Assessment Process
Animal toxicology (dose/response)
Risk Model(Extrapolation to
humans)
SS
RR
Effects in Humans ?
Replace default Replace default assumptions assumptions
about variabilityabout variability
Hazard/Risk Assessment
Chemical Chemical XX
Cancer - Yes/NoCancer - Yes/No
Dose ?Dose ?
Extrapolate to Extrapolate to HumansHumans
Susceptible Susceptible human human
subgroup?subgroup?
• BiochemistryBiochemistry
• Mechanism of toxicityMechanism of toxicity
• Genes, pathwaysGenes, pathways
• Human geneticsHuman genetics
Incorporating Human Genetic Polymorphism Incorporating Human Genetic Polymorphism Information Into Risk Assessment Information Into Risk Assessment
Incorporating Genetics Into Risk Incorporating Genetics Into Risk Assessment: IssuesAssessment: Issues
A polymorphism may have different effects A polymorphism may have different effects depending on the chemical, the target organ/ depending on the chemical, the target organ/ disease, and the population being considered. disease, and the population being considered.
Thus, a protective allele for one chemical may Thus, a protective allele for one chemical may
convey risk for a different chemical. Similarly convey risk for a different chemical. Similarly one organ system may be protected at the risk of one organ system may be protected at the risk of another; e.g. immune system response could another; e.g. immune system response could increase DNA damage or neurotoxicity. increase DNA damage or neurotoxicity.
D.A.Bell NIEHS
Ethylene oxide
HCHO
DetoxicationDetoxication
GSTT1 + Glutathione
ActivationActivation
Methylene chloride GSTT1
+ Glutathione
DNA
DNA
GST Theta 1 (GSTT1) - One gene with 2 effects
Glutathione
H2C
HO
CH2
Cl
Glutathione
Cl- CH2
+
(Unstable)
DNA DNA
ReactiveReactive
Inactive Inactive
(also Methyl chloride)
Activation vs. Detoxication Activation vs. Detoxication
Effects of polymorphism dependent on Effects of polymorphism dependent on chemical and toxicity pathway:chemical and toxicity pathway:
ActivationActivation - If the activation pathway is missing (null - If the activation pathway is missing (null genotypes), some individuals may have zero risk genotypes), some individuals may have zero risk even if they have exposure. even if they have exposure.
DetoxicationDetoxication - Since this process will never be - Since this process will never be 100% efficient, both functional and low activity 100% efficient, both functional and low activity genotypes will exhibit risk associated with exposure.genotypes will exhibit risk associated with exposure.
The Effect of GSTT1 Genotype on Metabolism of Methyl Chloride
From Lof, A. et al, Pharmacogenetics 10:645, 2000.
T1 + T1 + Metabolism to Metabolism to DNA reactive DNA reactive formsforms
T1 Null No T1 Null No MetabolismMetabolism
Measure Measure exhaled exhaled methyl methyl chloride chloride
D.A.Bell NIEHS
Smoking, GSTT1 Polymorphism, and Markers Smoking, GSTT1 Polymorphism, and Markers of Genotoxicity in Erythrocytes of Genotoxicity in Erythrocytes
Background: Background: Ethylene oxide –hemoglobin adducts are a Ethylene oxide –hemoglobin adducts are a good measure of smoking exposure in blood.good measure of smoking exposure in blood.
Experiment:Experiment: To test if GST genotypes modulated effects To test if GST genotypes modulated effects of smoking in erythrocytes, we measured ethylene oxide of smoking in erythrocytes, we measured ethylene oxide hemoglobin adducts in freshly collected human hemoglobin adducts in freshly collected human erythrocytes from nonsmokers and smokers.erythrocytes from nonsmokers and smokers.
Results:Results: Ethylene oxide adducts (HEV) were ~50% higher in Ethylene oxide adducts (HEV) were ~50% higher in
GSTT1 null individuals.GSTT1 null individuals.
GSTT1GSTT1 null genotypes have null genotypes have higherhigher levels of levels of smoking-induced hemoglobin adductssmoking-induced hemoglobin adducts
Effect of GSTT1 null Genotype:Ethylene Oxide-Hemoglobin Adducts Vs
Cotinine
0
100
200
300
400
500
600
700
800
0 200 400 600
Plasma Cotinine (ng/ml)
HE
Va
l Ad
du
cts
(fm
ol)
Series1
Series2
Linear (Series1)
Linear (Series2)
GSTT1 null
GSTT1 +
GST T1 Null
GST T1 +
Study Design: Study Design: 16 nonsmokers16 nonsmokers32 smokers 32 smokers
HEVal hemoglobin HEVal hemoglobin adducts measure by adducts measure by mass spectrometrymass spectrometry
P = 0.001 for difference P = 0.001 for difference in slopes;in slopes;Nonparametric analysisNonparametric analysis similar.similar.
Fennel et al CEBP 9:705,2000Fennel et al CEBP 9:705,2000
Incorporating Genetics Into Risk Assessment Incorporating Genetics Into Risk Assessment Needs: Needs: Identify genes involved in toxicological response.Identify genes involved in toxicological response. Detailed population genetic information including:Detailed population genetic information including:
Identify polymorphisms.Identify polymorphisms. Determine frequency in populations.Determine frequency in populations. Population-based risk estimates in large studies (n=2000).Population-based risk estimates in large studies (n=2000).
Determine functional relationship between genotype and Determine functional relationship between genotype and phenotypephenotype BiochemicalBiochemical In vitro, in vivo quantitative measurements of a cellular phenotype In vitro, in vivo quantitative measurements of a cellular phenotype
(tumors, adducts, mutation, cell death, gene expression).(tumors, adducts, mutation, cell death, gene expression).
Consider role of multiple genes, multiple pathways, etc.Consider role of multiple genes, multiple pathways, etc. Incorporate kinetic or other functional data into risk model.Incorporate kinetic or other functional data into risk model.
Environmental GenomicsEnvironmental Genomics
Discovery:Discovery:Phenotype-directedPhenotype-directedGenotype-directedGenotype-directed
FunctionalFunctionalAnalysisAnalysis
Disease Risk Disease Risk CharacterizationCharacterization
PhenotypePhenotypeGenotypeGenotype
CTTATGT A/C GGGTAT
Altered Binding
Effects in Populations
Polymorphism and FunctionPolymorphism and Function
Gene Deletions, DuplicationsGene Deletions, Duplications
Coding region changes:Coding region changes:aa subs, deletions, stops.aa subs, deletions, stops.
Transcription Transcription FactorsFactors
Effects of Polymorphism:
Altered function
Quantity of protein
Regulatory polymorphisms alter Regulatory polymorphisms alter transcription factor binding and transcription factor binding and mRNA/protein level.mRNA/protein level.
Exon 1 Exon 2Promoter 3’ UTR
e.g. GSTM1, CYP2D6e.g. GSTM1, CYP2D6
C TGGGCCCCGCCCCCTTATGTAGGGTATAAAGCCC …. CCCGTCACC ATG SP1/Oct
Phenotype—Directed Approach to Find SNPs Phenotype—Directed Approach to Find SNPs That Alter Gene Expression LevelThat Alter Gene Expression Level
Liu, X. et al Liu, X. et al
Sequence-Directed Approaches to Sequence-Directed Approaches to Catalogue All Significant SNPs In The Catalogue All Significant SNPs In The Human PopulationHuman Population
Resequencing Projects: Describing Resequencing Projects: Describing candidate gene polymorphisms in diverse candidate gene polymorphisms in diverse populations.populations.
~9 million SNPs in dbSNP now,~9 million SNPs in dbSNP now, by 2006, expect ~20 million human SNPs.by 2006, expect ~20 million human SNPs.
A SNP every ~100 bases.
Haplotype Map: Describing which SNPs Haplotype Map: Describing which SNPs occur together on chromosomes in occur together on chromosomes in populations (haplotypes).populations (haplotypes).
SNP Discovery ProjectsSNP Discovery Projects
The SNP Consortium – ~1 million SNPs The SNP Consortium – ~1 million SNPs across genome across genome
NIEHS – Environmental/toxicology NIEHS – Environmental/toxicology genesgenes
NHLBI – Heart disease genes, NHLBI – Heart disease genes, inflammation inflammation
NIGMS – Pharmacogenetic genesNIGMS – Pharmacogenetic genes
SNP data is entered into the NCBI dbSNP database SNP data is entered into the NCBI dbSNP database
HapmapHapmap
UCSCUCSC
U Wash EGP U Wash EGP WebsiteWebsite
Characterize the large scale genetic Characterize the large scale genetic structure across the genome.structure across the genome.
Genotyping SNPs at 1 kb interval across Genotyping SNPs at 1 kb interval across the genome in European, African, and the genome in European, African, and Asian populations.Asian populations.
HapMap WebsiteHapMap Website
HapMap WebsiteHapMap Website Seattle SNPs or EGP websiteSeattle SNPs or EGP website Many other freely available programsMany other freely available programs
Bioinformatic Tools Available For Bioinformatic Tools Available For Picking Haplotype Tagging SNPs Picking Haplotype Tagging SNPs
NIEHS Environmental Genome NIEHS Environmental Genome ProjectProject
Resequencing of candidate Resequencing of candidate environmental disease genesenvironmental disease genes
Accomplishments:Accomplishments: Total genes sequenced = 437Total genes sequenced = 437 Total kilobases sequenced = 11,001 kbTotal kilobases sequenced = 11,001 kb Total SNPs identified = 59,475 Total SNPs identified = 59,475
NIEHS’s Environmental Genome ProjectNIEHS’s Environmental Genome ProjectSummary: Summary:
Gene-environment interaction affects disease risk.Gene-environment interaction affects disease risk. Effects of G x E interactions can be complex.Effects of G x E interactions can be complex. Resequencing projects are providing many new Resequencing projects are providing many new
candidate gene polymorphism.candidate gene polymorphism. Determining the important functional SNPs that affect Determining the important functional SNPs that affect
disease risk is a difficult challenge.disease risk is a difficult challenge.
Strategies For Incorporating SNPs Strategies For Incorporating SNPs Into Epidemiology StudiesInto Epidemiology Studies
1.1. Whole genome association studies Whole genome association studies
Test 10,000-100,000 SNPs in case control studies.Test 10,000-100,000 SNPs in case control studies. Identify candidate regions, genes, followup with candidate Identify candidate regions, genes, followup with candidate
gene studies.gene studies.
2. High resolution candidate gene studies.2. High resolution candidate gene studies. Test functional SNPs and additional haplotype tagging SNPs in Test functional SNPs and additional haplotype tagging SNPs in
case/control or other design.case/control or other design. Bioinformatics to identify 1500 SNPs, 150 genes (10 SNPs/gene). Coding SNPs, regulatory SNPs, haplotype tag SNPs.
Application to p53 response elementsApplication to p53 response elements Application to NRF2 response Application to NRF2 response
elementselements
Bioinformatic Identification of SNPs Bioinformatic Identification of SNPs That Affect Gene ExpressionThat Affect Gene Expression
p53p53
p53 inducible genes contain p53 p53 inducible genes contain p53 RResponse esponse EElements.lements.
RRRCWWGYYY RRRCWWGYYY
RNA PolRNA Pol
Using bioinformatic methods, identify SNPs that Using bioinformatic methods, identify SNPs that
disrupt p53 response elements.disrupt p53 response elements.
SEI1SEI1mRNA mRNA
ATGATG
p53p53 p53p53 p53p53
SEI1 gene SEI1 gene
Following UV exposureFollowing UV exposurep53 binds RE of target gene.p53 binds RE of target gene.
RRRCWWAYYY
Test SNPs Against p53 Response Element Consensus
Filter:Filter:Best HitsBest Hits
Access Access databasedatabase
Build Table of Build Table of All Promoter SNPsAll Promoter SNPs
RRRCWWGYYYRRRCWWGYYYAAAGGACAAGTTGAAACTTGCACAAGCAGCCTCCATTCTG
DNA ambiguity codeDNA ambiguity code
R = A or GR = A or G
Y = C or TY = C or T
W = A or TW = A or T
dbSNP dbSNP DataData
Binding SiteBinding SiteConsensusConsensus
NCBI/EnsembleNCBI/EnsembleGenome DataGenome Data
Dan TomsoDan Tomso
Mismatch Mismatch with with
consensusconsensusCCWWWWGG
motifmotif
Dan TomsoDan Tomso
Do SNPs in putative p53 response elements Do SNPs in putative p53 response elements affect p53 induced expression in Saos2 affect p53 induced expression in Saos2 cells? cells?
0
5
10
15
20
25
p21-5' ADARB1 DCC ARHGEF7 RRM1 TLR8 EOMES SEI-1 SCGB1D2
REL
ATI
VE IN
DU
CTI
ON
Strong
Weak
WeakWeak
StronStrongg
Mike Resnick, Alberto Inga, Daniel Menendez
Saos2 Osteosarcoma Cells (p53 null)Saos2 Osteosarcoma Cells (p53 null)
Environmental Genomics Environmental Genomics SectionSection
Douglas A. BellDouglas A. BellGary S. PittmanGary S. Pittman
Merrill ‘Chip’ Miller, IIIMerrill ‘Chip’ Miller, IIIDaniel J. TomsoDaniel J. Tomso
Michelle R. CampbellMichelle R. CampbellXuemei LiuXuemei Liu
Xuting WangXuting WangMonica HorvathMonica Horvath
~4000 Human ~4000 Human ARE containing ARE containing
genesgenes
Phylogenetic Footprinting of NRF2/ARE GenesPhylogenetic Footprinting of NRF2/ARE Genes
~2100 Rat ARE ~2100 Rat ARE containing genes containing genes
~4000 Mouse ~4000 Mouse ARE containing ARE containing
genesgenes
Human/ Human/ mouse/ratmouse/rat
~380 ~380
1000 1000 human/mouse human/mouse
Gene x Environment Interaction Gene x Environment Interaction
Pharmacogenetics: Pharmacogenetics: Adverse drug reactions (toxicity)Adverse drug reactions (toxicity) Reduced efficacy Reduced efficacy
Environmental diseaseEnvironmental disease Modification of exposure-induced toxicityModification of exposure-induced toxicity Modification of exposure-induced diseaseModification of exposure-induced disease
Can we generalize about risk associated with a Can we generalize about risk associated with a specific gene?specific gene?