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Sequence variants in COL4A1 and COL4A2 genes in Ecuadorianfamilies with keratoconus
Justyna A. Karolak,1 Karolina Kulinska,1,2 Dorota M. Nowak,1 Jose A. Pitarque,3 Andrea Molinari,3Malgorzata Rydzanicz,1 Bassem A. Bejjani,4 Marzena Gajecka1,2
1Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland; 2Basic Medical Sciences Program, WWAMI(Washington, Wyoming, Alaska, Montana, and Idaho), Washington State University, Spokane, WA; 3Department of Ophthalmology,Hospital Metropolitano, Quito, Ecuador; 4Signature Genomics, Spokane, WA
Purpose: Keratoconus (KTCN) is a non-inflammatory, usually bilateral disorder of the eye which results in the conicalshape and the progressive thinning of the cornea. Several studies have suggested that genetic factors play a role in theetiology of the disease. Several loci were previously described as possible candidate regions for familial KTCN; however,no causative mutations in any genes have been identified for any of these loci. The purpose of this study was to evaluaterole of the collagen genes collagen type IV, alpha-1 (COL4A1) and collagen type IV, alpha-2 (COL4A2) in KTCN inEcuadorian families.Methods: COL4A1 and COL4A2 in 15 Ecuadorian KTCN families were examined with polymerase chain reactionamplification, and direct sequencing of all exons, promoter and intron-exon junctions was performed.Results: Screening of COL4A1 and COL4A2 revealed numerous alterations in coding and non-coding regions of bothgenes. We detected three missense substitutions in COL4A1: c.19G>C (Val7Leu), c.1663A>C (Thr555Pro), and c.4002A>C (Gln1334His). Five non-synonymous variants were identified in COL4A2: c.574G>T (Val192Phe), c.1550G>A(Arg517Lys), c.2048G>C (Gly683Ala), c.2102A>G (Lys701Arg), and c.2152C>T (Pro718Ser). None of the identifiedsequence variants completely segregated with the affected phenotype. The Gln1334His variant was possibly damaging toprotein function and structure.Conclusions: This is the first mutation screening of COL4A1 and COL4A2 genes in families with KTCN and linkage toa locus close to these genes. Analysis of COL4A1 and COL4A2 revealed no mutations indicating that other genes areinvolved in KTCN causation in Ecuadorian families.
Keratoconus (KTCN, OMIM 148300) is a non-inflammatory, usually bilateral disorder of the eye,characterized by progressive thinning and protrusion of thecentral cornea which results in altered refractive powers andloss of visual acuity [1]. The prevalence of the disease isestimated to be 1 in 2,000 individuals, and is the most commonectatic disorder of the cornea [1]. KTCN afflicts males andfemales in all ethnic groups [1]. Signs and symptoms dependon the stage of disease, with the first signs usually appearingin the third decade of life [1,2]. The cause of KTCN is stillunknown; both genetic and environmental factors seem toplay a role in its etiology. Although most cases of KTCN areisolated, an association with many syndromes, such as Downsyndrome [3], Ehlers-Danlos syndrome [4], and Lebercongenital amaurosis [5] has been described. Furthermore,extensive studies have shown an association between KTCNand constant eye rubbing [6], contact lens wear [7], or atopy[8]. Usually, KTCN is a sporadic disorder, but positive familyhistory has been observed in 6%–8% of cases [1]. An
Correspondence to: Marzena Gajecka, Ph.D., Institute of HumanGenetics, Polish Academy of Sciences, Strzeszynska 32, Poznan,60-479, Poland; Phone: (061) 657-9160; FAX: (061) 823-3235;email: [email protected]
autosomal dominant inheritance pattern with reducedpenetrance has been suggested in 90% of patients with familialKTCN [9,10].
Genomewide linkage analyses have indicated several lociinvolved in the etiology of familial KTCN at 16q22.3-q23.1(KTCN2; OMIM 608932), 3p14-q13 (KTCN3; OMIM608586), 2p24 (KTCN4; OMIM 609271), 1p36.23–36.21,5q14.3-q21.1, 5q21.2, 5q32-q33, 8q13.1-q21.11, 9q34,14q11.2, 14q24.3, 15q2.32, 15q22.33-q24.2, 17p13, and20q12 [10-20]. However, no mutations in any genes at any ofthese loci have been associated with KTCN.
We have demonstrated an evidence of linkage to a novellocus at 13q32 [21]. Collagen type IV, alpha-1 (COL4A1;OMIM 120130) and collagen type IV, alpha-2 (COL4A2;OMIM 120090) are mapped in close proximity to that locus.The COL4A1 and COL4A2 genes are organized in a head-to-head conformation [22]. These gene pairs share a commonpromoter, and each gene is transcribed in opposite directions[23]. The COL4A1 gene is placed on the minus strand andconsists of 52 exons, while the COL4A2 gene is on theopposite strand and consists of 48 exons. They encode two ofsix collagen type IV chains – α1 and α2 (1,669 and 1,712amino acids, respectively) – forming a heterotrimeric proteinmolecule of collagen type IV (α1α1α2), which is found in the
Molecular Vision 2011; 17:827-843 <http://www.molvis.org/molvis/v17/a94>Received 2 February 2011 | Accepted 22 March 2011 | Published 30 March 2011
© 2011 Molecular Vision
827
structure of the basement membrane (BM) [22,23]. Eachchain contains three domains: an NH2-terminal 7S domain, amajor collagenous domain with Gly-X-Y repeats (the Xposition is frequently occupied by proline, whereas the Yposition is often occupied by 4-hydroxyproline) and a non-collagenous domain (NC1) at the COOH-terminus.Repetitions of the Gly-X-Y motif determine the formation ofthe triple-helical structure of collagen [22].
Collagens are the major protein components of the humancornea, and several types of collagen, including collagen typeIV, have been identified [24]. Biochemical studies haverevealed thinning of corneas from patients with KTCN, whichmay occur as a result of a reduced amount of total collagenproteins [25] and changes in collagen fibers orientation [26].Moreover, a cornea affected by KTCN contains defects in BMand alterations in the BM composition [27]. The presence ofcollagen type IV in normal human cornea has remainedunclear [28]. Results from expression arrays have shown anexpression of COL4A1 in transplant-quality human donorcorneas [29] and a downregulation of COL4A1 in keratoconuscorneas [30]. Immunohistochemical studies have foundcollagen type IV α1/α2 chains in keratoconus corneas in largedefect sites [28]. In light of these results, we recognizeCOL4A1 and COL4A2 as candidate genes for KTCN.
The purpose of this study was to screen COL4A1 andCOL4A2 genes and determine whether sequence variants inthese genes are involved in the causation of KTCNin Ecuadorian families.
METHODSSubjects: Twenty-three individuals from family KTCN-014,25 affected individuals from other Ecuadorian families withKTCN, and 64 Ecuadorian control subjects were included inthe study. The pedigrees of these families have been describedelsewhere [21]. All individuals were examined in the HospitalMetropolitano in Quito, Ecuador, undergoing a completeophthalmic evaluation as previously described [21]. Thepossible consequences of the study were explained andinformed consent was obtained from all family members,according to the Declaration of Helsinki. Study protocol wasapproved by both the Institutional Review Board atWashington State University Spokane, Spokane, WA andPoznan University of Medical Sciences (Poland).Sequencing analyses: Oligonucleotide primers were designedto amplify all coding sequences and intron-exon junctions,promoter, and UTRs of both COL4A1 and COL4A2 (Table 1).PCR amplifications were performed using Taq DNAPolymerase (Fermentas Inc., Glen Burnie, MD). PCRproducts were purified with ExoSAP-IT® (USB Corporation,Cleveland, OH) or Montage® PCR Filter Units (Millipore,Jaffrey, NH) and sequenced using the BigDye® Terminatorv3.1 Cycle Sequencing Kit (Applied Biosystems, Inc. [ABI],Foster City, CA). Sequencing was visualized on an ABI
PRISM® 3100 Genetic Analyzer (ABI) and a 3730xl DNAAnalyzer (ABI). The DNA sequences of study subjects werecompared with the reference sequences of COL4A1 andCOL4A2 (GRCh37/hg19, GenBank accession numbers forthe mRNA NM_001845.4 and NM_001846.2, respectively)using Sequencher® 4.1.4. Software (Gene CodesCorporation, Ann Arbor, MI).Haplotype analysis: PEDSTATS [31] was used to verify thestructure of KTCN-014 family and identify potentialMendelian inconsistencies in the inheritance of singlenucleotide polymorphisms (SNPs) in COL4A1 and COL4A2.For that region, to determine the full haplotypes inheritedalong with the substitutions occurring in affected individuals,a reconstruction of observed sequence variants was preparedusing SimWalk2 [32,33]. Allele frequencies were set as equal.The location of genetic markers was determined on the basisof the Rutgers combined linkage-physical map of the humangenome [34], either directly or by interpolation. Haplotypewas generated with HaploPainter [35].Statistical analysis for Gln1334His substitution: Thedifference in distribution of Gln1334His substitution betweenaffected and unaffected individuals in family KTCN-014 wasanalyzed by Fisher's Exact Test for Count Data. Similarly, 25affected individuals from the remaining KTCN familiesversus 64 Ecuadorian control individuals were comparedusing Fisher's Exact Test. The difference between theexamined groups was considered significant if the value ofprobability (p) did not exceed 0.05.Prediction of effect of amino acid substitutions on proteinfunction: The potential impact of amino acid substitutions onthe COL4A1 and COL4A2 proteins was examined usingPolyPhen, SIFT, PMUT, PANTHER, and SNAP tools.
The PolyPhen tool predicts which missense substitutionaffects the structure and function of protein, and uses Position-Specific Independent Counts software to assign profile scores.These scores are the likelihood of the occurrence of a givenamino acid at a specific position, compared to the likelihoodof this amino acid occurring at any position (backgroundfrequency) [36].
The SIFT analytic tool, on the basis of gene sequenceshomology, evaluates conserved positions, and calculates ascore for the amino acid change at a particular position. Ascore of <0.05 is considered as pathogenic and has aphenotypic effect on protein structure [37].
The PMUT calculates the pathological significance ofnon-synonymous amino acid substitution using neuralnetworks (NN). NN output >0.5 is considered to be deleterious[38]. PANTHER estimates the likelihood of a particularamino acid’s change affecting protein function. On the basisof an alignment of evolutionarily related proteins, it generatesthe substitution Position-Specific Evolutionary Conservation(subPSEC). The subPSEC could achieve values from 0(neutral) to about −10 (most likely to be deleterious). The
Molecular Vision 2011; 17:827-843 <http://www.molvis.org/molvis/v17/a94> © 2011 Molecular Vision
828
TAB
LE 1
. PR
IMER
SEQ
UEN
CES
AN
D A
NN
EALI
NG
TEM
PER
ATU
RE
USE
D T
O P
CR
AM
PLIF
ICA
TIO
NS O
F CO
L4A1
AN
D C
OL4
A2 FR
AG
MEN
TS.
Nam
eFo
rwar
dR
ever
seA
nnea
ling
Tem
pera
ture
(°C
)A
mpl
icon
Size
(bp)
CO
L4A
1.1
CA
CC
CTC
CC
CC
TTTC
TAC
TCG
CC
CA
GA
GA
ATG
CA
CC
TG59
837
CO
L4A
1.2
TTG
GG
CTG
AG
TAA
CA
CTT
GG
GC
CTG
GTT
TGG
CTT
CA
TTTG
5845
9C
OL4
A1.
3–4
GG
GC
AA
CA
GA
ATG
AG
AC
TCC
TGTG
AG
CTG
GG
AG
AG
GA
GA
T66
477
CO
L4A
1.5–
6TG
CTC
TGTC
TGC
TTTG
TGTG
AC
AA
GC
TGTG
CTA
CTG
GG
TA60
698
CO
L4A
1.7–
8C
CA
AC
AA
ATG
AA
GG
GTA
GG
GTG
TGC
CA
AG
TGTC
TGA
AC
G58
578
CO
L4A
1.9–
10C
CTT
TGC
TTTG
CC
GTC
TCTA
TCA
TCA
TCC
CTT
TCC
CA
CA
G60
691
CO
L4A
1.11
GG
AG
ATG
GA
TTG
GTA
TTG
GT
GA
CTA
AG
GG
ATG
GA
TGA
AA
G58
451
CO
L4A
1.12
GG
GA
CA
AA
GC
TATT
GC
CTG
AG
AC
ATT
GA
TCC
AA
AG
GTG
GG
5823
9C
OL4
A1.
13G
CA
GA
GG
CA
AG
GA
TGA
TTA
GG
GG
GC
TCG
TATT
TTA
TGG
AC
5839
3C
OL4
A1.
14–1
5C
CC
TGC
CC
TGC
TTA
CA
TTG
TCC
CTA
CG
AG
CC
TTTT
CTG
6050
5C
OL4
A1.
16–1
7TT
AG
TGG
AG
AC
GG
GA
TTTC
GA
AC
TGC
CTG
CTT
GTG
TATG
C60
725
CO
L4A
1.18
GA
TGG
GA
CA
AG
TATC
TGG
GC
CA
TCTC
CTC
TCC
TTC
CTC
TC60
459
CO
L4A
1.19
GC
TAC
CA
TTG
CTG
CTA
CTT
CA
CA
ATA
GA
AA
GC
GTG
GG
GA
GA
G62
447
CO
L4A
1.20
GTC
AC
AA
CA
GG
CTT
CA
GG
AG
CC
CA
GG
AG
AG
AC
ATA
AG
GG
T60
486
CO
L4A
1.21
CA
GTG
ATG
GTC
TGG
TTG
GA
TA
TGC
CA
GG
AG
TCTC
AG
AG
GT
6053
2C
OL4
A1.
22TG
GG
TGG
TGTG
TGG
TGA
TTA
GA
GA
AG
GG
GC
AA
AA
CTC
TGA
6051
6C
OL4
A1.
23TT
CC
AC
CC
ATT
AG
CA
GA
GA
GG
CC
AA
CA
CA
CC
AA
AG
CA
A60
304
CO
L4A
1.24
GTC
CG
TCTT
GG
GC
ATT
TTA
GA
TTTG
GG
CTC
TGTG
GG
TAA
C60
431
CO
L4A
1.25
GTG
CC
CA
AA
GC
CA
CA
CTA
TATG
TTC
AG
TTC
CC
CC
AA
ATG
C60
718
CO
L4A
1.26
CC
TGG
GA
GG
GTA
GA
TGA
AG
TG
AA
AG
GG
AG
GC
AC
AA
AA
GG
6248
8C
OL4
A1.
27–2
8A
AG
TGG
AG
AA
CA
CA
GG
CA
GA
TCTT
CC
CA
AC
CA
AA
CC
CTA
C56
636
CO
L4A
1.29
AG
GTG
CTG
GA
AG
AG
AC
AG
CA
GC
TGA
GG
CTG
AG
AA
AC
CA
TC60
678
CO
L4A
1.30
GC
TTG
AA
AA
GG
GTT
GA
GC
AG
GG
CC
TCTA
AG
ATT
TGC
ATC
G64
315
CO
L4A
1.31
CA
GA
GC
CC
CTA
CC
GA
GTA
TAC
AG
TGG
GTG
GG
AG
AA
GA
ATC
6148
3C
OL4
A1.
32–3
3C
ATT
CA
AG
TTC
CC
AG
TGTG
GG
CC
TTC
TGC
TTG
ATG
TTC
CT
6065
3C
OL4
A1.
34C
TCA
TTTA
CC
TGG
GG
TTG
GA
TATG
GA
GG
AC
CC
GA
TAA
CC
C60
411
CO
L4A
1.35
–36
TGTG
CC
TTTC
CTG
GG
TTA
TCA
ATG
TCA
TCC
ATC
CC
TGA
GC
6459
4C
OL4
A1.
37G
GG
GG
ATT
CA
CG
TTC
TTG
TATC
CC
TGTG
TGTT
ATG
GC
TCA
5836
4C
OL4
A1.
38–3
9TG
GC
AG
GTA
GA
AA
CC
AG
ATG
TGA
AG
ATG
GG
AG
AC
AG
GA
CA
6164
1C
OL4
A1.
40G
AC
CTC
AG
GA
AA
AC
CA
GG
TGG
TAG
TTG
CA
GG
GA
TGTG
CA
G60
359
CO
L4A
1.41
TGG
TGG
TTC
TGA
GC
TGA
AA
GC
ATG
TGTC
TTG
CA
GG
CA
TTG
6044
7C
OL4
A1.
42TA
AA
GA
GA
AG
GA
GG
GA
TCG
GTC
TTC
AC
CA
GA
AC
CC
AC
AA
G60
673
CO
L4A
1.43
CC
TGC
CTC
GA
TTTC
TGTC
TCTA
GTG
GG
GA
TGTG
GG
AG
TGT
6043
5C
OL4
A1.
44C
CA
CA
AG
GC
AC
CA
TTTG
TTC
TAC
AA
ATT
GG
GC
TGC
CA
CA
C60
376
CO
L4A
1.45
GG
AC
CA
AA
AA
CA
GTG
CC
CTA
GA
GC
CTT
GG
GA
AG
TTTC
TGA
6079
0C
OL4
A1.
46C
CA
GA
ATG
CA
GTG
GG
AA
GTT
TTC
CTG
GG
TTTT
CTT
CTG
GA
6059
0C
OL4
A1.
47A
CA
GC
AA
GA
AA
CC
AG
GG
AG
AG
GC
TGC
CTT
TCA
AC
AA
CA
TC60
591
CO
L4A
1.48
TGA
AG
GA
GG
TAG
GC
TGC
TGT
CG
CA
GTG
TTTC
AC
TCG
CTA
C60
516
CO
L4A
1.49
TGTT
GTG
AA
AG
AC
ATT
GC
CC
GC
CC
AG
CC
AA
CTG
AC
TTTT
A60
650
CO
L4A
1.50
AA
AA
CC
AA
CG
GG
GA
GG
TAC
TTA
AG
CA
GC
GA
GA
TGC
AG
AG
A59
407
CO
L4A
1.51
GG
AA
GC
AG
CC
ATT
AG
AC
GA
TA
AA
TCG
TCTC
GG
TCA
TCTG
C60
573
Molecular Vision 2011; 17:827-843 <http://www.molvis.org/molvis/v17/a94> © 2011 Molecular Vision
829
TAB
LE 1
. CO
NTI
NU
ED.
Nam
eFo
rwar
dR
ever
seA
nnea
ling
Tem
pera
ture
(°C
)A
mpl
icon
Size
(bp)
CO
L4A
1.52
.1TA
CC
AG
GTT
GA
GG
CC
TGA
TGA
CC
TCC
TAG
CA
CC
CTT
TGG
T65
530
CO
L4A
1.52
.2G
AA
AA
CC
AA
AG
GG
TGC
TAG
GC
CG
AA
TGTG
CTT
AC
GTG
TGA
6579
3C
OL4
A1.
52.3
CC
TGG
CTT
GA
AA
AA
CA
GC
TCA
ATC
AC
CC
CC
AG
TCTG
TGA
C60
429
CO
L4A
2.1
TCG
TGG
GA
AA
GC
TCA
GA
TAC
AG
AC
AA
AG
CG
AG
TTTA
GC
GC
6014
54C
OL4
A2.
2G
CTT
CTG
GA
AG
GG
CC
AA
TG
GG
AA
AG
GG
AG
GA
AG
AG
AG
A60
587
CO
L4A
2.3
CC
TCA
TCC
TGC
GC
TAA
AC
TCA
CA
CTT
TCC
TGG
CC
TCTA
CG
6062
5C
OL4
A2.
4A
TTTC
AG
GG
GTG
GG
AG
AG
AC
CG
GC
CA
TCTA
GG
TTTG
TGTG
6046
7C
OL4
A2.
5–6
TTC
TTTC
ATC
CC
AA
CC
CA
GT
TCC
CC
AC
GTG
TTTT
ATG
TCA
5966
3C
OL4
A2.
7A
GA
CA
GA
AG
AA
AC
CC
CG
AC
ATC
TTG
GG
CG
TCA
AC
ATA
CA
G60
515
CO
L4A
2.8
TCA
GA
ATA
AC
CC
CC
ATC
AG
CA
AC
AG
ATC
AG
CC
CTA
TCA
GG
AC
6056
8C
OL4
A2.
9–10
AG
GTC
CTG
ATA
GG
GC
TGA
TCT
TAA
CTG
GC
AG
AG
AG
CTG
GTG
5955
1C
OL4
A2.
11G
CA
TCA
GA
AA
CC
TCC
ATG
CA
CA
TTG
GC
CTC
CC
TAC
AA
CA
5955
6C
OL4
A2.
12TC
CA
ATC
TCA
GC
TCC
CA
CTC
TGTC
CTC
AC
CTC
CA
CC
TTC
T60
548
CO
L4A
2.13
GG
AA
AC
AA
CC
CC
AC
AG
AA
AC
GG
AG
GA
CC
CG
GTT
ATG
TTTT
5952
4C
OL4
A2.
14G
TAA
AC
ATC
TGC
CTG
GA
AC
GC
TATG
GA
CA
AG
GG
GA
TGA
GA
5846
9C
OL4
A2.
15TG
TCA
CTG
CC
TGTC
CTC
AG
AC
CC
CA
GTG
CTA
GA
TGTT
CG
T61
513
CO
L4A
2.16
ATT
ATT
TCC
CA
TCC
CC
AC
CT
GC
AA
AA
ATG
AG
AG
CC
AA
GG
T59
473
CO
L4A
2.17
CC
CA
GTG
TCTT
CA
AC
AA
CC
ATG
TCA
GA
GG
CC
GTG
TATT
TG59
505
CO
L4A
2.18
AG
CA
CA
GTC
TCC
TGG
CA
TTC
CA
GG
CA
AC
ATG
AA
GG
TCTC
C60
569
CO
L4A
2.19
TTC
GA
GC
TTTG
GA
CTC
AC
CT
CTG
TGA
AG
GTG
TCC
AA
AG
CA
6052
1C
OL4
A2.
20A
CC
CA
TCG
GA
GTT
ATT
GA
CG
TAC
AG
GG
CTT
CA
GC
TTC
CA
T60
490
CO
L4A
2.21
CC
TGC
ATC
TGTG
GTT
GTC
TCA
AG
TTC
GC
CTC
CTC
ATC
AA
C59
609
CO
L4A
2.22
CC
TCTG
AA
TGTG
GTC
CC
AG
TA
AA
GTC
CG
CC
TTG
GG
GTA
T59
602
CO
L4A
2.23
–24
ATC
GC
AG
AA
AG
TGC
TCC
TTG
ATG
AG
CA
GC
CTG
TCC
TATG
C60
545
CO
L4A
2.25
TGG
CA
CTA
GG
TTC
CTG
TTC
AA
CA
GG
AG
AG
GC
TGC
ATG
TTT
5955
3C
OL4
A2.
26A
AA
CA
TGC
AG
CC
TCTC
CTG
TTT
CTG
AC
AA
GA
GG
GG
TTTG
G60
492
CO
L4A
2.27
CC
AG
AA
TGG
TAG
CC
GG
TTT
GC
AA
GA
CC
AG
TTTG
TGC
TGA
6031
8C
OL4
A2.
28TA
AG
CC
TGG
AG
GTG
CTG
TTT
CC
GA
AA
CA
CC
TGTC
TCC
TTT
5949
9C
OL4
A2.
29G
CG
AA
GG
TTG
TAG
GTT
CC
AA
TGC
CA
AG
AC
AA
AC
AG
TGA
GC
6070
8C
OL4
A2.
30G
AA
TAG
AC
AA
GG
GC
AG
GA
AG
GC
AG
AG
GA
TGA
GC
CG
ATG
TCT
6058
1C
OL4
A2.
31C
AC
AG
CC
TCA
AC
CTC
CA
GA
TC
AG
GC
AG
GA
GC
AG
TTTG
TCT
6064
3C
OL4
A2.
32TG
CTC
CTC
TGC
CTT
TGTC
TTTG
TTG
AG
GC
AG
GG
ATA
AA
GC
6065
6C
OL4
A2.
33TG
GTC
TCTC
TCC
AA
GG
CTT
CA
CC
GA
GG
TTA
CTC
AG
GC
ATC
5944
2C
OL4
A2.
34A
CA
GC
AC
GTA
GG
AC
AG
CA
AA
AC
ATC
TGC
ATG
GTG
TCC
AA
G59
470
CO
L4A
2.35
GC
TAA
GC
AA
AC
CG
CC
TATG
AA
CA
GG
AC
TTTC
CA
CTG
GG
AC
T60
416
CO
L4A
2.36
GG
GA
GTC
CA
CA
ATT
CA
GA
GC
GA
CC
CTT
CG
CTG
TTTC
TGA
G59
629
CO
L4A
2.37
CC
CA
TGC
TTC
TCTC
CA
ATT
CA
TGC
CTC
TCTC
CA
TTC
CTG
A60
446
CO
L4A
2.38
CTG
CTG
CTG
CTT
TCTG
TGTT
CC
TGTG
CTG
CTA
TGTT
GG
TG60
626
CO
L4A
2.39
GTG
CTG
TCC
CA
CA
CA
TGA
AA
AG
TCC
ATT
CA
AC
CC
AG
CA
AC
6151
0C
OL4
A2.
40A
TGG
GC
CTC
GA
TCC
TCTT
AT
AA
AC
CA
GC
TCTT
TCC
TGC
AC
6048
4C
OL4
A2.
41C
CC
AC
CA
TGA
GA
TGTT
CC
TTA
TGA
CA
CA
GG
AG
GA
GC
CA
TC60
427
Molecular Vision 2011; 17:827-843 <http://www.molvis.org/molvis/v17/a94> © 2011 Molecular Vision
830
TAB
LE 1
. CO
NTI
NU
ED.
Nam
eFo
rwar
dR
ever
seA
nnea
ling
Tem
pera
ture
(°C
)A
mpl
icon
Size
(bp)
CO
L4A
2.42
–43
AG
TCA
TTC
CA
TGC
CA
CA
GA
CTA
AG
CTC
TCC
ATT
CC
CC
AA
G60
666
CO
L4A
2.44
–45
CC
CG
TTA
GTG
TCTG
GC
TCA
TA
GG
TGTT
CTG
CTG
GG
CA
TAG
6074
4C
OL4
A2.
46G
AA
AC
TGC
CC
TGC
AC
TCC
TTA
GA
TGG
AC
CC
TTC
CG
TCA
G60
664
CO
L4A
2.47
CA
CTC
CC
TGG
TGA
TCC
AA
CT
CC
AA
CTA
CC
CTT
GTG
CA
GTG
6067
5C
OL4
A2.
48.1
GG
ATG
CC
TCA
TGTC
CG
TATT
TAC
ATG
GG
TGTG
TGC
GA
AG
T60
689
CO
L4A
2.48
.2C
ATC
CA
GC
AG
CA
GC
AC
TTA
GA
GG
TCTC
CA
CTT
CTG
CC
TGA
5953
0C
OL4
A2.
48.3
CC
TGC
TTTC
TAC
GC
CA
ATG
TC
TGG
TTG
GG
GTG
TTTT
CTG
T60
573
In
the
tabl
e, A
mpl
icon
Siz
e re
pres
ents
leng
th o
f the
PC
R p
rodu
ct in
bas
e pa
irs (b
p) a
nd A
nnea
ling
Tem
pera
ture
repr
esen
ts th
e an
neal
ing
tem
pera
ture
of t
he p
rimer
s
use
d fo
r PC
R a
mpl
ifica
tions
.
Molecular Vision 2011; 17:827-843 <http://www.molvis.org/molvis/v17/a94> © 2011 Molecular Vision
831
value −3 is the cutoff point for functional significance, andcorresponds to a Pdeleterious of 0.5. If the substitution occurs ata position not appearing in the multiple sequence alignment,a subPSEC score cannot be calculated and change is not likelyto be pathogenic [39,40].
The SNAP tool predicts the functional consequences ofexchanging amino acids using evolutionary conservation andstructure/function relationships. The SNAP output showsprediction neutral or non-neutral, and the expected accuracy[41].
RESULTSForty eight members of 15 Ecuadorian families and 64Ecuadorian control subjects were included in the study.Twenty-three individuals from family KTCN-014, twoaffected individuals from each of the families KTCN-011,015, 019, 020, 021, 024, 025, 030, 031, 034, and 035, and onepatient from each of KTCN-05, 013, and 017 were examined.
COL4A1 and COL4A2 sequence analyses: Screening ofCOL4A1 (NM_001845.4) coding regions revealed 12sequence variants, three of which were amino acidsubstitutions: c.19G>C (Val7Leu), c.1663A>C (Thr555Pro),and c.4002A>C (Gln1334His). We identified one novelsynonymous change, c.3693G>A (Thr1231Thr), and eightpreviously reported sequence variants: c.432T>A(Ala144Ala), c.1257T>C (Pro419Pro), c.1815T>C(Pro605Pro), c.2130G>A (Pro710Pro), c.3183G>A(Gly1061Gly), c.3189A>T (Arg1063Arg), c.4470C>T(Ala1490Ala), and c.4800C>T (Ser1600Ser). In the 5′untranslated region (5′ UTR), one novel sequence variant, c.84+124T>A, was identified. In the 3′ untranslated region (3′UTR), two previously reported variants, c.*587C>A andc.*975A>C, were detected.
Sequencing analyses of COL4A2 (NM_001846.2) codingregions revealed 13 previously reported sequence variants,including five non-synonymous substitutions: c.574G>T(Val192Phe), c.1550G>A (Arg517Lys), c.2048G>C(Gly683Ala), c.2102A>G (Lys701Arg), and c.2152C>T(Pro718Ser), and eight synonymous substitutions: c.297G>A(Thr99Thr), c.1008C>T (Pro336Pro), c.1095G>A(Pro365Pro), c.1179C>T (Ile393Ile), c.1488G>A(Pro496Pro), c.4089G>A (Ala1363Ala), c.4290T>C(Phe1430Phe), c.4515A>G (Pro1505Pro). In the 5′ UTR, fiveknown nucleotide changes, c.-277A>C, c.-232C>G,c.-215C>T, c.-203T>C, and c.-133A>G, were identified. Inthe 3′ UTR, eight previously reported sequence variants,c.*76T>C, c.*101_*102del2, c.*417C>G, c.*541C>T,c.*557A>G, c.*650T>C, c.*663T>C, and c.*727G>C weredetected.
Screening of exon/intron junctions in COL4A1 andCOL4A2 revealed numerous sequence variants in thesurrounding non-coding sequences, 71 and 86, respectively,including single nucleotide changes, insertions, and deletions.All screening results are summarized in Table 2.
The sequencing of the genomic region containing thecommon promoter of COL4A1 and COL4A2 revealed nosequence changes.
Statistical analysis and in silico predictions: PolyPhenanalyses of non-synonymous changes in COL4A1 andCOL4A2 predicted that only the Gln1334His variant inCOL4A1 was possibly damaging for protein function andstructure (Table 3). The multiple sequence alignment ofCOL4A1 orthologs shows that the amino acid glutamine atposition 1,334 is conserved throughout the analyzed species(Figure 1). Gln1334His substitution was observed morefrequently in patients than in healthy individuals in familyKTCN-014 (p=0.056). There was no difference in the c.4002A>C allele distribution between the analyzed affectedindividuals from the remaining KTCN families and theEcuadorian control subjects (p=0.17).
The SIFT, PMUT, PANTHER, and SNAP analysesdefined all missense amino acid substitutions in COL4A1 andCOL4A2 as neutral/tolerated and lacking any effect on proteinfunction. All prediction results are summarized in Table 3.
Haplotype reconstruction: Haplotypes of sequencevariants observed in family KTCN-014 are shown in Figure2. The coding sequence variants in COL4A1 are surroundedby markers rs13260 and col4a1_snp2. Exons of COL4A2 arelocalized between rs35466678 and rs422733.
KTCN-014 consists of two family branches. Distincthaplotypes in the branches were identified (Figure 2). In thefirst one, initiated by parents KTCN-93 and KTCN-01, sixsubjects with KTCN had the same haplotype in the COL4A1region, extending from rs13260 to col4a1_snp1. Threeunaffected individuals, KTCN-13, KTCN-14, and KTCN-22,share that part of the haplotype with their affected relatives.One of four variants in this region, rs3742207, causes a changein the protein sequence, replacing Gln in position 1334 withHis (Gln1334His). That haplotype region, from rs13260 tocol4a1_snp1, represents a short fragment of the haplotypewhich covers the whole COL4A1 and COL4A2 sequence inKTCN-03, KTCN-05, KTCN-06, and KTCN-14. In addition,individuals KTCN-07, KTCN-09, KTCN-13, KTCN-22, andKTCN-23 share the rs874203-rs422733 region (Figure 2 –pink bars). For markers rs13260-col4a1_snp1, a differenthaplotype was observed in the second family branch, initiatedby parents KTCN-92 and KTCN-16. This haplotype coveredthe entire length of the analyzed region, and was identified inall affected individuals and KTCN-21, whose phenotype wasunknown. Subject KTCN-17 had the same allele pattern formarkers s13260-col4a1_snp1, as individuals from the firstbranch of the family. However, in this case, analysis indicatedthat these markers are inherited from KTCN-92, who isunrelated to KTCN-93 and KTCN-01.
DISCUSSIONTo our knowledge, this is the first report describing completesequence analysis of the coding regions and the exon-intron
Molecular Vision 2011; 17:827-843 <http://www.molvis.org/molvis/v17/a94> © 2011 Molecular Vision
832
TAB
LE 2
. SEQ
UEN
CE
VA
RIA
NTS
FOU
ND
IN C
OL4
A1 A
ND
CO
L4A2
GEN
ES.
Aff
ecte
dK
TC
N-0
14
(n=1
0)
Una
ffec
ted
KT
CN
-014
(n
=11)
Unk
now
nK
TC
N-0
14
(n=
2)A
ll K
TC
N-0
14
(n
=23)
Oth
er K
TC
N
fa
mili
esaf
fect
ed (n
=25)
All
(n=4
8)
Exo
ndb
SNP
refI
DC
hrom
osom
ePo
sitio
nA
llele
Cha
nge
Res
idue
Cha
nge
no.
%no
.%
no.
%no
.%
no .%
no.
%
CO
L4A1
(NM
_001
845.
4)-
1109
5946
4c.
-90G
>T-
550
763
.60
012
52.2
1560
2756
.31
rs95
1518
511
0959
356
c.19
G>C
Val
7Leu
880
654
.51
5015
65.2
1872
3368
.8
-11
0959
167
c.84
+124
T>A
-10
100
1110
0.0
210
023
100.
025
100
4810
0.0
rs
7527
0666
1108
9520
0c.
85–1
19C
>T-
00
218
.21
503
13.0
520
816
.7
rs41
2751
0611
0895
150
c.85
–69T
>C-
110
327
.30
04
17.4
416
816
.7
rs95
2165
011
0866
265
c.23
4+8C
>T-
220
19.
10
03
13.0
520
816
.7
rs37
3732
811
0866
065
c.27
9+64
G>A
-5
505
45.5
150
1147
.810
4021
43.8
7rs
5326
2511
0864
225
c.43
2T>A
Ala
144A
la7
708
72.7
210
017
73.9
1456
3164
.6
rs71
8053
6611
0863
985,
1108
6398
9c.
468+
5_46
8+9d
el5
-3
302
18.2
00
521
.74
169
18.8
rs
7657
4181
1108
6275
0c.
469–
191C
>T-
330
436
.41
508
34.8
416
1225
.0
rs21
6620
811
0862
686
c.46
9–12
7C>T
-3
304
36.4
150
834
.84
1612
25.0
rs
9521
649
1108
6230
3c.
615+
24C
>T-
330
654
.51
5010
43.5
1664
2654
.2
rs21
6620
711
0862
268
c.61
5+59
T>G
-7
709
81.8
210
018
78.3
2184
3981
.3
rs64
5114
1108
6178
5c.
616–
11G
>C-
1010
011
100.
02
100
2310
0.0
2510
048
100.
0
rs73
3320
411
0861
671
c.65
1+68
A>G
-3
306
54.5
150
1043
.516
6426
54.2
rs
7332
120
1108
6167
0c.
651+
69C
>T-
330
654
.51
5010
43.5
1664
2654
.2
rs10
6876
4211
0861
652,
1108
6165
3c.
651+
86_6
51+8
7ins
2-
330
654
.51
5010
43.5
1664
2654
.2
rs
5583
3821
1108
6164
9c.
651+
90C
>G-
330
654
.51
5010
43.5
1664
2654
.2
rs35
6382
9411
0861
620,
1108
6162
1c.
651+
118_
651+
119i
ns4
-3
306
54.5
150
1043
.516
6426
54.2
rs
7333
008
1108
6156
0c.
651+
179A
>G-
330
654
.51
5010
43.5
1664
2654
.2
rs59
8893
1108
5974
3c.
780+
7G>A
-7
708
72.7
210
017
73.9
1768
3470
.8
rs59
8819
1108
5969
0c.
780+
60T>
C-
1010
011
100.
02
100
2310
0.0
2510
048
100.
0
rs95
8811
611
0859
069
c.80
8–7C
>G-
770
981
.82
100
1878
.321
8439
81.3
rs
6777
2891
1108
5932
6c.
781–
88de
lT-
1010
011
100.
02
100
2310
0.0
2510
048
100.
0
rs67
7877
1108
5789
5c.
859–
10T>
C-
770
545
.52
100
1460
.917
6831
64.6
rs
4827
5711
0857
823
c.90
3+18
G>A
-7
705
45.5
210
014
60.9
1768
3164
.6
rs66
5713
1108
5750
2c.
957+
198T
>C-
1010
011
100.
02
100
2310
0.0
1976
4287
.5
rs64
8735
1108
5618
0c.
958–
226T
>C-
770
872
.72
100
1773
.917
6834
70.8
rs
6487
0511
0856
153
c.95
8–19
9T>G
-7
708
72.7
210
017
73.9
1768
3470
.8
rs73
2772
811
0856
094
c.95
8–14
0T>A
-0
00
0.0
00
00.
01
41
2.1
rs
6482
6311
0856
085
c.95
8–13
1T>C
-7
708
72.7
210
017
73.9
1768
3470
.8
-11
0855
997
c.95
8–43
delT
-0
00
0.0
00
00.
01
41
2.1
-
1108
5303
2c.
1085
–205
A>T
-10
100
1110
0.0
210
023
100.
022
8845
93.8
rs
9952
2311
0851
036
c.11
21–5
8A>G
-3
306
54.5
150
1043
.517
6827
56.3
rs
4969
1611
0851
014
c.11
21–3
6C>G
-10
100
763
.61
5018
78.3
832
2654
.221
rs99
5224
1108
5084
2c.
1257
T>C
Pro4
19Pr
o3
306
54.5
150
1043
.517
6827
56.3
rs
6833
0911
0850
770
c.12
85+4
4A>G
-10
100
1110
0.0
210
023
100.
025
100
4810
0.0
rs
9588
112
1108
4756
6c.
1286
–101
G>A
-3
304
36.4
150
834
.84
1612
25.0
rs
5050
5011
0847
227
c.13
81+1
43C
>A-
1010
010
90.9
150
2191
.321
8442
87.5
rs
9521
643
1108
4721
7c.
1381
+153
T>C
-4
406
54.5
150
1147
.818
7229
60.4
rs
2241
966
1108
4719
0c.
1381
+180
A>G
-4
406
54.5
150
1147
.818
7229
60.4
rs
6858
8411
0845
314
c.13
82–5
4C>T
-9
9011
100.
02
100
2295
.721
8443
89.6
rs
2241
967
1108
4472
1c.
1466
–90G
>A-
880
763
.61
5016
69.6
1144
2756
.3
Molecular Vision 2011; 17:827-843 <http://www.molvis.org/molvis/v17/a94> © 2011 Molecular Vision
833
TAB
LE 2
. CO
NTI
NU
ED.
25rs
5361
7411
0839
550
c.16
63A
>CTh
r555
Pro
1010
011
100.
02
100
2310
0.0
2510
048
100.
0
rs95
2163
811
0839
428
c.17
28+5
7T>C
-7
707
63.6
150
1565
.219
7634
70.8
26rs
6174
9897
1108
3881
4c.
1815
T>C
Pro6
05Pr
o3
304
36.4
150
834
.84
1612
25.0
rs
2305
080
1108
3870
3c.
1897
+29A
>G-
770
763
.61
5015
65.2
1976
3470
.8
rs56
5470
1108
3864
6c.
1897
+86T
>C-
990
1090
.92
100
2191
.325
100
4695
.8
rs72
6541
1211
0835
460
c.19
91–1
6G>A
-0
00
0.0
00
00.
04
164
8.3
rs
7329
411
1108
3519
5c.
2095
+145
G>T
-7
707
63.6
150
1565
.219
7634
70.8
29rs
1697
5492
1108
3370
2c.
2130
G>A
Pro7
10Pr
o7
707
63.6
150
1565
.217
6832
66.7
rs
1697
5491
1108
3356
4c.
2193
+75G
>A-
770
763
.61
5015
65.2
1768
3266
.7
rs10
4924
9711
0831
866
c.21
94–9
8A>G
-3
304
36.4
150
834
.86
2414
29.2
rs
2131
939
1108
3183
7c.
2194
–69C
>T-
00
00.
00
00
0.0
416
48.
3
rs50
3053
1108
3145
1c.
2345
–68A
>G-
770
763
.61
5015
65.2
1768
3266
.7
-11
0830
612
c.26
26–3
4T>C
-0
00
0.0
00
00.
01
41
2.1
rs
2305
081
1108
3009
0c.
2716
+99C
>T-
550
436
.41
5010
43.5
936
1939
.6
rs15
6217
311
0828
922
c.29
68+5
1C>T
-7
707
63.6
150
1565
.217
6832
66.7
rs
1975
514
1108
2889
1c.
2969
–31A
>G-
770
763
.61
5015
65.2
1768
3266
.737
rs87
4204
1108
2758
0c.
3183
G>A
Gly
1061
Gly
770
763
.61
5015
65.2
1768
3266
.737
rs87
4203
1108
2757
4c.
3189
A>T
Arg
1063
Arg
770
763
.61
5015
65.2
1768
3266
.7
-11
0826
231
c.35
05+1
6C>T
-0
00
0.0
00
00.
01
41
2.1
rs
1751
7598
1108
2526
4c.
3506
–147
C>A
-1
101
9.1
00
28.
74
166
12.5
rs
2289
799
1108
2497
4c.
3556
+93G
>C-
770
763
.61
5015
65.2
1768
3266
.7
rs22
7584
511
0823
178
c.35
57–9
9C>T
-3
304
36.4
150
834
.86
2414
29.2
42-
1108
2294
3c.
3693
G>A
Thr1
231T
hr0
02
18.2
150
313
.07
2810
20.8
-
1108
2265
3c.
3742
+231
C>T
-0
00
0.0
00
00.
02
82
4.2
rs
5899
8511
0819
586
c.38
77–9
C>T
-9
9010
90.9
210
021
91.3
2496
4593
.8
rs17
7881
711
0819
460
c.39
49+4
5C>T
-9
9010
90.9
210
021
91.3
2496
4593
.8
rs65
2572
1108
1945
7c.
3949
+48T
>C-
990
1090
.92
100
2191
.324
9645
93.8
rs
1213
026
1108
1936
2c.
3949
+143
T>C
-9
9010
90.9
210
021
91.3
2496
4593
.8
-11
0818
760:
1108
1876
3c.
3950
–110
_395
0–11
3del
4-
00
00.
00
00
0.0
14
12.
1
45rs
3742
207
1108
1859
8c.
4002
A>C
Gln
1334
His
880
436
.41
5013
56.5
1040
2347
.9
rs18
1688
411
0817
171
c.41
50+3
8C>G
-4
406
54.5
210
012
52.2
1456
2654
.2
rs22
9824
111
0816
097
c.41
51–1
89C
>G-
220
654
.50
08
34.8
520
1327
.1
rs22
9824
011
0815
673
c.42
49+1
37G
>C-
00
00.
00
00
0.0
28
24.
2
rs16
9754
2411
0814
923
c.42
50–1
34T>
C-
110
545
.50
06
26.1
520
1122
.949
rs11
3321
911
0813
709
c.44
70C
>TA
la14
90A
la3
305
45.5
210
010
43.5
1040
2041
.7
rs22
7584
311
0813
532
c.46
40+7
C>T
-2
206
54.5
00
834
.85
2013
27.1
-
1108
1353
1c.
4640
+8G
>A-
440
218
.20
06
26.1
00
612
.5
rs22
7584
211
0813
523
c.46
40+1
6G>A
-2
206
54.5
00
834
.85
2013
27.1
rs
6171
1111
0807
776
c.46
41–3
2G>A
-4
402
18.2
00
626
.10
06
12.5
rs
6818
8411
0805
062
c.47
56–2
09C
>T-
1010
011
100.
02
100
2310
0.0
2496
4797
.9
-11
0804
970
c.47
56–1
17G
>C-
110
19.
10
02
8.7
00
24.
251
rs65
0724
1108
0480
9c.
4800
C>T
Ser1
600S
er2
204
36.4
00
626
.111
4417
35.4
rs
1326
011
0802
123
c.*5
87C
>A-
220
436
.40
06
26.1
1144
1735
.4
rs28
3625
1511
0801
735
c.*9
75A
>C-
110
19.
10
02
8.7
00
24.
2
Molecular Vision 2011; 17:827-843 <http://www.molvis.org/molvis/v17/a94> © 2011 Molecular Vision
834
Aff
ecte
dK
TC
N-0
14
(n=1
0)
Una
ffec
ted
KT
CN
-014
(n
=11)
Unk
now
nK
TC
N-0
14
(n=
2)A
ll K
TC
N-0
14
(n
=23)
Oth
er K
TC
N
fa
mili
esaf
fect
ed (n
=25)
All
(n=4
8)
Exo
ndb
SNP
refI
DC
hrom
osom
eA
llele
Cha
nge
Res
idue
Cha
nge
no.
%no
.%
no.
%no
.%
no%
no.
%
TAB
LE 2
. CO
NTI
NU
ED.
CO
L4A2
(NM
_001
846.
2)
rs
7989
823
1109
5964
3c.
-277
A>C
7
709
81.8
00
1669
.625
100
4185
.4
rs79
9000
911
0959
688
c.-2
32C
>G
550
763
.60
012
52.2
1768
2960
.4
rs79
9001
711
0959
705
c.-2
15C
>T
770
981
.80
016
69.6
2392
3981
.3
rs79
9133
211
0959
717
c.-2
03T>
C
550
763
.60
012
52.2
1560
2756
.3
rs35
4666
7811
0959
787
c.-1
33A
>G
330
327
.30
06
26.1
1456
2041
.7
rs73
2752
811
0960
044
c.-4
4–16
3G>C
0
00
0.0
00
00.
04
164
8.3
rs
7653
6922
1109
6016
4c.
-44–
43C
>T
00
00.
00
00
0.0
416
48.
3
rs47
7314
311
0960
685
c.99
+215
T>C
7
707
63.6
150
1565
.221
8436
75.0
rs
4773
144
1109
6071
2c.
99+2
42A
>G
770
763
.61
5015
65.2
2184
3675
.0
rs12
8765
1711
1009
643
c.10
0–17
6G>A
6
609
81.8
210
017
73.9
1976
3675
.0
rs47
7167
811
1076
940
c.18
1–14
1T>C
8
8010
90.9
210
020
87.0
2080
4083
.3Ex
5rs
4238
272
1110
7719
7c.
297G
>ATh
r99T
hr10
100
1110
0.0
210
023
100.
022
8845
93.8
rs
7496
7960
1110
7723
4c.
315+
19T>
C
00
00.
00
00
0.0
14
12.
1
rs73
3498
611
1080
609
c.36
1–20
5G>A
7
704
36.4
150
1252
.28
3220
41.7
-
1110
8096
4c.
477+
34C
>T
330
436
.41
508
34.8
00
816
.7
rs39
2975
811
1082
157
c.47
8–75
C>A
9
9010
90.9
210
021
91.3
2288
4389
.6Ex
9rs
6262
1885
1110
8277
2c.
574G
>TV
al19
2Phe
00
00.
00
00
0.0
14
12.
1
rs60
2120
7211
1086
650
c.68
5–98
G>A
0
03
27.3
00
313
.06
249
18.8
rs
4127
5108
1110
8845
6c.
727–
160A
>T
440
218
.20
06
26.1
28
816
.7
rs79
8348
711
1090
854
c.86
2–11
1A>G
1
105
45.5
00
626
.112
4818
37.5
rs
7984
937
1110
9090
9c.
862–
56T>
C
110
545
.50
06
26.1
1352
1939
.6
rs79
8410
011
1090
924
c.86
2–41
G>A
1
105
45.5
00
626
.113
5219
39.6
rs
7983
979
1110
9102
4c.
912+
9C>T
0
03
27.3
00
313
.06
249
18.8
rs
4771
680
1110
9801
7c.
958–
159T
>C
110
218
.20
03
13.0
1248
1531
.3
rs74
8970
511
1098
110
c.95
8–66
C>T
10
100
1110
0.0
210
023
100.
021
8444
91.7
Ex17
rs41
0311
1098
226
c.10
08C
>TPr
o336
Pro
1010
011
100.
02
100
2310
0.0
1560
3879
.2
rs59
9057
4711
1099
045
c.10
12–1
00C
>G
550
545
.51
5011
47.8
520
1633
.3
rs45
6128
3311
1099
057
c.10
12–8
8G>A
10
100
1110
0.0
210
023
100.
021
8444
91.7
rs
7326
449
1110
9912
2c.
1012
–23G
>A
1010
011
100.
02
100
2310
0.0
2184
4491
.7
rs56
6761
8111
1101
931
c.10
79–9
5C>T
5
506
54.5
150
1252
.27
2819
39.6
rs
7508
2326
1111
0195
2c.
1079
–74A
>G
550
654
.51
5012
52.2
728
1939
.6Ex
19rs
7642
5569
1111
0204
2c.
1095
G>A
Pro3
65Pr
o5
506
54.5
150
1252
.27
2819
39.6
Ex19
rs74
9417
9811
1102
126
c.11
79C
>TIle
393I
le5
506
54.5
150
1252
.27
2819
39.6
rs
3473
4302
1111
0218
3c.
1189
+47A
>G
990
981
.82
100
2087
.013
5233
68.8
-
1111
0285
3c.
1339
+52C
>G
00
00.
00
00
0.0
14
12.
1
rs72
6579
3411
1102
865
c.13
39+6
4G>A
0
02
18.2
00
28.
73
125
10.4
rs
9515
218
1111
0985
9c.
1432
+77A
>G
990
1090
.92
100
2191
.316
6437
77.1
rs
9555
703
1111
0988
2c.
1432
+100
A>G
6
604
36.4
210
012
52.2
520
1735
.4
rs95
1521
911
1109
960
c.14
32+1
78T>
C
990
1090
.92
100
2191
.316
6437
77.1
rs
9521
781
1111
1102
3c.
1433
–95T
>C
990
1090
.92
100
2191
.316
6437
77.1
rs
9521
782
1111
1104
3c.
1433
–75G
>A
990
1090
.92
100
2191
.315
6036
75.0
Ex22
rs79
9021
411
1111
173
c.14
88G
>APr
o496
Pro
990
1090
.92
100
2191
.316
6437
77.1
Ex22
rs79
9038
311
1111
235
c.15
50G
>AA
rg51
7Lys
990
1090
.92
100
2191
.316
6437
77.1
rs
4773
186
1111
1138
2c.
1596
+101
G>A
10
100
1110
0.0
210
023
100.
022
8845
93.8
rs
4127
5110
1111
1455
4c.
1669
+21G
>A
440
654
.50
010
43.5
624
1633
.3
rs79
9233
011
1114
751
c.17
76+2
0G>A
6
604
36.4
210
012
52.2
520
1735
.4
Molecular Vision 2011; 17:827-843 <http://www.molvis.org/molvis/v17/a94> © 2011 Molecular Vision
835
Aff
ecte
dK
TC
N-0
14
(n=1
0)
Una
ffec
ted
KT
CN
-014
(n
=11)
Unk
now
nK
TC
N-0
14
(n=
2)A
ll K
TC
N-0
14
(n
=23)
Oth
er K
TC
N
fa
mili
esaf
fect
ed (n
=25)
All
(n=4
8)
Exo
ndb
SNP
refI
DC
hrom
osom
eA
llele
Cha
nge
Res
idue
Cha
nge
no.
%no
.%
no.
%no
.%
no%
no.
%
TAB
LE 2
. CO
NTI
NU
ED.
rs
3803
237
1111
1766
8c.
1777
–84G
>A
440
654
.50
010
43.5
624
1633
.3
rs38
0323
611
1117
745
c.17
77–7
C>T
9
9010
90.9
210
021
91.3
1664
3777
.1
rs38
2549
011
1117
984
c.19
78+3
1C>T
4
404
36.4
150
939
.18
3217
35.4
rs
7265
7953
1111
1807
3c.
1978
+120
C>T
2
202
18.2
00
417
.44
168
16.7
rs
1983
931
1111
1810
2c.
1978
+149
G>A
9
9010
90.9
210
021
91.3
1664
3777
.1
rs19
8393
211
1118
221
c.19
79–1
29T>
C
330
327
.30
06
26.1
728
1327
.1
rs41
2751
1211
1118
450
c.20
38+4
1C>T
0
00
0.0
00
00.
01
41
2.1
rs
1927
350
1111
1854
6c.
2038
+137
T>G
3
303
27.3
00
626
.17
2813
27.1
rs
3803
232
1111
1929
6c.
2039
–91A
>G
990
1090
.92
100
2191
.316
6437
77.1
rs
3803
231
1111
1934
2c.
2039
–45T
>C
220
218
.20
04
17.4
416
816
.7Ex
27rs
3803
230
1111
1939
6c.
2048
G>C
Gly
683A
la2
202
18.2
00
417
.44
168
16.7
rs
9559
813
1111
2144
4c.
2096
–120
C>A
8
809
81.8
210
019
82.6
1456
3368
.8
rs95
5981
411
1121
483
c.20
96–8
1A>G
9
9010
90.9
210
021
91.3
1664
3777
.1Ex
28rs
7882
9338
1111
2157
0c.
2102
A>G
Lys7
01A
rg1
103
27.3
150
521
.73
128
16.7
Ex28
rs95
8350
011
1121
620
c.21
52C
>TPr
o718
Ser
440
436
.40
08
34.8
520
1327
.1
rs95
1522
911
1121
717
c.22
03+4
6A>G
9
9010
90.9
210
021
91.3
1664
3777
.1
rs95
1523
011
1121
847
c.22
03+1
76T>
C
220
218
.20
04
17.4
416
816
.7
rs95
8817
811
1125
576
c.24
25+7
9G>A
0
00
0.0
00
00.
02
82
4.2
rs
1161
7206
1111
2560
6c.
2425
+109
A>G
7
707
63.6
150
1565
.222
8837
77.1
rs
9588
179
1111
2574
7c.
2425
+250
C>A
0
00
0.0
00
00.
02
82
4.2
rs
9559
818
1111
3022
6c.
2426
–124
G>A
8
807
63.6
150
1669
.621
8437
77.1
rs
2281
974
1111
3051
9c.
2587
+8C
>T
550
545
.51
5011
47.8
1872
2960
.4
rs93
0145
711
1130
599
c.25
87+8
8G>C
10
100
1110
0.0
210
023
100.
025
100
4810
0.0
rs
2281
973
1111
3067
4c.
2587
+163
C>T
4
405
45.5
150
1043
.515
6025
52.1
rs
7265
7977
1111
3241
3c.
2588
–154
C>T
4
403
27.3
00
730
.43
1210
20.8
rs
4773
194
1111
3249
0c.
2588
–77A
>G
1010
08
72.7
210
020
87.0
1768
3777
.1
rs95
2180
311
1132
556
c.25
88–1
1C>T
8
806
54.5
210
016
69.6
832
2450
.0
rs14
7543
811
1132
820
c.27
58+8
3G>A
4
406
54.5
00
1043
.511
4421
43.8
rs
5812
4222
1111
3294
7c.
2758
+210
G>A
0
03
27.3
00
313
.08
3211
22.9
rs
3803
229
1111
3478
0c.
2759
–83G
>A
660
763
.61
5014
60.9
936
2347
.9
rs38
0322
811
1134
858
c.27
59–5
T>C
0
03
27.3
00
313
.08
3211
22.9
rs
2296
853
1111
3724
0c.
2903
–12A
>G
330
327
.32
100
834
.86
2414
29.2
rs
2296
852
1111
3746
5c.
3025
+91G
>A
220
218
.20
04
17.4
1040
1 429
.2
rs11
8395
2711
1137
488
c.30
25+1
14G
>A
220
218
.20
04
17.4
1040
1429
.2
rs41
3150
4811
1137
975
c.30
26–2
7G>T
1
103
27.3
00
417
.42
86
12.5
rs
2296
851
1111
3825
5c.
3207
+72G
>A
220
218
.20
04
17.4
1040
1429
.2
rs35
1209
1811
1143
541
c.33
47–3
9G>A
0
01
9.1
150
28.
70
02
4.2
rs
4137
5611
1143
755
c.34
54+6
8T>C
8
8011
100.
01
5020
87.0
2288
4287
.5
rs40
2661
1111
4385
1c.
3454
+164
G>C
3
305
45.5
00
834
.810
4018
37.5
rs
4520
2011
1144
102
c.34
55–3
15T>
C
1010
011
100.
02
100
2310
0.0
2510
048
100.
0
rs40
3839
1111
4432
1c.
3455
–96G
>A
220
218
.20
04
17.4
728
1122
.9
-11
1144
382
c.34
55–3
5T>C
1
100
0.0
00
14.
31
42
4.2
rs
2296
849
1111
4441
2c.
3455
–5C
>G
110
327
.30
04
17.4
28
612
.5
rs42
1177
1111
4456
5c.
3562
+41C
>T
110
327
.30
04
17.4
28
612
.5
rs57
0035
8211
1145
456:
1111
4548
6c.
3563
–100
_356
3–70
del3
07
7011
100.
01
5019
82.6
2288
4185
.4
rs
2274
544
1111
4563
3c.
3634
+4C
>T
110
327
.30
04
17.4
28
612
.5
rs23
9183
311
1145
676
c.36
34+4
7G>C
8
8010
90.9
150
1982
.620
8039
81.3
rs
9559
826
1111
4577
9c.
3634
+150
C>T
2
202
18.2
00
417
.48
3212
25.0
-
1111
4763
7c.
3635
–52A
>G
330
436
.41
508
34.8
00
816
.7
Molecular Vision 2011; 17:827-843 <http://www.molvis.org/molvis/v17/a94> © 2011 Molecular Vision
836
Aff
ecte
dK
TC
N-0
14
(n=1
0)
Una
ffec
ted
KT
CN
-014
(n
=11)
Unk
now
nK
TC
N-0
14
(n=
2)A
ll K
TC
N-0
14
(n
=23)
Oth
er K
TC
N
fa
mili
esaf
fect
ed (n
=25)
All
(n=4
8)
Exo
ndb
SNP
refI
DC
hrom
osom
eA
llele
Cha
nge
Res
idue
Cha
nge
no.
%no
.%
no.
%no
.%
no%
no.
%
TAB
LE 2
. CO
NTI
NU
ED.
rs
3786
0111
1153
934
c.37
61–8
1G>A
10
100
1110
0.0
210
023
100.
025
100
4810
0.0
rs
3882
2211
1154
159
c.38
77+2
8C>T
8
808
72.7
210
018
78.3
2184
3981
.3
rs22
8196
811
1154
160
c.38
77+2
9G>A
5
508
72.7
150
1460
.918
7232
66.7
rs
4773
198
1111
5571
1c.
4040
–19C
>T
550
654
.51
5012
52.2
832
2041
.7Ex
43rs
4773
199
1111
5577
9c.
4089
G>A
Ala
1363
Ala
550
436
.41
5010
43.5
832
1837
.5
rs93
0146
011
1156
153
c.41
39–4
1G>A
5
506
54.5
150
1252
.28
3220
41.7
rs
4148
8111
1156
411
c.42
85+7
1G>A
7
708
72.7
150
1669
.621
8437
77.1
Ex45
rs47
7168
311
1156
499
c.42
90T>
CPh
e143
0Phe
1010
011
100.
02
100
2310
0.0
2510
048
100.
0Ex
46rs
4453
4811
1158
874
c.45
15A
>GPr
o150
5Pro
1010
011
100.
02
100
2310
0.0
2510
048
100.
0
rs24
7942
611
1164
198
c.48
82–8
3T>C
7
706
54.5
210
015
65.2
2080
3572
.9
rs42
2733
1111
6461
4c.
*76T
>C
770
654
.52
100
1565
.220
8035
72.9
rs
3074
455
1111
6463
9,11
1164
640
c.*1
01_*
102d
el2
7
706
54.5
210
015
65.2
2080
3572
.9
rs
1050
911
1164
955
c.*4
17C
>G
880
1110
0.0
210
021
91.3
2510
046
95.8
rs
1049
906
1111
6507
9c.
*541
C>T
4
408
72.7
150
1356
.518
7231
64.6
rs
1049
931
1111
6509
5c.
*557
A>G
6
609
81.8
150
1669
.620
8036
75.0
rs
1049
977
1111
6518
8c.
*650
T>C
6
609
81.8
150
1669
.619
7635
72.9
rs
7711
1111
6520
1c.
*663
T>C
6
609
81.8
150
1669
.619
7635
72.9
rs
1545
711
1165
265
c.*7
27G
>C
440
872
.71
5013
56.5
1768
3062
.5
d
bSN
P re
f ID
: ide
ntity
num
bers
of o
bser
ved
sequ
ence
var
iant
s; c
hrom
osom
e po
sitio
n (N
CB
I bui
ld 3
7.1)
.
Molecular Vision 2011; 17:827-843 <http://www.molvis.org/molvis/v17/a94> © 2011 Molecular Vision
837
Aff
ecte
dK
TC
N-0
14
(n=1
0)
Una
ffec
ted
KT
CN
-014
(n
=11)
Unk
now
nK
TC
N-0
14
(n=
2)A
ll K
TC
N-0
14
(n
=23)
Oth
er K
TC
N
fa
mili
esaf
fect
ed (n
=25)
All
(n=4
8)
Exo
ndb
SNP
refI
DC
hrom
osom
eA
llele
Cha
nge
Res
idue
Cha
nge
no.
%no
.%
no.
%no
.%
no%
no.
%
T AB
LE 3
. PR
EDIC
TIO
N O
F EFF
ECT
OF A
MIN
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CID
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STIT
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ON
S FO
UN
D IN
CO
L4A
1 A
ND
CO
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2.
Poly
Phen
SIFT
PMU
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RSN
AP
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rian
tPS
ICsc
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Pred
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Pred
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tole
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1.13
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lera
ted
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61ne
utra
l-
-92
%ne
utra
l
Gly
683A
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/Abe
nign
0.96
tole
rate
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4841
neut
ral
--
85%
neut
ral
Ly
s701
Arg
N/A
beni
gn0.
97to
lera
ted
0.01
66ne
utra
l-
-89
%ne
utra
l
Pro7
18Se
rN
/Abe
nign
0.98
tole
rate
d0.
2039
neut
ral
--
89%
neut
ral
T
he P
olyP
hen
tool
pre
dict
s w
hich
mis
sens
e su
bstit
utio
n af
fect
s th
e st
ruct
ure
and
func
tion
of p
rote
in, a
nd u
ses
Posi
tion-
Spec
ific
Inde
pend
ent C
ount
s so
ftwar
e to
a
ssig
n pr
ofile
sco
res.
The
SIFT
tool
eva
luat
es c
onse
rved
pos
ition
s, an
d ca
lcul
ates
a s
core
for t
he a
min
o ac
id c
hang
e at
a p
artic
ular
pos
ition
. A s
core
of <
0.05
is
con
side
red
as p
atho
geni
c fo
r the
pro
tein
stru
ctur
e. T
he P
MU
T ca
lcul
ates
the
path
olog
ical
sig
nific
ance
of n
on-s
ynon
ymou
s am
ino
acid
sub
stitu
tion
usin
g ne
ural
n
etw
orks
(NN
). N
N o
utpu
t >0.
5 is
cons
ider
ed to
be d
elet
erio
us. P
AN
THER
gen
erat
es th
e sub
stitu
tion
Posi
tion-
Spec
ific E
volu
tiona
ry C
onse
rvat
ion
scor
e. T
he v
alue
−
3 is
cut
off p
oint
for f
unct
iona
l sig
nific
ance
and
cor
resp
onds
to a
Pde
lete
rious
of 0
.5. I
f the
sub
stitu
tion
occu
rs a
t a p
ositi
on n
ot a
ppea
ring
in th
e m
ultip
le s
eque
nce
a
lignm
ent,
a su
bPSE
C s
core
can
not b
e ca
lcul
ated
and
cha
nge
is n
ot li
kely
to b
e pa
thog
enic
. The
SN
AP
outp
ut s
how
s pr
edic
tion
neut
ral o
r non
-neu
tral,
and
the
e
xpec
ted
accu
racy
.
Molecular Vision 2011; 17:827-843 <http://www.molvis.org/molvis/v17/a94> © 2011 Molecular Vision
838
boundaries of COL4A1 and COL4A2 in families with KTCN.Previous studies have revealed a correlation between KTCNdevelopment and histopathological alterations in the structureof the corneal stroma and basement membrane, including aloss of collagen concentration [42] and rearrangement ofcollagen fibers [26]. Moreover, several types of collagen,including collagen type IV have been identified in the cornea[24], and COL4A1 and COL4A2 expression has been detectedin the human cornea [29]. Finally, we had mapped a locus forKTCN to 13q32, in close proximity of which COL4A1 andCOL4A2 are localized [21]. Given that information, wehypothesized that COL4A1 and COL4A2 genes are goodcandidates for causing KTCN in families with linkage to thatlocus.
Different studies have revealed several loci and a fewcandidate genes for familial KTCN. The first gene proposedas playing a significant role in KTCN pathogenesis was theVSX1 (visual system homeobox 1, OMIM 605020) gene. Itwas suggested that a few disease-causing mutations werepresent in this gene [43,44], but recent studies have notconfirmed these findings [21,45-47]. Next, heterozygousgenomic 7-bp deletion in intron 2 of SOD1 (superoxidedismutase 1; OMIM 147450) was identified in two familieswith KTCN [48,49]. In contrast, other studies have shown thatmutations in this gene are not associated with KTCNpathogenesis [21,47]. Genetic analyses ofCOL4A3,COL4A4,COL8A1, and COL8A2 genes haverevealed no pathogenic mutations in patients with KTCN,indicating that other genetic factors cause the disease[50-52].
We identified several single base pair substitutions in thecoding regions of COL4A1 and COL4A2, including one novelheterozygous change, c.3693G>A in exon 42 of COL4A1.None of the detected alterations segregated fullywith the affected phenotype in the analyzed members of theEcuadorian KTCN families. Among the identified missensesubstitutions in COL4A1, one change, c.4002A>C (p.Gln1334His), was observed more frequently in KTCNpatients than in healthy individuals in family KTCN-014.However, no significant statistical association of this changewith familial disease could be proven (p=0.056), and nodifference in the c.4002A>C allele distribution between theanalyzed affected individuals from the remaining KTCNfamilies and the Ecuadorian control subjects was discovered
(p=0.17). To predict the impact of the substitutions on thestructure and function of the protein, we used different tools.All identified missense substitutions in COL4A1 andCOL4A2 were predicted by the SIFT, PMUT, PANTHER, andSNAP tools to have no effect, but PolyPhen defined theGln1334His change in COL4A1 as possibly damaging.Glutamine at this position is highly conserved in differentspecies. Moreover, this change is present in the collagenousdomain of the α1(IV) chain with Gly-X-Y repeats, whichplays a role in the assembly into a triple-helical structure ofthe protein [22]. Replacement of the neutral residue (Gln) withthe polar amino acid (His) at the Y position is likely to affectthe protein structure. Nevertheless, further studies should beperformed to determine the functional significance of thissubstitution.
To the best of our knowledge, no mutations in COL4A1were associated with corneal disease. The spectrum ofCOL4A1-related disorders included porencephaly (OMIM175780) [53-55], Hereditary Angiopathy with Nephropathy,Aneurysm and Muscle Cramps (HANAC; OMIM 611773)[56], and brain small vessel disease with hemorrhage (OMIM607595) [57]. Recent studies have also revealed an associationbetween mutations in exon 29 of COL4A1 and Axenfeld-Rieger anomaly with leukoencephalopathy and stroke [58]. Inour study, none of the previously reported COL4A1 mutationswere identified. The absence of these changes in patients withKTCN suggests that they are specific to the above-mentioneddisorders only, and are not associated with KTCN in the testedfamilies. To date, no mutations responsible for COL4A2-related human diseases have been reported.
Besides changes identified in the coding regions ofCOL4A1 and COL4A2, our study revealed numerousalterations in introns and UTRs of both genes, including singlebase pair substitutions, deletions, and insertions. Fourteen ofthese were novel and their clinical significance is not known.Each of the changes was observed in affected and healthyindividuals in the tested families. Because importantfunctional elements are located in non-coding regions ofgenes [59] and intronic alterations can result in a deleteriouseffect on pre-mRNA splicing [60], identification of thesesequence variants could be non-accidental. Further researchis needed to delineate the role of these sequence variants.
Recent studies have shown that a mouse with a mutationin a splice acceptor site of Col4a1 has ocular dysgenesis. The
Figure 1. Multiple sequence alignment of the amino acid sequences of COL4A1 orthologs in different species. Conservation of glutamine (Q)at the 1334 position is shown in gray.
Molecular Vision 2011; 17:827-843 <http://www.molvis.org/molvis/v17/a94> © 2011 Molecular Vision
839
mutation results in a lack of exon 40 from mice’s transcriptsand leads to the accumulation of mis-folded protein in the lens
epithelial cells. Col4a1∆ex40 mice show optic nerve hypoplasiaand anterior segment dysgenesis (ASD) including pigment
Figure 2. Pedigree of the family KTCN-014. Black-filled symbols: individuals with KTCN; open symbols: individuals without KTCN; gray-filled symbols: individuals with unknown KTCN status. Below each symbol the haplotypes are shown for the coding sequence in genesCOL4A1, COL4A2 and UTRs between them. In COL4A1, the coding regions are surrounded by the markers rs13260 and col4a2_snp, and byrs35466678 and rs422733 in COL4A2, which were marked by a black frame. Haplotype regions in different colors indicate patterns ofinheritance in the two branches in the pedigree.
Molecular Vision 2011; 17:827-843 <http://www.molvis.org/molvis/v17/a94> © 2011 Molecular Vision
840
dispersion, cataracts, and corneal opacifications [61]. Spliceacceptor sites are highly conserved regions in different species[56]. We detected no alterations in the splice acceptor site inintron 39 of human COL4A1.
Extended genetic studies executed in families withKTCN have shown a high level of genetic heterogeneity[62]. The presence of many putative loci supports thehypothesis that KTCN is an oligogeneic disease in whichaccumulation of sequence variants at several loci cause aspecific KTCN haplotype and may trigger the phenotypiceffect. The absence of mutations in COL4A1 and COL4A2genes indicates that other genes are involved in KTCNpathogenesis in Ecuadorian families.
ACKNOWLEDGMENTSSupported by the Polish Ministry of Science and HigherEducation, Grant NN 402097837. The authors thankGenomed Company (Warsaw, Poland) for support insequencing service.
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Articles are provided courtesy of Emory University and the Zhongshan Ophthalmic Center, Sun Yat-sen University, P.R. China.The print version of this article was created on 25 March 2011. This reflects all typographical corrections and errata to the articlethrough that date. Details of any changes may be found in the online version of the article.
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