Research Article

Detection of Mutation c.139G>A (D47N) in GJA8 and c.2036C>T in FYCO1

Gene in an Extended Family with Inheritance of Autosomal Dominant

Zonular Cataract without Pulverisation Pulverulent opacities by Exome

Sequencing

Padma Gunda1, Mamata Manne1, Syed Saifuddin Adeel2, Ravi Kumar Reddy

Kondareddy2, *Padma Tirunilai1

1. Department of Genetics, Osmania University, Hyderabad- 500007, India

2. Medivision Eye Care Center, Masab Tank, Hyderabad – 500028, India

Padma Gunda – padma.genetics@gmail.com

Mamata Manne – mamathamanne@gmail.com

Syed Saifuddin Adeel-saif_azeem@yahoo.co.in

Ravi Kumar Reddy Kondareddy – ravi_medivision@yahoo.co.in

*Corresponding Author:

Prof. T. Padma (Rtd)

Dept. of Genetics, Osmania University

Hyderabad, Andhra Pradesh, India

Phone: +91-9866229810;

Fax: 91-40-27095178;

email: padmatirunilai@gmail.com

Running Title: GJA8 mutation and autosomal dominant zonular cataract

Keywords: Autosomal dominant, Zonular cataract, Pulverisation, GJA8, Exome sequencing

Formatted: Font: Times New Roman, 13 pt, Bold, Font color:Text 1

Formatted: Left

Abstract

Purpose: To identify the gene causing bilateral autosomal dominant zonular congenital

cataract (ADZCC) without pulverulent opacities isation in an extended Muslim family by

exome sequencing and subsequent analysis.

Methods: An extended family of 37 members (14 affected and 23 unaffected) belonging to

different nuclear families were screened for causative gene. Proband and her unaffected son

were screened for causative variant by exome sequencing followed by Sanger sequencing of

the proband’s entire nuclear family. Remaining members were further screened for variants

detected, by PCR-RFLP and Tetra ARMS PCR.

Results: Review of exome sequencing data of the proband and her unaffected son for 40

known genes causing congenital non-syndromic cataracts revealed two variants viz.,

c.139G>A (p.Asp47Asn; D47N) in GJA8 gene and c.2036C>T in FYCO1 gene to be

potentially pathogenic. Further, validation of these two variants in the entire family showed

co-segregation of c.139G>A variant in GJA8 with ADZCC without pulverisationrulent

opacities. Variation c.2036C>T in FYCO1 was not associated with disease in the family.

Conclusion: The mutation c.139G>A in GJA8 gene detected in the present study was also

previously described in Caucasian and Chinese families but with different phenotypes i.e

nuclear and nuclear pulverulent cataracts. Thus, the mutation c.139G>A in GJA8 appears to

exhibit marked interfamilial phenotypic variability.

INTRODUCTION

Cataract, the opacification of lens, results from alteration in cellular architecture of lens, lens

proteins or both. It is a major ocular disease that causes blindness in millions of people

throughout the world. Opacification of the ocular lens is caused by a wide range of etiological

factors including exogenous factors like infections, chemicals, radiations etc. All these factors

have their effect right from intrauterine life to senescence. Further, lens opacity shows

considerable interfamilial and intrafamilial phenotypic variation in the expression of different

types of cataracts that are reported worldwide (Scott et al, 1994; Vanita, 1998; Amaya et al,

2003). Depending on the morphological, clinical and pathological profiles, the lens opacities

acquire an independent identity like cortical, nuclear, zonular, lamellar, crystalline, total and

other types. These different types may originate either congenitally or during infantile,

juvenile, presenile or senile stages of life. Congenital cataracts exhibit clinically and

genetically heterogenous lens disorders and account for approximately 10% of the world

wide cases of childhood blindness (Francis et al, 2000). They occur either as syndromic or

non-syndromic form with different modes of inheritance of which autosomal dominant mode

of transmission is most commonly reported (Amaya et al, 2003).

To date, about 40 genes have been reported to be associated with non-syndromic congenital

cataracts, among which twenty nine genes are known to cause autosomal dominant pattern of

inheritance (Cat-map; Table-1). Formation of these cataracts is caused by mutations in

different families of genes which include lens related crystalline genes (CRYAA, CRYAB,

CRYBB1, CRYBB2, CRYBB3,CRYBA1, CRYBA4, CRYGB, CRYGC, CRYGD, and CRYGS),

connexin genes (GJA3, GJA8), membrane protein genes (AGK, CHMP4B, EPHA2, MIP, and

LIM2), cytoskeleton-related genes (BFSP1 and BFSP2), transcription factor genes (HSF4,

MAF, PITX3) and others (CCA5, CCPSO, CTAA1, CTAA2, CTPL1, CTRCT29, CTRCT35,

FYCO1, GCNT2, LONP1, LSS, NHS, UNC45B, VIM, WDR87, WFS1 and WNT3). Some of

the genes viz., BFSP2, CRYAB, CRYAA, CRYBA1, CRYBB1, CRYBB3, EPHA2, GJA8, HSF4

and PITX3 are reported to cause both dominant as well as recessively inherited congenital

cataracts. Among the different genes involved, nearly 50% of the cataract types are caused by

mutations in crystalline genes followed by mutations in the genes encoding for connexins

(Sheils et al, 2010).

Connexins are gap junction proteins that play an important role in intercellular

communication and maintenance of lens homeostasis. They are composed of four

transmembrane domains linked by two extracellular loops that have highly conserved amino

acid sequence identity, and a highly variable intracellular loop and an intracytoplasmic NH2-

and COOH- terminal (Hertzberg et al, 1988; Bennett et al, 1991). The genes GJA3 and GJA8

encoding Connexin 46 and Connexin 50 respectively are known to account for 20% of non-

syndromic cataracts with different types of phenotypes reported worldwide (He and Li,

2000). To-date, more than 43 mutations have been reported in the coding region of GJA8

gene of which 19 are known to be associated with non- syndromic autosomal dominant

pattern of inheritance (Cat-map). GJA8 (NM_005267) gene comprising two exons is located

on chromosome 1q21. Only exon2 in the gene codes for a 50KDa protein. Mutations that are

reported in GJA8 gene leading to the formation of non-syndromic autosomal dominant

congenital cataracts are enlisted in Table-2. So far, 7 mutations causing congenital cataracts

(5-autosomal dominant & 2-autosomal recessive) are reported in GJA8 gene from India. Of

these, only 1 mutation was associated with non- syndromic autosomal dominant cataract

where the phenotype was full moon with Y-sutural opacities (Vanita et al, 2006). The

remaining mutations were associated with other phenotypes such as micro cornea with mild

myopia (Devi and Vijayalaxmi, 2006; Vanita et al, 2008) and nystagmus (Ponnam et al,

2007; Kumar et al, 2011).

In the present study, an extended Muslim family settled long back in India from Hyderabad,

India with bilateral autosomal dominant zonular cataracts with no traces of pulverisation

pulverulent opacities was analysed to detect the pathogenic mutation causing the condition by

exome sequencing of the affected proband and her unaffected son. Two pathogenic variants

namely c.139G>A (rs121434643) in GJA8 gene and c.2036C>T (rs3796375) in FYCO1 gene

were detected by exome sequencing followed by Sanger sequencing in the nuclear family.

PCR-RFLP and Tetra ARMS PCR methods were designed to screen the two variants among

rest of the family members to detect their co-segregation with the inherited dominant zonular

cataract.

MATERIALS AND METHODS

Clinical evaluation

An extended four generation Muslim family with inheritance of bilateral congenital zonular

cataracts with no traces of pulverisation pulverulent opacities registered at Medivision Eye

Care Center, Hyderabad, India was investigated to identify the causative gene segregating in

the family. The couple in the first generation were not alive but the male member of the

family was informed to be affected with cataract during his childhood. Among their progeny

and grand and great grand children, 37 members in the family, comprising 14 affected and 23

unaffected individuals were available for our study (Fig-1). 37 individuals belonged to

different nuclear families of which only one family was complete with 5 members including

parents and three children. The mother i.e. the proband in the nuclear family and her two

daughters were affected with congenital zonular cataract whereas her husband and son were

normal. In the entire family there was no history of consanguinity. Clinical and

ophthalmological examinations including screening for visual acuity, slit lamp and fundus

examination of all the 14 affected members did not reveal history of any other ocular

abnormalities apart from the zonular cataract. The peripheral embryonic and peripheral

infantile nuclei were found to be transparent and in foetal tissue the opaque zone was

detected. So the origin of zonular cataract among the affected members was considered as

foetal. The cataract phenotypes were documented by slit lamp photography (Fig-2). Written

consent was obtained from all the members who participated in the study and the study was

approved by the institutional ethical committee and adhered to the guidelines of the

Declaration of Helsinki.

Collection of Blood Samples

Peripheral venous blood samples were collected in EDTA vacutainers from all the members

who co-operated to participate in the study. Genomic DNA was extracted using conventional

chloroform method as described by Dahm (Dahm, 2008). The DNA was quantitated using

Nanodrop (Thermoscientific) by recording O.D at 260nm.

Whole exome sequencing

Whole exome sequencing was used to identify the pathogenic mutation causing autosomal

dominant zonular cataract in the extended family as it is a powerful and cost effective tool for

identifying the variants of various genes. We selected the female proband and her unaffected

son from the nuclear family within the extended family for exome sequencing. The criteria

for selecting only these two samples was (1) The entire family with five members was

Formatted: Font: (Default) Times New Roman

available for the study (2) The family consisted of both affected and unaffected offspring and

(3) Only one of the two parents was affected.

Exome sequencing was performed by a commercial service, Sandor Life Sciences Pvt Ltd.

Exome capture was carried out using an Illumina TruSeq Exome Enrichment Kit (62 M) that

covered about 97.2% consensus coding sequence. Exome-enriched DNA fragments were

sequenced by an Illumina HiSeq2000 with the average sequencing depth being 125-fold.

Over 99% base call accuracy was up to Q20, which means that the probability of an incorrect

base call is 0.01.

After the low quality reads were filtered, the clean data was aligned to the consensus

sequence (UCSC hg19) to detect variants by SAMtools. Additional bioinformatics analysis of

all the variants was obtained from dbSNP (http://www.ncbi.nlm.nih. gov/), OMIM

(http://www.omim.org/), 1000 Genome (http://browser.1000genomes.org/index.html),

PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/) and SIFT (http://sift.jcvi.org/).

Variant Analysis

The results of exome sequencing of the proband and her unaffected son were compared for

variants in 40 known causative genes reported in Cat-map database (http://cat-

map.wustl.edu/; Table-1) for non- syndromic congenital cataracts. Then we excluded the

variants which we didn’t consider as pathogenic using the following criteria: 1) Minor allele

frequency (MAF) ≥ 0.01 from1000 Human Genome Project database; 2) Located in non-

coding region without affecting splicing site; 3) Synonymous variants without affecting

splicing site and 4). Only one single heterozygous variation detected in recessive genes. Only

variants having possibly damaging effect on the protein were considered as potentially

pathogenic and were summarized for validation.

Sanger Sequencing

Sanger sequencing was done to confirm the potential pathogenic variants detected by exome

sequencing in the nuclear family through commercial source (Eurofins Genomics India Pvt

Ltd). The variants that were confirmed by Sanger sequencing in the five samples belonging to

nuclear family were further screened in the rest of the family members and 120 unrelated

normal healthy controls by Polymerase chain reaction Restriction fragment length

polymorphism (PCR-RFLP) and Tetra ARMS PCR. Primers to amplify the regions with each

variant for Sanger sequencing, PCR RFLP and Tetra ARMS PCR were designed using Batch

Primer 3 software (http://probes.pw.usda.gov/batchprimer3/; Table-23). RFLP protocol was

designed using NEB Cutter (http://nc2.neb.com/NEBcutter2/).

PCR RFLP

Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) was used

for screening c.139G>A variation in exon2 of GJA8 gene. PCR amplification was carried out

in a total reaction volume of 10µl containing 1μl PCR buffer, 200mMeach dNTP, 0.25U Taq

polymerase, and 2.5 pmol each of forward and reverse primers. The PCR conditions to

amplify the marker included initial denaturation at 94°C for 5 m, followed by 30 cycles of

denaturation at 94°C for 35 s, annealing at 54°C for 30 s, extension at 72°C for 35 s and a

final extension at 72°C for 7m. Restriction digestion was carried out by incubating 5 ml of

the PCR product with 2U of Fok1 restriction enzyme overnight. Then genotyping was done

by electrophoresis on polyacrylamide gels. Homozygous wild types (GG) were determined

by the presence of three fragments of 199bp, 56bp and 33bp and heterozygotes (GA) by four

fragments of 199bp, 89bp, 56bp and 33bp. None of the samples showed homozygosity for the

mutanat allele.

Tetra ARMS PCR

Tetra ARMS PCR was used to detect the presence of c.2036C>T variation in exon 8 of

FYCO1 gene. The tetra ARMS PCR method involves the use of two primer pairs to amplify

two different alleles in one PCR reaction. PCR reaction was carried out in a total reaction

volume of 10µl containing 1μl PCR buffer, 200mMeach dNTP, 0.25U Taq polymerase, and

primers at a ratio of 3:1 (inner to outer). PCR conditions included initial denaturation at 94°C

for 5 m, followed by 40 cycles of denaturation at 94°C for 1 m, annealing at 65°C for 1m,

extension at 72°C for 45 s and a final extension at 72°C for 7m. Genotyping was done by

agarose gel electrophoresis and the alleles were interpreted based on the size of the fragments

generated which was 208bp for allele T and 287bp for allele C.

RESULTS

Whole exome sequencing was used to identify the causative mutation segregating among the

members of the extended family affected with bilateral autosomal dominant zonular cataract

without pulverisation pulverulent opacities and other ophthalmic symptoms by testing two

samples one from affected mother (proband) and another from her unaffected son belonging

to the nuclear family.

The results of whole exome sequencing revealed the presence of 49 variants in the proband

and 65 variants in her unaffected son which are related to 40 known causative genes reviewed

for non-syndromic congenital cataracts. After excluding all the non-pathogenic variants and

comparing the variants detected in the proband and her unaffected son, two variants,

c.139G>A (p.Asp47Asn; D47N)) in GJA8 gene and the other c.2036C>T (p.Ala679Val)

variant in FYCO1 gene were chosen for follow up since they were considered as potentially

pathogenic. found to have possible damaging effect and therefore were considered potentially

pathogenic. Presence of these two variants in all the five members of the nuclear family was

confirmed by following Sanger sequencing.

Analysis of c.139G>A in GJA8 gene

Screening of c.139G>A variation in the nuclear family by Sanger sequencing (Fig-3)

revealed the proband and her two affected daughters to be heterozygous for c.139G>A

variant in GJA8 gene while her unaffected son and husband were found to be homozygous

normal. Screening of rest of the members of the extended family revealed co-segregation of

c.139G>A mutation with the inherited autosomal dominant zonular congenital cataract

without pulverisationpulverulent opacities. To confirm further, the causative nature of

c.139G>A, we genotyped 115 unrelated normal healthy individuals from the same population

and 222 cases with age related cataract. None of the controls and age related cataract cases

showed the variation detected in GJA8 gene supporting the causative nature of c.139G>A to

the development of congenital zonular cataract in the family.

Analysis of c.2036C>T in FYCO1 gene

Considering the variation c.2036C>T (rs3796375) in FYCO1 gene, Sanger sequence (Fig-4)

of all the 5 members of the nuclear family were found to be homozygous mutants. Analysis

of the remaining family members by Tetra ARMS PCR revealed that inheritance of FYCO1

variation was independent from the inheritance of congenital zonular cataract among the

members of the extended family. Further, analysis of the variation in 222 age related cataract

cases did not show any susceptibility of this variation to the development of cataract

formation. It is reported to be a common variant in various populations across the world.

Further screening of the variation in 115 unrelated normal healthy individuals revealed that

Formatted: Font: (Default) Times New Roman, 12 pt

the variation was polymorphic in nature with minor allele frequency >0.01. Further, analysis

of the variation in 222 age related cataract cases did not show any susceptibility of this

variation to the development of cataract formation.

DISCUSSION

Congenital cataracts are one of the most common eye disorders leading to blindness in

children worldwide. Of the mutations known to be associated with congenital cataracts,

approximately one quarter of them are located in the connexin genes. Both connexin 46

(Cx46) and connexin 50 (Cx50), encoded by GJA3 and GJA8 respectively, are reported to be

associated with autosomal dominant congenital cataracts (Jiang, 2010). Studies have shown

that these transport membrane proteins are vital for the proper embryological development of

the lens. They are essential for maintaining lens transparency, and GJA8 is required for

proper fiber cell proliferation and control of lens transparency (Gong et al, 2010). Moreover,

Cx50 is required for pH mediated gating of gap junction channels in differentiating fibers and

mutations in the gene could alter the electrical properties of gap junction channels. To date,

more than 43 mutations have been identified in different domains of GJA8 gene that

contribute to inherited congenital cataract with clear cut phenotypic variability.

The mutation c.139G>A (p.Asp47Asn; D47N) detected in the present study is located in the

interface between the first transmembrane domain and the first extracellular loop and replaces

the negatively charged aspartic acid at position 47 by the highly conserved polar, uncharged

asparagine by a negatively charged aspartic acid at position 47. The amino acid position at 47

in connexion 50 is a mutational hotspot comprising various variants viz., D47Y, D47H and

D47N reported at this position previously (Lin et al, 2008; Wang et al, 2011; Li et al, 2013;

Liang et al, 2015). D47N mutants result in loss of function and the A mutant protein of Cx50

is unable to form functional channels preventing its localization to the plasma membrane

(Arora et al, 2008). Previously this mutation was identified in a Caucasian family with

nuclear pulverulent cataract (Arora et al, 2008), and three different Chinese families with

nuclear cataracts (Wang et al, 2011; Liang et al, 2015; He et al, 2011). So far majority of

mutations reported in GJA8 gene were found to be associated with either nuclear pulverulent

or zonular pulverulent cataracts.

The present study differs from these reports as it is the first report indicating the co-

segregation of mutation c.139G>A in GJA8 gene causing replacement of asparagine by

aspartic acid at position 47 with autosomal dominant zonular cataract without pulverisation

pulverulent opacities and with foetal origin. This shows that mutations in Cx50 exhibit

significant interfamilial phenotypic variability.

Considering the mutation c.2036C>T (p.Ala679Val) in FYCO1 gene, it was inherited

independently from the disease in the present family. FYCO1 encodes a binding protein

involved in autophagosome trafficking and was found to be associated with congenital

cataracts (Chen et al, 2011). Recessive mutations in the gene are rare but are responsible for

10% of recessive type of cataract in Pakistan (Chen et al, 2011). Chen et al, (2011) reported 9

different pathogenic mutations in FYCO1 gene in 12 Pakistani families with bilateral nuclear

congenital cataract and 1 Arab Israeli family with posterior lenticunous affecting the

intracellular transport of phagocytic vesicles.. These mutations were shown to affect the

intracellular transport of phagocytic vesicles. Khan et al, (2015) identified 2 mutations

(c.2505del and c.449T>C in FYCO1 gene) in two families with bilateral posterior capsular

abnormalities. In addition, one splice variant was reported by Gillespie et al, (2014) in their

study using next generation sequencing to enhance the diagnosis of congenital cataracts.

It is interesting to note that the variation c.2036C>T in FYCO1 gene detected in the present

study was not associated with either congenital or age related cataracts though it was found to

be possibly pathogenic to have damaging effect on the protein. This variant lies 170bp away

from the c.2206C>T variant found to be associated with congenital cataracts in a Pakistani

family reported by Chen et al, (Chen et al, 2011). Among other diseases, the variation in

FYCO1 gene detected by us was found to be associated with microscopic colitis in a study by

Garner et al, (2014). This shows that phenotypic variability may exist in the expression of

FYCO1 gene variants and hence the mutations in the gene may not always cause cataract

formation. The members in the family studied did not report any other significant disease

condition.

In conclusion, we report a pathogenic heterozygous c.139G>A mutation in GJA8 (connexin

50) in an extended family with bilateral autosomal dominant zonular cataract without

pulveraisation pulverulent opacities that showed marked phenotypic difference from

previously reported cases viz., nuclear and nuclear pulverulent cataracts with the same

substitution. There is possibility of occurrence of digenic or modifier genes in the genome as

observed in case of non-syndromic hearing impairment (Pallares Ruiz et al, 2002; Bronya et

al, 2006) influencing the variability in the expression of lens opacification associated with

same gene mutations. Though c.139G>A mutation in GJA8 gene was a major cause of

development of autosomal dominantt zonular cataract in the present study, the mutation

detected in FYCO1 gene was also evaluated to verify if there was any interaction co-

segregation of between the two genes GJA8 and FYCO1, causing opacification of the lens in

the family studied.

Acknowledgements

We are thankful to all the members of the family who co-operated to participate in the study.

No conflict of interest exists.

References

Amaya L, Taylor D, Russell-Eggitt I, Nischal KK, Lengyel D. 2003. The morphology and

natural history of childhood cataracts. SurvOphthalmol; 48:125-44.

Arora A, Minogue PJ, Liu X, Addison PK, Russel-Eggitt I,Webster AR, Hunt DM, Ebihara

L, Beyer EC, Berthoud VM,Moore AT. 2008. A novel connexin50 mutation associated

withcongenital nuclear pulverulent cataracts. J Med Genet; 45:155-60.

Bennett MV, Barrio LC, Bargiello TA, Spray DC, Hertzberg E,Saez JC. 1991. Gap junctions:

new tools, new answers, new questions. Neuron; 6 (3):305-20.

Bronya JB, Keats Charles IB, Gregory P. 2006. Epidemiology of genetic hearing loss. Semin

Hear; 27:136–147.

Chen J, Ma Z, Jiao X, Fariss R, Kantorow WL, Kantorow M, Pras E, Frydman M, Pras E,

Riazuddin S, Riazuddin SA, Hejtmancik JF. 2011. Mutations in FYCO1 cause autosomal-

recessivecongenital cataracts. Am J Hum Genet; 88:827–838.

Dahm R. 2008. Discovering DNA: Friedrich Miescher and the early years of nucleic acid

research. Hum Genet; 122(6):565-81

Devi RR, Vijayalakshmi P. 2006. Novel mutations in GJA8 associatedwith autosomal

dominant congenital cataract andmicrocornea. Mol Vis; 12:190-5.

Francis PJ, Berry V, Bhattacharya SS, Moore AT. 2000. The genetics of childhood cataract. J

Med Genet ; 37:481-8.

Garner C, Ahn R, Ding YC, Steele L, Stoven S, Green PH, Fasano A, Murray JA, Neuhausen

SL. 2014. Genome-wide association study of celiac disease in north america confirms

FRMD4B as new celiac locus. PLoS One; 9(7): e101428.

Gillespie RL, O'Sullivan J, Ashworth J, Bhaskar S, Williams S, Biswas S, Kehdi E, Ramsden SC,

Clayton-Smith J, Black GC, Lloyd IC. 2014. Personalized diagnosis and management of congenital

cataract by next-generation sequencing. Ophthalmology; 121:2124-37.

Gong X, Cheng C, Xia CH. 2007. Connexins in lens development and cataractogenesis. J

MembrBiol; 218(1–3): 9–12.

He W, Li S. 2000. Congenital cataracts:gene mapping. Hum Genet; 106:1-13

He W, Li X, Chen J, Xu L, Zhang F, Dai Q, Cui H, Wang DM, Yu J, Hu S, Lu S. 2011.

Genetic linkage analyses and Cx50 mutation detection in a large multiplex Chinese family

with hereditary nuclear cataract. Ophthalmic Genet. Mar;32 (1):48-53.

Hertzberg EL, Disher RM, Tiller AA, Zhou Y, Cook RG. 1988. Topologyof the Mr 27,000

liver gap junction protein. Cytoplasmiclocalization of amino- and carboxyl termini and a

hydrophilicdomain which is protease-hypersensitive. J BiolChem; 263:19105-11.

Jiang JX. 2010. Gap Junctions or Hemichannel-Dependent and Independent Roles of Connexins in

Cataractogenesis and Lens Development. CurrMol Med; 10(9): 851–863.

Khan AO, Aldahmesh MA, Alkuraya FS. 2015. Phenotypes of Recessive Pediatric Cataract

in a Cohort of Children with Identified Homozygous Gene Mutations (An American

Ophthalmological Society Thesis). Trans ophthalmol Soc; T71-T715.

Kumar M, Agarwal T, Khokhar S, Kumar M, Kaur P, Roy TS, Dada R. 2011. Mutation

screening and genotype phenotype correlation of alpha-crystallin, gamma-crystallin and

GJA8 gene in congenital cataract. Mol Vis.; 17:693-707.

Li J, Wang Q, Fu Q, Zhu Y, Zhai Y, Yu Y, Zhang K, Yao K. 2013. A novel connexin 50

gene (gap junction protein, alpha 8) mutation associated with congenital nuclear and zonular

pulverulent cataract. Mol Vis; 19: 767-774.

Liang C, Liang H, Yang Y, Ping L, Jie Q. 2015. Mutation analysis of two families

withinherited congenital cataracts. Mol Med Rep; 12(3):3469-3475

Lin Y, Liu NN, Lei CT, Fan YC, Liu XQ, Yang Y, Wang JF, Liu B, Yang ZL. 2008. A

novel GJA8 mutation in a Chinese family with autosomal dominant congenital cataract.

Zhonghua Yi Xue Yi ChuanXueZaZhi; 25: 59-62.

Pallares-Ruiz N., Blanchet P., Mondain M., Claustres M. and Roux A-F. 2002. A large

deletion including most of GJB6 in recessive non syndromic deafness: a digenic effect? Eur.

J. Hum. Genet; 10:72–76.

Ponnam SPG, Ramesha K, Tejwani S, Ramamurthy B, Kannabiran C. 2007. Mutation of the

gap junction protein alpha 8 (GJA8) gene causes autosomal recessive cataract. J Med Genet;

44:e85.

Scott MH, Hejtmancik JF, Wozencraft LA, Reuter LM, ParksMM, Kaiser-Kupfer MI. 1994.

Autosomal dominant congenitalcataract: Interocular phenotypic variability. Ophthalmology;

101:866-71.

Shiels A, Bennett TM, Hejtmancik JF. 2010. Cat-Map: putting cataract on the map. Mol Vis;

16:2007-15

Vanita V, Singh JR, Hejtmancik JF, Nürnberg P, Hennies HC, Singh D, Sperling K. 2006. A

novel fan-shaped cataract microcornea syndrome caused by a mutation of CRYAA in an

Indian family. Mol Vis; 12:518-22.

Vanita V, Singh JR, Singh D, Varon R, Sperling K. 2008. A novel mutation in GJA8

associated with jellyfish-like cataract in a family of Indian origin. Mol Vis; 14:323-6.

Vanita. 1998. Genetical investigations in congenital cataract cases. Ph.D. thesis, Guru Nanak

Dev University, Amritsar, India.

Wang L, Luo Y, Wen W, Zhang S and Lu Y: 2011. Another evidence for a D47N mutation in

GJA8 associated with autosomal dominant congenital cataract. Mol Vis; 17: 2380-2385.

Figure legends

Figure-1: Pedigree showing the segregation of c.139G>A in GJA8 and c.2036C>T in

FYCO1 gene in an extended family with autosomal dominant zonular cataract without

pulverisation. Arrow denotes the affected proband in the nuclear family with 5 members

indicated by asterix. Black and open symbols denote affected and unaffected individuals

respectively. H denotes heterozygosity, N homozygous normal and M homozygous mutant

genotypes for both GJA8 and FYCO1 genes variants. Number in the male and female symbol

denotes no. of sibs.

Figure-2: Illustrates the appearance of zonular cataracts without pulverulent opacities among

affected members of the family. A) Diffused illumination that shows opacification in central

part of the lens A) Diffused illumination showing cataract development in central part of the

lens B) Slit lamp photograph of lens with clear central embryonic nucleus and clear

peripheral lens. Cataract development is seen only in the fetal nucleus.

Figure-3: Chromatogram showing partial genomic sequence of GJA8 gene. a) Seqence of an

unaffected member from nuclear family and b) Sequence of an affected member from the

nuclear family. Arrow indicates the mutation c.139G>A in GJA8 gene

Figure-4: Chromatogram showing partial genomic sequence of a member of nuclear family

with c.2036 C>T substitution in FYCO1 gene. Arrow indicates the mutation.