Germ-line and somatic EPHA2 coding variants in lens aging and cataract
Germ-line and somatic EPHA2 coding variants in lens aging and cataract
Editor: Alvaro Galli
Thomas M. Bennett 0
J. Fielding Hejtmancik
Alan Shiels 0
0 Department of Ophthalmology and Visual Sciences, Washington University School of Medicine , St. Louis , Missouri, United States of America , 2 Ophthalmic Genetics and Visual Function Branch , National Eye Institute, National Institutes of Health , Bethesda, Maryland , United States of America
Rare germ-line mutations in the coding regions of the human EPHA2 gene (EPHA2) have been associated with inherited forms of pediatric cataract, whereas, frequent, non-coding, single nucleotide variants (SNVs) have been associated with age-related cataract. Here we sought to determine if germ-line EPHA2 coding SNVs were associated with age-related cataract in a case-control DNA panel (> 50 years) and if somatic EPHA2 coding SNVs were associated with lens aging and/or cataract in a post-mortem lens DNA panel (> 48 years). Micro-fluidic PCR amplification followed by targeted amplicon (exon) next-generation (deep) sequencing of EPHA2 (17-exons) afforded high read-depth coverage (1000x) for > 82% of reads in the cataract case-control panel (161 cases, 64 controls) and > 70% of reads in the post-mortem lens panel (35 clear lens pairs, 22 cataract lens pairs). Novel and reference (known) missense SNVs in EPHA2 that were predicted in silico to be functionally damaging were found in both cases and controls from the age-related cataract panel at variant allele frequencies (VAFs) consistent with germ-line transmission (VAF > 20%). Similarly, both novel and reference missense SNVs in EPHA2 were found in the post-mortem lens panel at VAFs consistent with a somatic origin (VAF > 3%). The majority of SNVs found in the cataract case-control panel and post-mortem lens panel were transitions and many occurred at di-pyrimidine sites that are susceptible to ultraviolet (UV) radiation induced mutation. These data suggest that novel germ-line (blood) and somatic (lens) coding SNVs in EPHA2 that are predicted to be functionally deleterious occur in adults over 50 years of age. However, both types of EPHA2 coding variants were present at comparable levels in individuals with or without age-related cataract making simple genotype-phenotype correlations inconclusive.
Data Availability Statement: All relevant data are
available within the paper and its Supporting
Information files and from NIH Short Read
ArchiveAccession number (PRJNA384802).
Funding: This work was supported by NIH/NEI
grants EY012284 and EY023549 (to A.S.) and P30
EY02687 (Core Grant for Vision Research), and an
unrestricted grant to the Department of
Ophthalmology and Visual Sciences from Research
to Prevent Blindness (RPB). The Genome
Technology Access Center (GTAC) at Washington
University School of Medicine is partially supported
by NIH grants P30 CA91842 and UL1 TR000448.
Cataract(s) is a clinically heterogeneous disorder that causes clouding or opacification of the
crystalline lens and, thereby, impairs refraction and focusing of light onto the photosensitive
retina of the eye. Typically, cataract is acquired with aging (> 50 years) and, despite surgical
treatment, age-related cataract remains a leading cause of adult visual impairment (17%-33%)
The funders had no role in study design, data
collection and analysis, decision to publish, or
preparation of the manuscript.
and blindness (33%-51%) worldwide [1±3]. Besides aging, epidemiological studies have
identified multiple environmental or lifestyle risk factors for age-related cataract including, solar
UV-radiation exposure, tobacco smoking, and diabetes [4±6]. In addition, genetic factors are
believed to account for 35±58% of the risk for age-related cataract [
]. Beyond age-related
cataract, congenital, infantile and childhood forms of cataract that occur with relatively low
prevalence (1±15 cases/10,000) account for 1.4%-34% of pediatric visual impairment globally
[9±12]. Etiological studies of pediatric cataract reveal that genetic causes account for 10%-39%
of cases; however, this may represent an underestimate since 50%-60% of cases are deemed
]. So far, genetic studies have identified at least 30 genes underlying inherited
forms of pediatric cataract and several of these genes have also been implicated in the much
more common forms of age-related cataract [
EPH-receptor A2 (EPHA2) is a member of the erythropoietin-producing
hepatocellularcarcinoma (EPH) sub-family of receptor tyrosine kinases (RTKs) that play critical signaling
roles in embryonic development, adult tissue homeostasis, and cancer development and
progression [16±20]. Structurally, EPHA2 is a type-1 (single-pass) transmembrane glycoprotein
(~130kDa) with multiple functional domains including an extracellular (N-terminal) ligand
binding domain (LBD) for eph-receptor interacting (ephrin) ligands and cytoplasmic
(C-terminal) domains including a tyrosine kinase (TK) signaling domain and a sterile-α-motif
(SAM) domain implicated in receptor clustering and protein-protein interactions [
First identified as epithelial cell kinase (eck) , EPHA2 is widely expressed in epithelial
tissues and is surprisingly abundant in the plasma-membrane proteome of the ocular lens in
both humans and mice [
], where it is believed to function in lens cell migration and
Genetic studies have identified germ-line mutations in the human EPHA2 gene (EPHA2)
on chromosome 1p that underlie inherited forms of pediatric cataract exhibiting both
autosomal dominant and recessive modes of inheritance [30±43]. EPHA2-related cataract may
present at birth (congenital), during infancy or during childhood and displays variable clinical
morphology including posterior polar opacities, nuclear opacities, cortical opacities and total
lens opacities (https://sites.wustl.edu/catmap). Currently, the EPHA2 mutation spectrum
includes 14 missense mutations predicted to result in amino-acid substitutions, one nonsense
mutation, and five frame-shift mutations predicted to result in either C-terminally truncated
or extended proteins. Most of these mutations (13/20) occur in cytoplasmic domains of
EPHA2 with four mutations clustered within the SAM domain and two in the TK domain.
Ectopic overexpression studies in cultured cells suggest that mutations in the SAM domain
destabilize the receptor and/or impair targeting to the plasma-membrane [
Beyond rare mutations, single nucleotide polymorphisms/variants (SNPs/SNVs) across the
EPHA2 region have been variably associated with the much more prevalent forms of
agerelated cataract including cortical cataract, posterior sub-capsular cataract (PSC) and mixed
forms of lens opacities in Caucasian/European, Asian/Indian and Chinese populations [26,30,
46±50]. While most of the associated SNVs were located in non-coding or untranslated regions
(UTRs), at least one rare, non-synonymous (missense), coding SNV (rs116506614) predicted
to result in an amino-acid substitution (p.R721Q) has been associated with age-related cataract
]. Further, in silico prediction analysis suggests that several other missense SNVs in EPHA2
(e.g. rs229180, p.E825K) may have deleterious effects on receptor function [
] and expression
of several EPHA2 coding SNVs (rs1058371Ðp.I96F, p.E825K) in cultured lens epithelial cells
has been associated with receptor destabilization and increased susceptibility to oxidative
]. These observations suggest that rare coding SNVs in EPHA2 may increase
susceptibility to age-related forms of cataract. Here we sought to determine whether rare coding SNVs
2 / 19
in EPHA2, of either germ-line or somatic origin, were associated with lens aging and/or
Materials and methods
Ethical approval for this study was obtained from the University of Parma, the National
Eye Institute, and Washington University (IRB ID #: 201111056 and 00±0320), and written
informed consent was provided in accordance with the tenets of the Declaration of Helsinki.
Cataract case-control DNA panel
Genomic DNA was extracted using standard methods from blood samples donated by a
casecontrol cohort of unrelated individuals age 50 years form Northern Italy that were
ascertained from the Clinical Trial of Nutritional Supplements (CTNS) and Age-Related Cataract
]. Cataract status (nuclear, cortical, posterior sub-capsular, clear lens) was
evaluated by grading slit-lamp and retro-illumination lens photographs according to a modification
of the Age-Related Eye Disease Study (AREDS) cataract grading system as described .
Lens DNA panel
Post-mortem human donor lenses ( 48 years of age, with or without cataract) were obtained
(on dry-ice) from the National Disease Research Interchange (http://ndriresource.org/).
Lens genomic DNA was extracted using the DNeasy Kit (Qiagen, Valencia, CA) essentially
according to the manufacturer's protocol with the following modifications to mitigate the
high protein-to-DNA content of the lens. Each lens was homogenized (2 minÐsetting 8,
Bullet Blender 24, Next Advance, Averill park, NY) in buffer ATL (360 ul) then digested (16
hr, 56ÊC) with proteinase K (40 ul 15 mg/ml). Samples were then diluted with buffer ATL
(360ul) and re-digested (2 hr, 56ÊC) with proteinase K (40 ul) followed by centrifugation (5
min, 10,000 x g) to remove excess protein before processing through spin-columns according
to the manufacturer's instructions. DNA was eluted from the spin-columns in buffer AE
(200 ul) and quantified (OD260) using a spectrophotometer (ND-2000, NanoDrop,
Wilmington, DE). If necessary, samples were concentrated by air-drying in a laminar-flow hood and
re-suspended in ultrapure water to give a minimum concentration of 50 ng/μl required for
Targeted-amplicon deep-sequencing and variant calling
Targeted-amplicon deep-sequencing was performed using the Access Array Integrated
Fluidic Circuit (IFC) System with custom designed and validated gene-specific adaptor-primers
(Fluidigm, San Francisco, CA). Each IFC enables nanoliter-volume high-throughput PCR to
generate amplicons ( 200 bp) across 48 samples in a single run for subsequent
next-generation (deep)-sequencing (NGS). Briefly, DNA samples (50 ng) and primers were mixed
`onchip' (48.48 Access Array IFC/pre-PCR IFC Controller AX), and PCR amplified (FC1
Cycler). Amplicons for each sample were pooled on-chip (post-PCR IFC Controller AX)
then indexed with sample barcodes and NGS adaptors (Access Array Barcode kit) to produce
48 sequencer-ready libraries. Sequencing-by-synthesis was performed on the MiSeq platform
(Illumina, San Diego, CA). Paired-end reads were aligned to the human reference genome
(hg19) with Novoalign (www.novocraft.com) and processed using the Sequence Alignment/
Map (SAM) tools software package and Picard programs (http://samtools.sourceforge.net/).
Variants were called using the FreeBayes program (https://arxiv.org/abs/1207.3907) for
3 / 19
germ-line variants and the VarScan 2 program for somatic variants [
sourceforge.net). Finally, selected SNVs were confirmed in both directions by manual
inspection using the Integrative Genomics Viewer (IGV) browser  (http://software.
Genetic association analysis and logistic regression analysis of selected SNVs found in the
cataract case-control panel was performed using the Golden Helix SNP and Variation Suite 7
(Golden Helix, Bozeman, MT). Statistical comparison of somatic SNVs found in the
post-mortem lens panel was performed using Fisher's Exact Test by means of the online spreadsheet at
http://www.langsrud.com/fisher.htm. A probability (p) value of < 0.05 after correction for
multiple testing was considered significant.
The cataract case-control panel comprised 225 leukocyte DNA samples from 161 patients with
age-related cataract (age 50+) and 64 age-matched clear lens controls from the N. Italian
]. The cataract cases included 67 nuclear only, 43 cortical only, and two posterior
sub-capsular cataract (PSC) only. In addition to `pure' forms of cataract, there were multiple
cases of mixed cataract including 21 nuclear + cortical, 14 nuclear + PSC, 10 cortical + PSC,
and four nuclear + cortical + PSC. The mean age of cataract cases = 74.2 ± SD 6.54 years
(range 50±85 years) and the mean age of clear lens controls = 75.19 ± SD 4.2 years (range 57±
86), with no significant difference between cases and controls (p = 0.21). The sex distribution
was 50% female and 50% male in the cases and 44% female and 56% male in the controls.
There was no association between any cataract and sex in the case-control panel using
chisquare test (p = 0.51).
Post-mortem donor lenses were briefly examined at the time of procurement for the
presence or absence of obvious age-related cataract prior to cryopreservation. However, the donor
information report did not identify age-related cataract sub-types (e.g. nuclear, cortical).
Further, we cannot exclude the possibility that cataract in some of these donor lenses may have
been associated with causes other than aging (e.g. uveitis). The post-mortem lens panel
comprised 118 genomic DNA samples extracted from 74 clear lenses (37 pairs) and 44 cataract
lenses (22 pairs) all obtained from Caucasian donors (age 48+ years). Two of the clear lens
pairs failed amplicon sequencing and/or QC criteria leaving 114 lens samples (35 clear pairs,
22 cataract pairs) for variant analysis. The mean age of cataract lenses = 65.5 ± SD 6.67 (range
48±74 years) and the mean age of clear lenses = 64.06 ± SD 7.37 (range 48±78 years) with no
significant difference between the two groups (p = 0.45). The sex distribution was 23% female
and 77% male in the cataract lenses and 49% female and 51% male in the clear lenses. Despite
the numerical sex difference in the cataract lenses there was no significant association between
any cataract and sex in the post-mortem lens panel using chi-square test (p = 0.095).
Targeted-amplicon deep-sequencing of exonic variants
We performed targeted-amplicon deep-sequencing of the coding regions (exons) of the
human EPHA2 gene to identify germ-line single nucleotide variants (SNVs) in the cataract
case-control panel and somatic SNVs in the lens panel. EPHA2 (GeneID: 1969) spans ~31.8
Kbp on the short (p) arm of chromosome 1 (cytogenetic band region 1p36.1) [
] with a
physical location between nucleotides (nt) 16124337±16156104 (counted from the short-arm
4 / 19
telomere, ptel) on the complement strand [Annotation release 108, Genome Reference
Consortium Human Build 38 patch release 7 (GRCh38.p7)] (http://www.ncbi.nlm.nih.gov/gene/
1969). Currently, the gene reference sequence (NG_021396.1) comprises 17 coding exons
generating two transcript variants, NM_004431.4 and NM_001329090.1, encoding protein
isoforms of 976 amino acids (NP_004422.2) and 922 amino acids (NP_001316019), respectively.
For comparison with EPHA2, we simultaneously performed amplicon sequencing of the gene
coding for cellular tumor antigen p53 (TP53)Ða tumor suppressor gene that is known to
acquire somatic mutations in several cancers (e.g. cutaneous melanoma) (http://cancer.sanger.
ac.uk/cosmic). TP53 (Gene ID: 7157) spans ~19.15 Kbp on chromosome 17p13.1 (7668402±
7687550, complement) and the gene reference sequence (NG_017013.2) comprises 11 coding
exons giving rise to 8 transcript variants and 12 protein isoforms (a-l) ranging from 182±393
Optimal custom design of PCR primer pairs (Fluidigm) to amplify exons for
deepsequencing resulted in 35 amplicons for EPHA2 and 15 amplicons for TP53. Across the
cataract case-control panel the mean total number of reads was 418,214 with > 99% on target of
which > 82% attained 1000x coverage (S1 Table). Similarly, across the lens panel the mean
total number of reads was 456,286 with > 99% on target of which > 70% attained 1000x
coverage (S1 Table). All amplicons were fully sequenced in both directions with the exception of
amplicon 35 in EPHA2 (part of exon-1) likely due to its high G/C content.
Following sequencing, germ-line SNVs in the cataract case-control panel (blood leukocyte
DNA) were called using the FreeBayes program. Variant allele frequencies (VAFs) were
calculated as a percentage by dividing the number of individual variant reads by the total number of
amplicon reads and those SNVs with VAFs 20% were designated germ-line. Somatic
variants in the lens DNA panel were called using the VarScan 2 program that was originally
designed to call low-frequency (> 1%) somatic variants from deep-sequencing data derived
from matched tumor (case) versus control tissue samples [
]. For our purposes, we
compared left and right lenses from the same individual using the paired analysis or somatic mode.
Rare variants present in both lenses were designated as germ-line, whereas, those present in
only the left or the right lens (i.e. discordant SNVs) were designated as somatic. In order to
reduce the risk of false positives we excluded somatic SNVs with VAFs below 3% and/or
coverage depths below 600 reads as potential sequencing errors. For convenience, germ-line and
somatic SNVs were divided into novel and reference categories to denote their absence or
presence, respectively, in public genome databases including the Single Nucleotide
Polymorphism database (dbSNP build 138), Exome Variant Server (EVS), Exome Aggregation
Consortium (ExAC), 1000 Genomes project (1000G), and Catalogue of Somatic Mutations in Cancer
(COSMIC). Both categories predominantly contained synonymous and non-synonymous (i.e.
missense) SNVs with in silico predictions of damaging or deleterious effects at the protein level
determined using appropriate algorithms (e.g. SIFT and PolyPhen-2). Binary versions (.bam
files) of the Sequence Alignment/Map (.sam) files have been deposited with the NIH Short
Read Archive (SRA Accession no. PRJNA384802).
Germ-line EPHA2 variants in the cataract case-control panel
Exon deep-sequencing of EPHA2 in the cataract case-control panel detected 10 novel SNVs
(all transitions) and 20 reference SNVs (18 transitions) in the exon regions of EPHA2 at
VAFs >20%Ðconsistent with germ-line transmission (Table 1). Of the novel SNVs, two
were synonymous and eight were non-synonymousÐpredicted to result in missense
aminoacid substitutions. Two of the novel missense SNVs (p.I142T, p.W348R) occurred in controls
and both were predicted in silico to be damaging. Of the remaining six missense SNVs found
5 / 19
2 8 5
0 0 0 0 3 2 1 1 3 0 0 1 0 0 0 1 4 0 0 0 0 6 2 1 1 1 0 2 1 0
0 0 0 0 1 1 0 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0
3 0 4
0 0 0 0 4 4 2 0 4 0 1 1 2 2 3 0 6 1 0 1 1 2 0 0 0 0 1 3 0 1
7 6 0
1 0 1 1 2 2 1 0 3 0 0 1 0 0 2 0 6 0 1 1 0 5 0 1 0 0 0 2 0 0
8 8 1
0 1 0 0 3 3 0 0 4 1 0 1 0 1 1 0 9 0 1 0 0 6 0 2 2 0 0 3 1 1
M C C C P
ÐG ) 0
F 0 U 0
AM 001 (EC 0
.) E s
2 A a
p T ed
l IF r
r S (p
- -2 )n
s en ito
c h ic
t P d
c y e
r loP (rp
e ien ian
n ro om
d P D
fsouVSN i-onAmicdA eagnhC .14LpV I.241pT .171pEG .571pCR .091pAA .1L91Lp .612pAA .482pEE .923pPP .483pRW .363pSS .913pRG .384pEE .534pSS .32L6Lp .506pTA .61L6Lp .726pHH .217pRQ I.477pV .767pG .847pT .318pN .768pR .908pR .958Lp .329pA II.589p .968pQ .973pG i,)(gngDÐ .t00118988
S T D H H P T H G
g C T A A T A A T T A A A T G A A C A T C A a 10
ili.-rc2ee1dnonEPAHGm ...feeqnoSANDR eagnhC .rsc121979772741CG> l.vc524eoTCN> l.vc215eoANG> .rsc325453725641TC> .rsc0756871866AG> .rsc3756871666AG> .rsc84635746543TC> l.vc258eoANG> .rsc7895039722TC> l.vc2401eoTN> .rsc9801288647473C> .rsc171192915443G> .rsc413160070055G> .rsc953117402955C> .rsc698156551355G> l.vc8491eoNG> .rsc389130972201C> l.vc6102eoCN> .rsc2612415606611G> .rsc9322805592941G> l.vc6232eoNG> .rsc2532432285811C> l.vc1942eoAN> .rsc726250932253G> .rsc9662637289241G> l.vc4862eoTN> l.vc4972eoNG> .rsc47824533473C> .rsc4092948818831G> .rsc9192778495911G> li,r()ydababopngnebPmÐÐ lji....:////rreas1godoonpnuo17013
le n ) tp
b o 1 1 1 1 3 3 4 4 5 5 5 6 6 7 7 7 B th
a x 2 3 3 3 3 3 3 4 5 5 5 5 5 6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 (
.)(00D .)(00D .)(00D .)(00D yynonm yynonm yynonm yynonm yynonm .)(10D yynonm .)(11T yynonm yynonm yynonm .)(22T yynonm yynonm .(0000D .(0000D .)(000D ysynonm .)(000D .(0000D .(0000D .)(000D .)(001T yysnonm .(0001T yysnonm
0 0 0 0 s s s s s 0 s 0 s s s 0 s s
.()899D .()959D .()388P .()589D ysyonounm ysyonounm ysyonounm ysyonounm ysyonounm .()000D ysyonounm .()010B ysyonounm ysyonounm ysyonounm .()334B ysyonounm ysyonounm .()0001D .()9990D .()0001D ysysnonoum .()3790D .()9990D .()9990D .()0001D .()0000B yyssnonoum .()0310B yyssnonoum lttroeaedÐ
0 0 0 0 s s s s s 1 s 0 s s s 0 s s )
D D D D D D
B B B B B B
L L L L L L
K K K K K K K K K
T T T T T T T T T
1 0 0
3 0 0
1 0 0
in cases, two were predicted in silico to be benign (p.A650T, p.A932T) and four damaging (p.
G171E, p.G776S, p.N831D, p.L895P). Since nine of the novel SNVs occurred only once in
the panel, and the other only twice, we were unable to perform further statistical analysis.
Of the reference SNVs, 12 were synonymous and eight were predicted to result in missense
amino-acid substitutions (Table 1). Of the eight missense reference SNVs six were predicted to
result in damaging amino-acid substitutionsÐwith two occurring in cases only (p.L41V, p.
R175C) and three occurring in both cases and controls (p.R721Q, p.R876H, p.R890H). The
minor allele frequencies (MAFs) for all reference SNVs found in the cataract case-control
panel were similar to those reported in Caucasians by public genome variant databases
(Table 1). Four of the synonymous reference SNVs that were relatively common in the
Caucasian population (MAF 28%-44%) were also the most common in the cataract case-control
panel (S2a Table). However, only one of these SNVs (rs6678616) showed weak association
(p = 0.032) with nuclear cataract and nuclear cataract + PSC using Fisher's Exact Test (S2b
Table). Correcting for sex using logistic regression in the association analysis of rs6678616 did
not provide significant association with any type of cataract (p > 0.24). The remainder of
synonymous reference SNVs occurred in cases and/or controls but were comparatively rare in the
panel (MAF < 1%) hampering further statistical analysis.
Germ-line TP53 variants in the cataract-case control panel
Exon deep-sequencing of TP53 in the cataract case-control panel detected no novel SNVs and
only nine reference SNVs (5 transitions) of which five were also present in the COSMIC
database (Table 2 and S3a Table). Two of these SNVs (rs1042522, rs730882008) were
non-synonymous and predicted in silico (SIFT) to be damaging, with one (rs1042522, p.P72R) present at
relatively high frequency in Caucasians (MAF 0.25) and in multiple cases and controls.
However, rs1042522 was not associated with any type of cataract (p > 0.33) using Fisher's Exact
Test (S3b Table). Correcting for sex with logistic regression in the association analysis of
rs1042522 did not provide significant association with any type of cataract (p = 0.85). The
other SNV (rs730882008, p.R282L) occurred at unknown frequency in the population and in
only one case of cortical cataract preventing further statistical analysis.
Somatic EPHA2 variants in the post-mortem lens panel
Exon deep-sequencing of EPHA2 in the lens panel detected a total of 935 discordant SNVs
(VAF > 1%) in 35 pairs of clear lenses and 726 discordant SNVs in 22 pairs of cataract lenses
suggesting a somatic origin (S4 and S5 Tables). We arbitrarily selected a VAF cut-off threshold
value of 3% to minimize false-positive sequencing errors. In the clear lenses, 109 discordant
SNVs occurred with a VAF of 3% in 27 of the 35 clear lens pairs; however, 43 were excluded
due to low coverage (read-depth <600). The remaining 66 SNVs, each of which occurred only
once in the clear lens pairs, included 28 synonymous SNVs, 32 non-synonymous or missense
SNVs resulting in missense substitutions, 3 stop-gain or nonsense SNVs, 2 UTR SNVs, and
one splice-site SNV (Table 3). Of these SNVs, only 14 were listed in reference databases (e.g.
snp138, cosmic70, exac01) suggesting that 52 were novel somatic SNVs. Of the 32 missense
SNVs, only nine were listed in reference databases (e.g. snp138, cosmic70) and 29 were
predicted in silico (by the SIFT algorithm) to be damaging (Table 3). Surprisingly, 31 of the 32
missense SNVs involved C/T or G/A transitions and 17 of these occurred at di-pyrimidine
sites that are susceptible to UV-induced mutation [
]. Similarly, 18 of the 28 synonymous
SNVs along with two UTR SNVs and two nonsense SNVs occurred at UV-susceptible
dipyrimidine sites (Table 3).
7 / 19
1 4 3 1 2 1
4 6 5
1 3 1 1 1
2 6 1 7
A G T C G C T C > (
> > > > > > > > C ,
8G 5C 4A 8T 9A 9T 5G 5T 13 ign
0 1 5 5 3 8 4 8 1 n 2
.c1 .c2 .c3 .c5 .c6 .c7 .c8 .c8 .c1 eb .t00
61 3 85 0 2 (B 98
0052 2854 8942 7444 3354 lll.ee .e810n
M M M M M a o
S S S S S C l.p
O O O O O e an
C C C C C cn ru
3 1 8 8 7 0 re j/o
07 22 588 635 27 448 002 093 900 free 1731
03 25 97 27 03 59 88 07 53 to .10
08 40 15 57 08 07 03 00 56 rs /rg
rs1 rs1 rs7 rs3 rs1 rs7 rs7 rs2 rs7 free i.oo
1 M tt
4 4 4 5 6 8 8 8 1 * h
Chr Start/End Ref Alt ExonicFunc.
chr1 16451690 G* A* UTR-3
chr1 16451707 G* A* UTR-3
chr1 16451720 A
chr1 16451809 G* A* synonymous SNV
chr1 16451815 G
chr1 16455972 C* T* nonsynonymous
chr1 16456009 G* A* synonymous SNV
chr1 16456014 C
chr1 16456016 A
chr1 16456023 C* T* nonsynonymous
chr1 16456039 G* A* synonymous SNV
chr1 16456067 G* A* nonsynonymous
chr1 16456744 C* T* synonymous SNV
chr1 16456822 C
chr1 16458257 T
chr1 16458240 G* A* synonymous SNV
chr1 16458352 G* A* nonsynonymous
chr1 16458353 G* A* nonsynonymous
chr1 16458579 C* T* nonsynonymous
chr1 16458598 G
chr1 16458890 G* A* synonymous SNV
chr1 16458893 C* T* nonsynonymous
chr1 16458896 C* T* nonsynonymous
chr1 16458911 C* T* nonsynonymous
chr1 16458927 G* A* synonymous SNV
chr1 16459729 T
chr1 16459977 T
EPHA2:NM_004431:exon16:c. ID = COSM3934228
EPHA2:NM_004431:exon12:c. ID = COSM1727288
chr1 16464625 C* T* synonymous SNV
EPHA2:NM_004431:exon5:c. ID = COSM1205441
In the cataract lenses, 35 discordant EPHA2 SNVs occurred with a VAF 3% in 10 of the 22
cataract lens pairs with only two excluded due to low read-depth (S5 Table). The remaining 33
singly occurring SNVs included 12 synonymous SNVs, 19 non-synonymous or missense SNVs,
and two stop-gain or nonsense SNVs (Table 4). Of these SNVs, six were present in reference
databases suggesting that 27 were novel somatic SNVs and only one (at position 16460407 bp)
was present in both cataract and clear lenses (Tables 3 and 4). Of the 19 missense SNVs only
four were present in reference databases and 15 were predicted in silico (SIFT) to be damaging
(Table 4). All 19 missense SNVs involved C/T or A/G transitions and 12 of these occurred at
UV-susceptible di-pyrimidine sites. Ten of the 12 synonymous SNVs and both nonsense SNVs
also occurred at UV-susceptible di-pyrimidine sites. Overall for EPHA2, there was no
significant difference between the paired clear lens panel and the paired cataract lens panel with
respect to total SNVs (p = 0.48), damaging SNVs (p = 0.85), or novel SNVs (p = 0.64) using
Fisher's Exact Test (S6 Table). Correcting for sex in the lens panels using logistic regression
analysis did not provide any significant association for total EPHA2 SNVs (p = 0.62), damaging
EPHA2 SNVs (p = 0.63), or novel EPHA2 SNVs (p = 0.70).
Somatic TP53 variants in the post-mortem lens panel
Exon deep-sequencing of TP53 in the lens panel detected a total of 392 discordant SNVs
(VAF > 1%) in 35 clear lens pairs and 298 discordant SNVs in 22 cataract lens pairs (S7 and
11 / 19
Chr Start/End Ref Alt ExonicFunc.
chr1 16460407 C* T* synonymous SNV
chr1 16462261 G* A* synonymous SNV
chr1 16464353 T
chr1 16464600 T
chr1 16464608 G* A* nonsynonymous
chr1 16464609 G* A* nonsynonymous
chr1 16464610 G* A* synonymous SNV
chr1 16464614 G
chr1 16464617 C* T* stopgain
chr1 16464623 A
chr1 16464624 G* A* synonymous SNV
chr1 16464633 T
chr1 16464665 G* A* nonsynonymous
chr1 16464666 G* A* nonsynonymous
ID = COSM1185338
S8 Tables). In the clear lenses, 64 discordant SNVs were present at a VAF > 3% in 27 of
the 35 pairs; however, 12 of these SNVs were excluded due to low read-depth (<600). In
addition, three discordant SNVs occurred more than onceÐone non-synonymous SNV
(COSM1658764) occurred in nine lenses, one synonymous SNV (present in ExAC01)
occurred in 11 lenses, and one UTR SNV occurred in two lensesÐresulting in a total of 19
SNVs that were excluded for recurrence. The remaining 33 single occurrence SNVs included
nine synonymous SNVs (8 transitions), 16 non-synonymous or missense SNVs (15
transitions), seven UTR SNVs (all transitions), and one splicing SNV (transition). Of these SNVs, 18
were present in reference databases (e.g. cosmic, snp138, exac01) leaving 15 putatively novel
somatic SNVs (S7d Table). Of the 16 missense SNVs, 13 were present in reference databases,
seven were predicted in silico (SIFT) to be damaging and five occurred at UV-susceptible
dipyrimidine sites. Apart from the splicing SNV, none of the synonymous SNVs or UTR SNVs
occurred at di-pyrimidine sites (S7d Table).
In the cataract lenses, 18 discordant TP53 SNVs (all transitions) occurred with VAFs > 3%
in five of the 22 pairs of lenses including five synonymous SNVs, 12 non-synonymous or
missense SNVs, and one UTR-3' SNV (S8d Table). Of these single occurrence SNVs, 12 were
present in reference databases leaving six potentially novel somatic SNVs and only one (at position
7572892 bp) was present in both cataract and clear lenses (S7d and S8d Tables). Of the 12
missense SNVs, eight were present in reference databases, six were predicted to be damaging, and
11 occurred, along with the UTR SNV, at UV-susceptible di-pyrimidine sites (S8d Table).
Overall for TP53, there was no significant difference between the paired clear lens panel and
the paired cataract lens panel with respect to total SNVs (p = 0.73), damaging SNVs (p = 0.77),
or novel SNVs (p = 0.78) using Fisher's Exact Test (S9 Table). Correcting for sex in the lens
panels using logistic regression analysis did not provide any significant association for total
TP53 SNVs (p = 0.39), damaging TP53 SNVs (p = 0.71), or novel TP53 SNVs (p = 0.57).
In this study we utilized targeted-amplicon (exon) deep-sequencing to identify germ-line and
somatic variants of EPHA2Ðparticularly novel missense variants predicted in silico to result in
13 / 19
deleterious amino-acid substitutionsÐthat may be associated with lens aging and/or
agerelated cataract. First, we profiled germ-line SNVs (VAF > 20%) in EPHA2 for association
with age-related cataract in a Caucasian case-control panel that had previously revealed
association with common reference SNVs flanking EPHA2 [
]. Exon deep-sequencing detected six
novel missense SNVs and eight reference missense SNVs in the cataract case-control panel
that were predicted to be damaging (Table 1). However, the relatively small number of
individuals in the cataract case-control panel that harbored these damaging EPHA2 SNVs (n < 20)
limited the power of this study to detect disease association. For example, of two novel SNVs
located in the extracellular LBD of EPHA2 one (p.I142T) was present in a control, while the
other (p.G171E) occurred in a case with cortical cataract. Similarly, one of the reference
missense SNVs, rs116506614 (c.2162G>A, p.R721Q), located in the TK domain of EPHA2, that
has previously been associated with age-related cortical cataract [
], was present in a case
with cortical cataract and in a control from our cataract case-control panel. Overall, while it is
possible that such control individuals may be pre-symptomatic for age-related cataract, we
note that other putatively deleterious SNVs were found only in controls, whereas, putatively
benign SNVs were present in cases (Table 1) rendering simple genotype-phenotype
Second, we profiled putative somatic SNVs in EPHA2 (VAF 3%) that arose in
post-mortem lenses procured from Caucasian donors over 48 years of age (Tables 3 and 4). Paired
analysis of right and left lenses from the same individual for discordant SNVs, analogous to that of
matched tumor versus control tissues, detected 19 novel missense SNVs in a clear lens panel
(35 pairs) and 13 novel missense SNVs in a cataract lens panel (22 pairs) that were predicted to
be damaging (Tables 3 and 4). By comparison, the same paired-lens analysis of TP53 for
discordant SNVs yielded predominantly reference somatic SNVs found in the COSMIC database
and no novel SNVs that were predicted to be damaging (S7 and S8 Tables). This difference in
SNV profile between the two genes likely reflects the high frequency of somatic mutations
identified in TP53 versus EPHA2. Currently, the COSMIC database lists over 29,480 somatic
mutations in TP53 including 17,166 missense substitutions that have been detected in multiple
tumor samples (e.g. cutaneous melanoma) at relatively high frequencies (~27%). By contrast,
EPHA2 harbors some 275 somatic mutations including 164 missense substitutions that have
been detected in multiple tumor samples (e.g. stomach, intestine, skin), at relatively low
frequencies (typically < 5%) (http://cancer.sanger.ac.uk/cosmic). These observations suggest that
novel somatic variants in EPHA2 that are predicted to be functionally deleterious are
detectable in aging human lenses. Overall, our data are in agreement with a recent study that
employed targeted-hybridization deep-sequencing of human lens epithelial samples to identify
somatic variants in a panel of 151 cancer-related genes [
]. To the best of our knowledge, this
is the first report of putative somatic mutations in a lens-expressed gene causally implicated in
age-related cataract. However, since rudimentary statistical analysis confirmed that somatic
SNVs in EPHA2 were present at comparable frequencies in both clear lenses and those with
age-related cataract we are unable to determine if such variants are causative for disease.
A striking feature of both the germ-line and the somatic missense SNVs in EPHA2 detected
here was the high frequency of transitions (C/T, G/A) versus transversions (G/C, G/T, A/C,
A/T). Theoretically, transversions should occur twice as often as transitions; however, a review
of the germ-line variation annotated in the EPHA2 reference sequence reveals that the vast
majority of missense variants involve C/T or G/A transitions (http://www.ncbi.nlm.nih.gov/
variation/view/). The occurrence of somatic C>T transitions is of particular interest since they
may result from exposure to solar UV radiation [
]. Absorption of solar UV radiation (95%
UV-A, 5% UV-B) by DNA promotes the formation of photodimeric lesions, mostly
cyclobutane pyrimidine dimers (CPDs), at adjacent pyrimidine bases (C and T) that may escape
14 / 19
nucleotide excision repair leading to base substitution and generation of UV-signature
mutations (C>T or CC>TT) during DNA replication [
]. Among the somatic missense SNVs
detected in our lens panel (clear and cataract) many of the C>T changes (G>A on the
complementary strand) were present at di-pyrimidine (diPy) sites (CT, TC, CC) in both EPHA2 and
TP53 raising the possibility that they represent UV-signature mutations (Tables 3 and 4 and S7
and S8 Tables). While there was no significant association between these somatic SNVs and
cataract in our lens panel, epidemiological studies have established that lifetime exposure to
solar UV radiation (particularly UV-B) is a significant risk factor for cortical cataract
particularly within the lens nasal quadrant [
]. In addition, UV-A radiation has been implicated
in the increased prevalence of left-sided cortical cataract and facial skin cancer, likely in part,
due to increased exposure while operating left-hand drive vehicles . Further, it has been
suggested that oxidative stress secondary to solar UV exposure might contribute to age-related
]. However, since the cornea effectively absorbs most solar UV-B radiation (290±
320 nm) and the levels of CPDs in lens epithelia obtained from cataract patients has been
reported to be relatively low compared to those of oxidized purines, the cause-effect
relationship between solar UV exposure and age-related cataract remains unclear [
studies of somatic variants, including UV-signature mutations, in EPHA2 and over 30 other
known cataract genes, including those for crystallins (e.g. CRYAA), connexins (e.g. GJA8) and
ocular transcription factors (e.g. HSF4) [
] may provide new insights regarding the
molecular genetic mechanisms underlying age-related cataract.
S1 Table. Amplicon deep-sequencing coverage in the cataract case-control panel (a) and
the post-mortem lens panel (b).
S2 Table. Germ-line EPHA2 coding SNV frequency (a) and association (b) in the cataract
case-control panel (VAF >20%).
S3 Table. Germ-line TP53 coding SNV frequency (a) and association (b) in the cataract
case-control panel (VAF >20%).
S4 Table. Somatic EPHA2 coding SNVs found in the paired clear lens panel.
S5 Table. Somatic EPHA2 coding SNVs found in the paired cataract lens panel.
S6 Table. Fisher's exact test of EPHA2 coding SNVs found in the post-mortem lens panel.
S7 Table. Somatic TP53 coding SNVs found in the paired clear lens panel.
S8 Table. Somatic TP53 coding SNVs found in the paired cataract lens panel.
S9 Table. Fisher's exact test of TP53 coding SNVs found in the post-mortem lens panel.
15 / 19
We thank the Genome Technology Access Center (GTAC) at Washington University School
of Medicine for help with amplicon deep-sequencing and variant analysis.
Conceptualization: J. Fielding Hejtmancik, Alan Shiels.
Data curation: Thomas M. Bennett, Alan Shiels.
Formal analysis: Thomas M. Bennett, Oussama M'Hamdi, J. Fielding Hejtmancik, Alan
Funding acquisition: Alan Shiels.
Investigation: Thomas M. Bennett, Alan Shiels.
Project administration: Alan Shiels.
Resources: J. Fielding Hejtmancik, Alan Shiels.
Supervision: J. Fielding Hejtmancik, Alan Shiels.
Validation: Thomas M. Bennett, Alan Shiels.
Visualization: Thomas M. Bennett, Oussama M'Hamdi, J. Fielding Hejtmancik, Alan Shiels.
Writing ± original draft: Alan Shiels.
Writing ± review & editing: J. Fielding Hejtmancik, Alan Shiels.
16 / 19
17 / 19
18 / 19
1. Pascolini D , Mariotti SP . Global estimates of visual impairment: 2010. Br J Ophthalmol . 2012 ; 96 ( 5 ): 614 ± 618 . PMID: 22133988 . https://doi.org/10.1136/bjophthalmol-2011 -300539
2. Bourne RR , Stevens GA , White RA , Smith JL , Flaxman SR , Price H , et al. Causes of vision loss worldwide , 1990 ±2010: a systematic analysis . Lancet Glob Health . 2013 ; 1 ( 6 ):e339± 349 . PMID: 25104599 . https://doi.org/10.1016/ S2214 -109X( 13 ) 70113 -X
3. Khairallah M , Kahloun R , Bourne R , Limburg H , Flaxman SR , Jonas JB , et al. Number of People Blind or Visually Impaired by Cataract Worldwide and in World Regions, 1990 to 2010. Invest Ophthalmol Vis Sci. 2015 ; 56 ( 11 ): 6762 ± 6769 . PMID: 26567788 . https://doi.org/10.1167/iovs.15- 17201
4. West SK , Valmadrid CT . Epidemiology of risk factors for age-related cataract . Surv Ophthalmol . 1995 ; 39 ( 4 ): 323 ± 334 . PMID: 7725232 .
5. Hodge WG , Whitcher JP , Satariano W. Risk factors for age-related cataracts . Epidemiol Rev . 1995 ; 17 ( 2 ): 336 ± 346 . PMID: 8654515 .
6. Taylor HR . Epidemiology of age-related cataract . Eye (Lond) . 1999 ; 13 (Pt 3b): 445 ± 448 .
7. McCarty CA , Taylor HR . The genetics of cataract . Invest Ophthalmol Vis Sci . 2001 ; 42 ( 8 ): 1677 ± 1678 . PMID: 11431427 .
8. Sanfilippo PG , Hewitt AW , Hammond CJ , Mackey DA . The heritability of ocular traits . Surv Ophthalmol . 2010 ; 55 ( 6 ): 561 ± 583 . PMID: 20851442 . https://doi.org/10.1016/j.survophthal. 2010 . 07 .003
9. Foster A , Gilbert C , Rahi J . Epidemiology of cataract in childhood: a global perspective . J Cataract Refract Surg . 1997 ; 23 Suppl 1 : 601 ± 604 . PMID: 9278811 .
10. Haargaard B , Wohlfahrt J , Fledelius HC , Rosenberg T , Melbye M. A nationwide Danish study of 1027 cases of congenital/infantile cataracts: etiological and clinical classifications . Ophthalmology . 2004 ; 111 ( 12 ): 2292 ± 2298 . PMID: 15582089 . https://doi.org/10.1016/j.ophtha. 2004 . 06 .024
11. Lim Z , Rubab S , Chan YH , Levin AV . Pediatric cataract: the Toronto experience-etiology . Am J Ophthalmol . 2010 ; 149 ( 6 ): 887 ± 892 . PMID: 20430363 . https://doi.org/10.1016/j.ajo. 2010 . 01 .012
12. Kong L , Fry M , Al-Samarraie M , Gilbert C , Steinkuller PG . An update on progress and the changing epidemiology of causes of childhood blindness worldwide . J AAPOS . 2012 ; 16 ( 6 ): 501 ± 507 . PMID: 23237744 . https://doi.org/10.1016/j.jaapos. 2012 . 09 .004
13. Rahi JS , Dezateux C . Congenital and infantile cataract in the United Kingdom: underlying or associated factors . British Congenital Cataract Interest Group. Invest Ophthalmol Vis Sci . 2000 ; 41 ( 8 ): 2108 ± 2114 . PMID: 10892851 .
14. Shiels A , Bennett TM , Hejtmancik JF . Cat-Map: putting cataract on the map . Mol Vis . 2010 ; 16 : 2007 ± 2015 . PMID: 21042563 .
15. Shiels A , Hejtmancik JF . Mutations and mechanisms in congenital and age-related cataracts . Exp Eye Res . 2016 ; 156 : 95 ± 102 . PMID: 27334249 . https://doi.org/10.1016/j.exer. 2016 . 06 .011
16. Pasquale EB . Eph-ephrin bidirectional signaling in physiology and disease . Cell . 2008 ; 133 ( 1 ): 38 ± 52 . PMID: 18394988 . https://doi.org/10.1016/j.cell. 2008 . 03 .011
17. Pasquale EB . Eph receptors and ephrins in cancer: bidirectional signalling and beyond . Nat Rev Cancer . 2010 ; 10 ( 3 ): 165 ± 180 . PMID: 20179713 . https://doi.org/10.1038/nrc2806
18. Lisabeth EM , Falivelli G , Pasquale EB . Eph receptor signaling and ephrins . Cold Spring Harbor Perspect Biol . 2013 ; 5:a009159 . PMID: 24003208 . https://doi.org/10.1101/cshperspect.a009159
19. Barquilla A , Pasquale EB . Eph receptors and ephrins: therapeutic opportunities . Ann Rev Pharmacol Toxicol . 2015 ; 55 : 465 ± 487 . PMID: 25292427 . https://doi.org/10.1146/annurev-pharmtox- 011112 - 140226
20. Kania A , Klein R . Mechanisms of ephrin-Eph signalling in development, physiology and disease . Na Rev Mol Cell Biol . 2016 ; 17 ( 4 ): 240 ± 256 . PMID: 26790531 . https://doi.org/10.1038/nrm. 2015 .16
21. Stapleton D , Balan I , Pawson T , Sicheri F. The crystal structure of an Eph receptor SAM domain reveals a mechanism for modular dimerization . Nat Struct Biol . 1999 ; 6 ( 1 ): 44 ± 49 . PMID: 9886291 . https://doi. org/10.1038/4917
22. Lee HJ , Hota PK , Chugha P , Guo H , Miao H , Zhang L , et al. NMR structure of a heterodimeric SAM: SAM complex: characterization and manipulation of EphA2 binding reveal new cellular functions of SHIP2 . Structure. 2012 ; 20 ( 1 ): 41 ± 55 . PMID: 22244754 . https://doi.org/10.1016/j.str. 2011 . 11 .013
23. Lindberg RA , Hunter T. cDNA cloning and characterization of eck, an epithelial cell receptor proteintyrosine kinase in the eph/elk family of protein kinases . Mol Cell Biol . 1990 ; 10 ( 12 ): 6316 ± 6324 . PMID: 2174105 .
24. Bassnett S , Wilmarth PA , David LL . The membrane proteome of the mouse lens fiber cell . Mol Vis . 2009 ; 15 : 2448 ± 2463 . PMID: 19956408 .
25. Wang Z , Han J , David LL , Schey KL . Proteomics and phosphoproteomics analysis of human lens fiber cell membranes . Invest Ophthalmol Vis Sci . 2013 ; 54 ( 2 ): 1135 ± 1143 . PMID: 23349431 . https://doi.org/ 10.1167/iovs.12- 11168
26. Jun G , Guo H , Klein BE , Klein R , Wang JJ , Mitchell P , et al. EPHA2 is associated with age-related cortical cataract in mice and humans . PLoS Genetics . 2009 ; 5(7):e1000584 . PMID: 19649315 . https://doi. org/10.1371/journal.pgen.1000584
27. Cheng C , Gong X . Diverse roles of Eph/ephrin signaling in the mouse lens . PLoS One . 2011 ; 6 ( 11 ) : e28147 . PMID: 22140528 . https://doi.org/10.1371/journal.pone.0028147
28. Shi Y , De Maria A , Bennett T , Shiels A , Bassnett S . A role for epha2 in cell migration and refractive organization of the ocular lens . Invest Ophthalmol Vis Sci . 2012 ; 53 ( 2 ): 551 ± 559 . PMID: 22167091 . https:// doi.org/10.1167/iovs.11- 8568
29. Cheng C , Ansari MM , Cooper JA , Gong X. EphA2 and Src regulate equatorial cell morphogenesis during lens development . Development . 2013 ; 140 ( 20 ): 4237 ± 4245 . PMID: 24026120 . https://doi.org/10. 1242/dev.100727
30. Shiels A , Bennett TM , Knopf HL , Maraini G , Li A , Jiao X , et al. The EPHA2 gene is associated with cataracts linked to chromosome 1p . Mol Vis . 2008 ; 14 : 2042 ± 2055 . PMID: 19005574 .
31. Zhang T , Hua R , Xiao W , Burdon KP , Bhattacharya SS , Craig JE , et al. Mutations of the EPHA2 receptor tyrosine kinase gene cause autosomal dominant congenital cataract . Hum Mutat . 2009 ; 30 ( 5 ): E603± 611 . PMID: 19306328 . https://doi.org/10.1002/humu.20995
32. Kaul H , Riazuddin SA , Shahid M , Kousar S , Butt NH , Zafar AU , et al. Autosomal recessive congenital cataract linked to EPHA2 in a consanguineous Pakistani family . Mol Vis . 2010 ; 16 : 511 ± 517 . PMID: 20361013 .
33. Aldahmesh MA , Khan AO , Mohamed JY , Hijazi H , Al-Owain M , Alswaid A , et al. Genomic analysis of pediatric cataract in Saudi Arabia reveals novel candidate disease genes . Genet Med . 2012 ; 14 ( 12 ): 955 ± 962 . PMID: 22935719 . https://doi.org/10.1038/gim. 2012 .86
34. Shentu XC , Zhao SJ , Zhang L , Miao Q. A novel p.R890C mutation in EPHA2 gene associated with progressive childhood posterior cataract in a Chinese family . Int J Ophthalmol . 2013 ; 6 ( 1 ): 34 ± 38 . PMID: 23447127 . https://doi.org/10.3980/j.issn. 2222 - 3959 . 2013 . 01 .07
35. Dave A , Laurie K , Staffieri SE , Taranath D , Mackey DA , Mitchell P , et al. Mutations in the EPHA2 gene are a major contributor to inherited cataracts in South-Eastern Australia . PLoS One . 2013 ; 8(8):e72518 . PMID: 24014202 . https://doi.org/10.1371/journal.pone.0072518
36. Reis LM , Tyler RC , Semina EV . Identification of a novel C-terminal extension mutation in EPHA2 in a family affected with congenital cataract . Mol Vis . 2014 ; 20 : 836 ± 842 . PMID: 24940039 .
37. Gillespie RL , O'Sullivan J , Ashworth J , Bhaskar S , Williams S , Biswas S , et al. Personalized diagnosis and management of congenital cataract by next-generation sequencing . Ophthalmology . 2014 ; 121 ( 11 ): 2124 ± 2137 e1± 2 . PMID: 25148791 . https://doi.org/10.1016/j.ophtha. 2014 . 06 .006
38. Sun W , Xiao X , Li S , Guo X , Zhang Q . Exome sequencing of 18 Chinese families with congenital cataracts: a new sight of the NHS gene . PLoS one . 2014 ; 9(6):e100455 . PMID: 24968223 . https://doi.org/ 10.1371/journal.pone.0100455
39. Bu J , He S , Wang L , Li J , Liu J , Zhang X. A novel splice donor site mutation in EPHA2 caused congenital cataract in a Chinese family . Ind J Ophthalmol . 2016 ; 64 ( 5 ): 364 ± 368 . PMID: 27380975 . https://doi.org/ 10.4103/ 0301 - 4738 . 185597
40. Li D , Wang S , Ye H , Tang Y , Qiu X , Fan Q , et al. Distribution of gene mutations in sporadic congenital cataract in a Han Chinese population . Mol Vis . 2016 ; 22 : 589 ± 598 . PMID: 27307692 .
41. Patel N , Anand D , Monies D , Maddirevula S , Khan AO , Algoufi T , et al. Novel phenotypes and loci identified through clinical genomics approaches to pediatric cataract . Hum Genet . 2017 ; 136 : 205 ± 225 . PMID: 27878435 . https://doi.org/10.1007/s00439-016-1747-6
42. Musleh M , Hall G , Lloyd IC , Gillespie RL , Waller S , Douzgou S , et al. Diagnosing the cause of bilateral paediatric cataracts: comparison of standard testing with a next-generation sequencing approach . Eye (Lond) 2016 ; 30 : 1175 ± 1181 . PMID: 27315345 . https://doi.org/10.1038/eye. 2016 .105
43. Chen J , Wang Q , Cabrera PE , Zhong Z , Sun W , jiao X , et al. Molecular genetic analysis of Pakistani families with autosomal recessive congenital cataracts by homozygosity screening . Invest Ophthalmol Vis Sci . 2017 ; 58 : 2207 ± 2217 . PMID: 28418495 . https://doi.org/10.1167/iovs.17- 21469
44. Park JE , Son AI , Hua R , Wang L , Zhang X , Zhou R . Human cataract mutations in EPHA2 SAM domain alter receptor stability and function . PLoS One . 2012 ; 7(5):e36564 . PMID: 22570727 . https://doi.org/10. 1371/journal.pone.0036564
45. Dave A , Martin S , Kumar R , Craig JE , Burdon KP , Sharma S. Epha2 Mutations Contribute to Congenital Cataract through Diverse Mechanisms . Mol Vis . 2016 ; 22 : 18 ± 30 . PMID: 26900323 .
46. Tan W , Hou S , Jiang Z , Hu Z , Yang P , Ye J . Association of EPHA2 polymorphisms and age-related cortical cataract in a Han Chinese population . Mol Visi . 2011 ; 17 : 1553 ± 1558 . PMID: 21686326 .
47. Sundaresan P , Ravindran RD , Vashist P , Shanker A , Nitsch D , Talwar B , et al. EPHA2 polymorphisms and age-related cataract in India . PLoS One . 2012 ; 7(3):e33001 . PMID: 22412971 . https://doi.org/10. 1371/journal.pone.0033001
48. Yang J , Luo J , Zhou P , Fan Q , Luo Y , Lu Y. Association of the ephreceptor tyrosinekinase-type A2 (EPHA2) gene polymorphism rs3754334 with age-related cataract risk: a meta-analysis . PLoS One . 2013 ; 8(8):e71003 . PMID: 23976972 . https://doi.org/10.1371/journal.pone.0071003
49. Celojevic D , Abramsson A , Seibt Palmer M , Tasa G , Juronen E , Zetterberg H , et al. EPHA2 Polymorphisms in Estonian Patients with Age-Related Cataract . Ophthal Genet . 2016 ; 37 ( 1 ): 14 ± 18 . PMID: 24673449 . https://doi.org/10.3109/13816810. 2014 .902080
50. Zhang H , Zhong J , Bian Z , Fang X , Peng Y , Hu Y. Association between polymorphisms of OGG1, EPHA2 and age-related cataract risk: a meta-analysis . BMC Ophthalmol . 2016 ; 16 ( 1 ): 168 . PMID: 27681698 . https://doi.org/10.1186/s12886-016-0341-y
51. Masoodi TA , Shammari SA , Al-Muammar MN , Almubrad TM , Alhamdan AA . Screening and structural evaluation of deleterious Non-Synonymous SNPs of ePHA2 gene involved in susceptibility to cataract formation . Bioinformation . 2012 ; 8 ( 12 ): 562 ± 567 . PMID: 22829731 . https://doi.org/10.6026/ 97320630008562
52. Yang J , Li D , Fan Q , Cai L , Qiu X , Zhou P , et al. The Polymorphisms with Cataract Susceptibility Impair the EPHA2 Receptor Stability and Its Cytoprotective Function . J Ophthalmol . 2015 ; 2015 : 401894 . PMID: 26664742 . https://doi.org/10.1155/ 2015 /401894
53. Clinical Trial of Nutritional S, Age-Related Cataract Study G , Maraini G , Williams SL , Sperduto RD , Ferris F , et al. A randomized, double-masked, placebo-controlled clinical trial of multivitamin supplementation for age-related lens opacities. Clinical trial of nutritional supplements and age-related cataract report no. 3 . Ophthalmology . 2008 ; 115 ( 4 ): 599 ± 607 e1. PMID: 18387406 . https://doi.org/10.1016/j. ophtha. 2008 . 01 .005
54. Maraini G , Hejtmancik JF , Shiels A , Mackay DS , Aldigeri R , Jiao XD , et al. Galactokinase gene mutations and age-related cataract. Lack of association in an Italian population . Mol Vis . 2003 ; 9 : 397 ± 400 . PMID: 12942049 .
55. Age-Related Eye Disease Study Research G. The age-related eye disease study (AREDS) system for classifying cataracts from photographs : AREDS report no. 4 . Am J Ophthalmol . 2001 ; 131 ( 2 ): 167 ± 175 . PMID: 11228291 .
56. Koboldt DC , Zhang Q , Larson DE , Shen D , McLellan MD , Lin L , et al. VarScan 2: somatic mutation and copy number alteration discovery in cancer by exome sequencing . Genome Res . 2012 ; 22 ( 3 ): 568 ± 576 . PMID: 22300766 . https://doi.org/10.1101/gr.129684.111
57. Koboldt DC , Larson DE , Wilson RK . Using VarScan 2 for Germline Variant Calling and Somatic Mutation Detection . Current Protoc Bioinform . 2013 ; 44 : 15 .4.1± 15 .4.17. PMID: 25553206 . https://doi.org/10. 1002/0471250953.bi1504s44
58. Robinson JT , Thorvaldsdottir H , Winckler W , Guttman M , Lander ES , Getz G , et al. Integrative genomics viewer . Nat Biotechnol . 2011 ; 29 ( 1 ): 24 ± 26 . PMID: 21221095 . https://doi.org/10.1038/nbt.1754
59. Sulman EP , Tang XX , Allen C , Biegel JA , Pleasure DE , Brodeur GM , et al. ECK, a human EPH-related gene, maps to 1p36.1, a common region of alteration in human cancers . Genomics . 1997 ; 40 ( 2 ): 371 ± 374 . PMID: 9119409 . https://doi.org/10.1006/geno. 1996 .4569
60. Brash DE. UV signature mutations . Photochem Photobiol . 2015 ; 91 ( 1 ): 15 ± 26 . PMID: 25354245 . https:// doi.org/10.1111/php.12377
61. Mesa R , Tyagi M , Harocopos G , Vollman D , Bassnett S. Somatic Variants in the Human Lens Epithelium: A Preliminary Assessment . Invest Ophthalmol Vis Sci . 2016 ; 57 ( 10 ): 4063 ± 4075 . PMID: 27537255 . https://doi.org/10.1167/iovs.16- 19726
62. Besaratinia A , Yoon JI , Schroeder C , Bradforth SE , Cockburn M , Pfeifer GP . Wavelength dependence of ultraviolet radiation-induced DNA damage as determined by laser irradiation suggests that cyclobutane pyrimidine dimers are the principal DNA lesions produced by terrestrial sunlight . FASEB J . 2011 ; 25 ( 9 ): 3079 ± 3091 . PMID: 21613571 . https://doi.org/10.1096/fj.11- 187336
63. McCarty CA , Taylor HR . A review of the epidemiologic evidence linking ultraviolet radiation and cataracts . Dev Ophthalmol . 2002 ; 35 : 21 ± 31 . PMID: 12061276 .
64. Abraham AG , Cox C , West S. The differential effect of ultraviolet light exposure on cataract rate across regions of the lens . Invest Ophthalmol Vis Sci . 2010 ; 51 ( 8 ): 3919 ± 3923 . PMID: 20375345 . https://doi. org/10.1167/iovs.09- 4557
65. Weiss JS . UV-A protection from auto glass, cataracts, and the ophthalmologist . JAMA Ophthalmol . 2016 ; 134 ( 7 ): 776 ± 777 . PMID: 27258328 . https://doi.org/10.1001/jamaophthalmol. 2015 .5101
66. Spector A . Oxidative stress-induced cataract: mechanism of action . FASEB J . 1995 ; 9 ( 12 ): 1173 ± 1182 . PMID: 7672510 .
67. Lombardo M , Pucci G , Barberi R , Lombardo G . Interaction of ultraviolet light with the cornea: clinical implications for corneal crosslinking . J Cataract Refract Surg . 2015 ; 41 ( 2 ): 446 ± 459 . PMID: 25542349 . https://doi.org/10.1016/j.jcrs. 2014 . 12 .013
68. Osnes-Ringen O , Azqueta AO , Moe MC , Zetterstrom C , Roger M , Nicolaissen B , et al. DNA damage in lens epithelium of cataract patients in vivo and ex vivo . Acta Ophthalmol . 2013 ; 91 ( 7 ): 652 ± 656 . PMID: 22994213 . https://doi.org/10.1111/j.1755- 3768 . 2012 . 02500 .x