Post on 04-Jul-2020
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
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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
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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
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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.
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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.