1040-5488/15/9203-0337/0 VOL. 92, NO. 3, PP. 337Y342 OPTOMETRY AND VISION SCIENCE Copyright * 2015 American Academy of Optometry
ORIGINAL ARTICLE
Identification of a Novel GJA3 Mutation in Congenital Nuclear Cataract Lamei Yuan*, Yi Guo†, Junhui Yi†, Jingjing Xiao‡, Jinzhong Yuan†, Wei Xiong†, Hongbo Xu*, Zhijian Yang§, Jianguo Zhang‡, and Hao Deng†
ABSTRACT Purpose. Congenital cataract is a visual impairment that needs correction as early as possible after birth. This study aimed to identify whether genetic defects exist in a Chinese Han pedigree with congenital nuclear cataract. Methods. A family consisting of six members and three patients with nuclear cataract spanning three generations and 100 unrelated ethnically matched normal subjects were recruited in this study. Exome sequencing was performed in the 24-yearold proband, and Sanger sequencing was then conducted in other family members and 100 normal controls. Results. A novel missense variant, c.428G>A (p.G143E), in the gap junction protein-alpha 3 gene (GJA3) was identified in three patients of the family but unidentified in three family members without lens opacity and 100 normal controls. Conclusions. A novel missense mutation, c.428G>A (p.G143E), in the GJA3 gene, localized to the cytoplasmic loop, was suggested to be the genetic cause of congenital nuclear cataract, which further expands the gene mutation spectrum. Our findings suggest that exome sequencing is a powerful and cost-effective tool to discover mutation(s) in disorders with high genetic and clinical heterogeneity. Further functional studies in the GJA3 gene mutations may help uncover pathogenic mechanisms of congenital cataract and therefore provide a possible genetic therapy for this disorder. (Optom Vis Sci 2015;92:337Y342) Key Words: congenital nuclear cataract, exome sequencing, the GJA3 gene, mutation, p.G143E
C
ongenital cataract is defined as opacification of the normally transparent crystalline lens, which results in impairment of vision at birth or during early childhood.1,2 The disorder is the most common treatable cause of visual impairment, and it accounts for about 10% of poor vision or blindness in childhood worldwide.1,3,4 The incidence is estimated to be about 1 to 15 per 10,000 children worldwide.1 Hereditary cataract usually accounts for between 8.3 and 25% of congenital cataract.2 Good outcomes have been reported in children undergoing surgery before 6 or 10 weeks of age in unilateral or bilateral cases, showing that early diagnosis followed by appropriate intervention is important for obtaining good visual function.5 *MD † PhD ‡ MS § RA Center for Experimental Medicine and Department of Neurology (LY, YG, HX, ZY, HD), Department of Ophthalmology (J Yi), Department of Nephrology (J Yuan), the Third Xiangya Hospital, Central South University, Changsha, China; Department of Medical Information (YG), Cancer Research Institute (WX), Xiangya School of Medicine, Central South University, Changsha, China; and BGI-Shenzhen, Shenzhen, China (JX, JZ).
Several clinical classification systems have been developed, and it can be classified as polar/subcapsular, nuclear, lamellar, sutural, cortical, membranous/capsular, and total cataract according to the anatomic location of opacities in the lens.1,2,4 Nuclear cataract, the most common type of hereditary congenital cataract, refers to the opacification limited to the embryonic and/or fetal nuclei of the lens with transparent cortex.1,4 Cataract can be isolated, accompanied with other ocular abnormalities, or associated with metabolic diseases and genetic syndromes.1,6 Most congenital cataract is inherited as a Mendelian trait with high penetrance. Although autosomal recessive and X-linked inheritance patterns have been reported, hereditary Mendelian cataract is most frequently inherited as an autosomal dominant trait.1,2,4 At least 23 disease loci and 18 disease-causing genes for congenital nuclear cataract are described, including eight crystallin genes (the alpha-A-crystallin gene, CRYAA; the alpha-B-crystallin gene, CRYAB; the beta-A1-crystallin gene, CRYBA1; the beta-B1crystallin gene, CRYBB1; the beta-B2-crystallin gene, CRYBB2; the beta-B3-crystallin gene, CRYBB3; the gamma-C-crystallin gene, CRYGC; and the gamma-D-crystallin gene, CRYGD), one cytoskeletal protein gene (the beaded filament structural protein 2 gene, BFSP2), three membrane protein genes (the gap junction
Optometry and Vision Science, Vol. 92, No. 3, March 2015
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338 Novel GJA3 Mutation in Congenital Nuclear CataractVYuan et al.
protein-alpha 3 gene, GJA3; the gap junction protein-alpha 8 gene, GJA8; and the major intrinsic protein of lens fiber gene, MIP), one growth and transcription factor gene (the v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog gene, MAF ), and the ferritin light chain gene (FTL), the galactokinase 1 gene (GALK1), the FYVE and coiled-coil domain containing 1 gene (FYCO1), the Nance-Horan syndrome gene (NHS ), and the Wolfram gene (WFS1).1,4,7 Mutations in the crystallins are responsible for about half of families with identified causative genes, followed by connexins.1,6 Because of the wide phenotypic and genetic heterogeneity in congenital nuclear cataract,4 we performed exome sequencing to identify the disease gene responsible for a Chinese Han family with autosomal dominant inheritance form of congenital nuclear cataract. A novel heterozygous c.428G>A transition (p.G143E) in the GJA3 gene (MIM 121015) was identified and the variant cosegregated with subjects with the disorder in the family, whereas it was unidentified in unaffected family members and normal controls, indicating that it may be a pathogenic mutation for congenital nuclear cataract in the family.
METHODS Study Subjects and Clinical Evaluation A three-generation, six-member Chinese Han family with autosomal dominant nuclear cataract from Hunan province, China, was recruited from the Third Xiangya Hospital, Central South University (Fig. 1). There were three patients (I:1, II:1, and III:1) across three generations of the pedigree with congenital nuclear cataract. Four subjects (I:2, II:1, II:3, and III:1) were previously
reported to have X-linked Alport syndrome with a novel mutation in the collagen type IV alpha-5 gene (COL4A5). All subjects underwent detailed ophthalmic examinations, including visual acuity, slit lamp examination, fundus examination with the dilated pupils, and intraocular pressure measurement. The phenotypes were evaluated by slit lamp photography.4 One hundred unrelated ethnically matched subjects (male/female: 50/50; mean [TSD] age, 40.2 [T8.3] years) without diagnostic features or family history of congenital cataract were randomly recruited to serve as normal controls. Ethylenediaminetetraacetic acidYanticoagulated venous blood samples were collected from all the participants. Written informed consent was obtained from all the participants, and the protocol of this study was approved by the Ethics Committee of the Third Xiangya Hospital of Central South University, China.
Exome Capture Isolation of genomic DNA from venous blood leukocytes was performed by standard phenol-chloroform extraction method.4 Three micrograms of genomic DNA was used to generate the exome library. DNA of the proband (II:1, Fig. 1) was sheared by sonication and then hybridized to the Nimblegen SeqCap EZ Library for enrichment, according to the manufacturers’ instructions. Sequencing of the enriched library targeting the exome was performed on the HiSeq 2000 platform (Illumina, San Diego, CA) to get 90-bp paired-end reads.8 A mean exome coverage of 81.65 was obtained, which allowed each selected region of the genome to be checked and provided sufficient depth to accurately call variants at 99.41% of targeted exome.9
Read Mapping and Variant Analysis
FIGURE 1. Pedigree of the family with congenital nuclear cataract showing affected cases (fully shaded). N, normal; M, the GJA3 c.428G>A (p.G143E) mutation. Arrow indicates the proband.
The human reference genome was obtained from the University of California, Santa Cruz database (http://genome.ucsc.edu/), version hg19 (build 37.1). The obtained sequence reads from the patient were aligned using the program SOAPaligner, and single nucleotide polymorphisms (SNPs) were called using SOAPsnp set with the default parameters after the duplicated reads (produced mainly in the polymerase chain reaction [PCR] step) were deleted.8Y10 Short insertions or deletions (indels) altering coding sequence or splicing sites were also identified by GATK.11,12 We filtered candidate SNPs with the following criteria: SNP quality greater than or equal to 20, sequencing depth greater than or equal to 4, estimated copy number less than or equal to 2, and distance between two SNPs greater than 5 (the quality score is a Phred score, generated by the program SOAPsnp1.03, quality score 20 represents 99% accuracy of a base call). Based on the assumption that the variant of interest is rare within the normal population, all variants were filtered against the SNP database (dbSNP132, http://www.ncbi.nlm.nih.gov/projects/SNP/snp_summary.cgi), 1000 genomes data (1000 genomes release_20100804, http:// www.1000genomes.org/), HapMap (2010-08_phase II + III, http://hapmap.ncbi.nlm.nih.gov/), YanHuang1 (YH1) project, as well as synonymous substitutions, and the variants were kept as candidates with the frequency less than 0.50%.4,7Y9 Variants that were not annotated in the above public databases were further
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Novel GJA3 Mutation in Congenital Nuclear CataractVYuan et al.
filtered by in-house database from BGI-Shenzhen with 2375 ethnically matched controls. Sorting intolerant from tolerant (SIFT) prediction was performed to evaluate whether the amino acid substitutions affect protein function.13
Mutation Validation Locus-specific PCR amplification primers were designed. Sanger sequencing was then performed in all available family members and 100 ethnically matched normal controls to validate the presence and identity of potential disease-causing variants. Sequences of primers used for the causative variation were as follows: 5¶-GGAGGAGCAGCTGAAGAGAG-3¶ and 5¶-ATGAAGCAGTCCACCGTGTT-3¶. Polymerase chain reaction amplification was conducted as described previously,14 and PCR products were sequenced using ABI3500 genetic analyzer (Applied Biosystems, Foster City, CA) following the standard procedures.15
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regions or the splicing sites. A prioritization scheme was applied to identify the pathogenic mutation in the patient, similar to recent studies.4,11,18,21,22 We excluded known variants identified in dbSNP132, 1000 genomes project, HapMap, and YH1. After this, we reduced the number of candidate genes by more than 90.33%. Sequence variants that were not annotated in any of the above public databases were further filtered by in-house database from BGI-Shenzhen, and only 56 novel variants predicted to affect protein function were identified. Except for the GJA3 p.G143E mutation, other known disease-causing gene mutations for congenital cataract were excluded. After validation by Sanger sequencing, a c.428G>A variant (p.G143E) in the GJA3 gene was identified in the patient, and no other known disease-causing gene mutations for congenital cataract were found (Fig. 2B). The same heterozygous variant was subsequently identified in both her affected father and daughter. The variant cosegregated with disease in the family and was unidentified in three unaffected family members and in the 100 ethnically matched unrelated controls.
Bioinformatics Analysis of the Mutation Multiple sequence alignment was performed using the Basic Local Alignment Search Tool (http://blast.st-va.ncbi.nlm.nih.gov/ Blast.cgi). Online tools including Polymorphism Phenotyping version 2 (PolyPhen-2, http://genetics.bwh.harvard.edu/pph2/), SIFT (http://sift.jcvi.org/, scores less than 0.05 are deleterious), and MutationTaster (http://www.mutationtaster.org/) were used to evaluate the possible effects of amino acid substitution on protein structure and function in terms of sequence conservation, chemical change, and likelihood of pathogenicity.13,16Y18 A threedimension model of the protein was created by PyMOL 1.7 based on the CPHmodels-3.2.19,20
RESULTS Clinical Findings According to the medical records, three patients (I:1, II:1, and III:1, Fig. 1) were diagnosed as having bilateral nuclear cataract by slit lamp examination. All patients presented with cataract at birth. The proband (II:1) also presented with bilateral myopia and had phacoemulsification and intraocular lens implantation performed at 21 years. Her father (I:1) had cataract extraction performed but had no medical records available. Her daughter (III:1) had mild nuclear opacities. Both her mother (I:2) and brother (II:3) were normal based on ophthalmic evaluation.
Mutation Screening We sequenced the exome of the proband (II:1) of the Chinese Han family with congenital nuclear cataract and attained an average of 8.14 billion bases of 90-bp paired-end read sequence. Among the 8.14 billion bases, 7.88 billion (96.81%) passed the quality assessment, 7.37 billion (93.53%) were aligned to the human reference sequence, and 3.60 billion bases (48.85%) were mapped to the targeted regions with an average coverage of 81.65-fold.8 A total of 105,963 genetic variants, including 14,723 nonsynonymous variants, were identified in either the coding
Bioinformatics Analysis of the Mutation The glycine at position 143 is phylogenetically conserved among various species (Fig. 2C) and human gap junctions (Fig. 2D) by multiple sequence alignment.23 PolyPhen-2 analysis produced a score of 0.98 on the HumVar database (sensitivity, 0.55; specificity, 0.94) (Fig. 2E), which is predicted to be probably damaging. The SIFT prediction revealed a score of 0.00, indicating that the substitution is predicted to affect protein function. MutationTaster predicted that the alteration was disease causing with a probability value close to 1, which indicates the high security of prediction. Cartoon representation of the model structure of the protein was shown in Fig. 3. Computer-based protein analysis of the variant indicated that the p.G143E variant in the GJA3 gene was likely deleterious and the disease-causing mutation for congenital cataract in our family.
DISCUSSION The GJA3 gene, located on 13q12.11, consists of two exons, one of which encodes a 435Yamino acid protein. The GJA3 gene belongs to the connexin gene family. GJA3 protein, also known as connexin 46, is predominantly expressed in the lens fiber cells and functions in gap junction communication. GJA3 is essential for maintaining lens transparency.1 Each connexin includes cytoplasmic amino and carboxyl termini as well as four transmembrane domains linked by a single cytoplasmic loop (CL) and two extracellular domains.23 GJA3, a structurally related transmembrane protein, functions as a gap junction allowing intercellular passage of lowYmolecular weight metabolites, ions, and second messengers between adjacent cells.4,20 Diverse gap junction channels formed by GJA3 and GJA8 subunits are important for differentiation, elongation, and maturation of lens fiber cells.24 Gap junction coupling largely contributes to the avascular lens homeostasis and the maintenance of transparency.23 Mutations in the GJA3 gene have been considered to lead to nuclear cataract in both humans and mice.4
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340 Novel GJA3 Mutation in Congenital Nuclear CataractVYuan et al.
FIGURE 2. Mutation analysis of c.428G>A (p.G143E) mutation in the GJA3 gene. (A) Unaffected member (II:3) of the family. (B) Heterozygous p.G143E mutation patient (II:1). (C, D) Glycine at position 143 is highly conserved in different animal species and human gap junction proteins (>-connexins). (E) The p.G143E is predicted to be probably damaging by PolyPhen-2, with a score of 0.98. A color version of this figure is available online at www.optvissci.com.
FIGURE 3. Cartoon representation of the model structure of the GJA3 protein. (A) Overall structure of the protein is displayed in three dimensions. E1 and E2 stand for two extracellular domains, and M1, M2, M3, and M4 mean four transmembrane domains of the protein. (B, C) Amino acids at position 143, the glycine (B) and the mutated glumatic acid (C), are shown as sticks. A color version of this figure is available online at www.optvissci.com. Optometry and Vision Science, Vol. 92, No. 3, March 2015
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Novel GJA3 Mutation in Congenital Nuclear CataractVYuan et al.
Gja3j/j mice developed nuclear cataract associated with the proteolysis of crystallins,25 and the Gja3 gene maintained cellular differentiation.26 Genetic background influenced the cataract phenotype of Gja3j/j mice, consistent with clinical findings and genetic studies in humans.4,27 The missense p.E42K mutation in the Gja3 gene was found to be responsible for a rat model of autosomal recessive congenital nuclear cataract.28 Knocked-in Gja3 replacing the endogenous wild-type Gja8 gene could suppress nuclear cataract caused by Crygb p.S11R mutation, suggesting that elevated Gja3 can prevent or delay nuclear cataract formation.29 To our knowledge, at least 29 mutations, including nucleotide substitution, deletion, and insertion mutations involving different domains, have been described to contribute to assorted phenotypic cataracts in humans (the Human Gene Mutation Database, http://www.hgmd.cf.ac.uk/ac/).4,20,30Y32 Several mutations (p.L11S, p.V44M, p.D47N, p.P59L, and p.R76H) were reported in at least two families,4 and founder effect or mutation hotspot should be considered. Most of these mutations occur in extracellular loop and transmembrane domains, which are associated with gap junction formation and pore structure/gating, respectively.23,33 Mutations in the GJA3 gene tend to produce phenotypically similar autosomal dominant nuclear and especially zonular pulverulent cataract,2 although there are some interfamilial and intrafamilial differences regarding the appearance and location of opacities within the lens.4,34 Radically different GJA3-related cataract morphologies and severities were reported in different families or even in the same family, which may be explained by the interaction of modifier genes or background environmental factors affecting the expression of the gene and hence resulting in various phenotypes.2,34 In 2011, the GJA3 p.V139M variant, the first report about an amino acid change in the CL domain, was found in a 76-year-old sporadic Chinese patient with cortical cataract, and the variant was predicted to be benign, making it difficult to assess the real role of the p.V139M variant in the development of the disease.35 However, another GJA3 mutation, p.G143R, in the CL domain was identified in a five-generation Chinese pedigree with congenital Coppock-like cataract,23 and the mutation enhanced its interaction with the carboxyl terminus, resulting in reduced gap junction channel function, increased hemichannel function, and cell death, a possible underlying mechanism for cataract formation in a dominant negative manner.33 Intriguingly, a different amino acid substitution, p.G143E, at the same position, potentially changing the normal protein function, was identified in our family with congenital nuclear cataract. The variant cosegregated with patients with congenital nuclear cataract in our family and was unidentified in unaffected family members and in unrelated ethnically matched controls. The c.428G>A transition led to the replacement of a neutral amino acid residue, glycine, by a negatively charged residue, glutamic acid. A multiple sequence alignment of the amino acid sequence of GJA3 showed that glycine at position 143 in the CL domain is phylogenetically conserved in various animal species and also in different human gap junctions, although the CL and carboxylterminal domains are highly variable between gap junction family members.23 The recurring variant at position p.G143 further supported the pathogenicity of this missense variant and the essential
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role of the CL domain in the GJA3 gene function.23,33 PolyPhen-2, SIFT, and MutationTaster analyses predicted the variant to be ‘‘damaging’’ or intolerant, further supporting the variant to be the disease-causing mutation in this family. Interestingly, in our family, the paternal allele carries the cataract-related GJA3 gene mutation, and the maternal allele harbors the COL4A5 gene mutation, c.499delC (p.P167Qfs*36), responsible for X-linked Alport syndrome in the proband.36 Both mutations were found in the proband’s daughter. Alport syndrome is associated with typical ocular abnormalities, including dot-and-fleck retinopathy, anterior lenticonus, and posterior polymorphous corneal dystrophy, and other rare changes, including cataract, can be observed.37 However, we identified that the congenital nuclear cataract in these three patients was caused by the GJA3 p.G143E mutation instead of being a symptom of Alport syndrome. The genetic basis of cataract and glomerulopathy in the proband and her daughter appears to be independent. In summary, our data support that the novel missense mutation, c.428G>A (p.G143E), in the GJA3 gene is probably the genetic cause of congenital nuclear cataract in this family. Our finding extends the spectrum of GJA3 mutations. Our strategy of using exome sequencing for genetic diagnosis is highly efficient and cost-effective for disorders with high genetic and clinical heterogeneity and even in small pedigrees.18,36 Further functional studies in the GJA3 gene mutations may help uncover pathogenic mechanisms of congenital cataract and therefore provide a possible genetic therapy for this disorder in the future.
ACKNOWLEDGMENTS We thank the participating patients and investigators for their cooperation and their efforts in collecting the genetic information and DNA specimens. This work was supported by grants from the National Natural Science Foundation of China (81271921, 81101339, and 81441033); the Sheng Hua Scholars Program of Central South University, China (HD); the Research Fund for the Doctoral Program of Higher Education of China (20110162110026); the Natural Science Foundation of Hunan Province, China (10JJ5029); the Construction Fund for Key Subjects of the Third Xiangya Hospital, Central South University, China (HD); the Hunan Provincial Innovation Foundation for Postgraduate, China (7138000008); the Students Innovative Pilot Scheme of Central South University, China (YC12417); and the Top-notch Innovative Doctoral Scholarship of Central South University, China (2014bjjxj039). Received August 25, 2014; accepted October 16, 2014.
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Hao Deng Center for Experimental Medicine The Third Xiangya Hospital, Central South University Tongzipo Rd 138 Hunan Changsha 410013 P.R. China e-mail: [email protected]
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ORIGINAL ARTICLE
Identification of a Novel GJA3 Mutation in Congenital Nuclear Cataract Lamei Yuan*, Yi Guo†, Junhui Yi†, Jingjing Xiao‡, Jinzhong Yuan†, Wei Xiong†, Hongbo Xu*, Zhijian Yang§, Jianguo Zhang‡, and Hao Deng†
ABSTRACT Purpose. Congenital cataract is a visual impairment that needs correction as early as possible after birth. This study aimed to identify whether genetic defects exist in a Chinese Han pedigree with congenital nuclear cataract. Methods. A family consisting of six members and three patients with nuclear cataract spanning three generations and 100 unrelated ethnically matched normal subjects were recruited in this study. Exome sequencing was performed in the 24-yearold proband, and Sanger sequencing was then conducted in other family members and 100 normal controls. Results. A novel missense variant, c.428G>A (p.G143E), in the gap junction protein-alpha 3 gene (GJA3) was identified in three patients of the family but unidentified in three family members without lens opacity and 100 normal controls. Conclusions. A novel missense mutation, c.428G>A (p.G143E), in the GJA3 gene, localized to the cytoplasmic loop, was suggested to be the genetic cause of congenital nuclear cataract, which further expands the gene mutation spectrum. Our findings suggest that exome sequencing is a powerful and cost-effective tool to discover mutation(s) in disorders with high genetic and clinical heterogeneity. Further functional studies in the GJA3 gene mutations may help uncover pathogenic mechanisms of congenital cataract and therefore provide a possible genetic therapy for this disorder. (Optom Vis Sci 2015;92:337Y342) Key Words: congenital nuclear cataract, exome sequencing, the GJA3 gene, mutation, p.G143E
C
ongenital cataract is defined as opacification of the normally transparent crystalline lens, which results in impairment of vision at birth or during early childhood.1,2 The disorder is the most common treatable cause of visual impairment, and it accounts for about 10% of poor vision or blindness in childhood worldwide.1,3,4 The incidence is estimated to be about 1 to 15 per 10,000 children worldwide.1 Hereditary cataract usually accounts for between 8.3 and 25% of congenital cataract.2 Good outcomes have been reported in children undergoing surgery before 6 or 10 weeks of age in unilateral or bilateral cases, showing that early diagnosis followed by appropriate intervention is important for obtaining good visual function.5 *MD † PhD ‡ MS § RA Center for Experimental Medicine and Department of Neurology (LY, YG, HX, ZY, HD), Department of Ophthalmology (J Yi), Department of Nephrology (J Yuan), the Third Xiangya Hospital, Central South University, Changsha, China; Department of Medical Information (YG), Cancer Research Institute (WX), Xiangya School of Medicine, Central South University, Changsha, China; and BGI-Shenzhen, Shenzhen, China (JX, JZ).
Several clinical classification systems have been developed, and it can be classified as polar/subcapsular, nuclear, lamellar, sutural, cortical, membranous/capsular, and total cataract according to the anatomic location of opacities in the lens.1,2,4 Nuclear cataract, the most common type of hereditary congenital cataract, refers to the opacification limited to the embryonic and/or fetal nuclei of the lens with transparent cortex.1,4 Cataract can be isolated, accompanied with other ocular abnormalities, or associated with metabolic diseases and genetic syndromes.1,6 Most congenital cataract is inherited as a Mendelian trait with high penetrance. Although autosomal recessive and X-linked inheritance patterns have been reported, hereditary Mendelian cataract is most frequently inherited as an autosomal dominant trait.1,2,4 At least 23 disease loci and 18 disease-causing genes for congenital nuclear cataract are described, including eight crystallin genes (the alpha-A-crystallin gene, CRYAA; the alpha-B-crystallin gene, CRYAB; the beta-A1-crystallin gene, CRYBA1; the beta-B1crystallin gene, CRYBB1; the beta-B2-crystallin gene, CRYBB2; the beta-B3-crystallin gene, CRYBB3; the gamma-C-crystallin gene, CRYGC; and the gamma-D-crystallin gene, CRYGD), one cytoskeletal protein gene (the beaded filament structural protein 2 gene, BFSP2), three membrane protein genes (the gap junction
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338 Novel GJA3 Mutation in Congenital Nuclear CataractVYuan et al.
protein-alpha 3 gene, GJA3; the gap junction protein-alpha 8 gene, GJA8; and the major intrinsic protein of lens fiber gene, MIP), one growth and transcription factor gene (the v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog gene, MAF ), and the ferritin light chain gene (FTL), the galactokinase 1 gene (GALK1), the FYVE and coiled-coil domain containing 1 gene (FYCO1), the Nance-Horan syndrome gene (NHS ), and the Wolfram gene (WFS1).1,4,7 Mutations in the crystallins are responsible for about half of families with identified causative genes, followed by connexins.1,6 Because of the wide phenotypic and genetic heterogeneity in congenital nuclear cataract,4 we performed exome sequencing to identify the disease gene responsible for a Chinese Han family with autosomal dominant inheritance form of congenital nuclear cataract. A novel heterozygous c.428G>A transition (p.G143E) in the GJA3 gene (MIM 121015) was identified and the variant cosegregated with subjects with the disorder in the family, whereas it was unidentified in unaffected family members and normal controls, indicating that it may be a pathogenic mutation for congenital nuclear cataract in the family.
METHODS Study Subjects and Clinical Evaluation A three-generation, six-member Chinese Han family with autosomal dominant nuclear cataract from Hunan province, China, was recruited from the Third Xiangya Hospital, Central South University (Fig. 1). There were three patients (I:1, II:1, and III:1) across three generations of the pedigree with congenital nuclear cataract. Four subjects (I:2, II:1, II:3, and III:1) were previously
reported to have X-linked Alport syndrome with a novel mutation in the collagen type IV alpha-5 gene (COL4A5). All subjects underwent detailed ophthalmic examinations, including visual acuity, slit lamp examination, fundus examination with the dilated pupils, and intraocular pressure measurement. The phenotypes were evaluated by slit lamp photography.4 One hundred unrelated ethnically matched subjects (male/female: 50/50; mean [TSD] age, 40.2 [T8.3] years) without diagnostic features or family history of congenital cataract were randomly recruited to serve as normal controls. Ethylenediaminetetraacetic acidYanticoagulated venous blood samples were collected from all the participants. Written informed consent was obtained from all the participants, and the protocol of this study was approved by the Ethics Committee of the Third Xiangya Hospital of Central South University, China.
Exome Capture Isolation of genomic DNA from venous blood leukocytes was performed by standard phenol-chloroform extraction method.4 Three micrograms of genomic DNA was used to generate the exome library. DNA of the proband (II:1, Fig. 1) was sheared by sonication and then hybridized to the Nimblegen SeqCap EZ Library for enrichment, according to the manufacturers’ instructions. Sequencing of the enriched library targeting the exome was performed on the HiSeq 2000 platform (Illumina, San Diego, CA) to get 90-bp paired-end reads.8 A mean exome coverage of 81.65 was obtained, which allowed each selected region of the genome to be checked and provided sufficient depth to accurately call variants at 99.41% of targeted exome.9
Read Mapping and Variant Analysis
FIGURE 1. Pedigree of the family with congenital nuclear cataract showing affected cases (fully shaded). N, normal; M, the GJA3 c.428G>A (p.G143E) mutation. Arrow indicates the proband.
The human reference genome was obtained from the University of California, Santa Cruz database (http://genome.ucsc.edu/), version hg19 (build 37.1). The obtained sequence reads from the patient were aligned using the program SOAPaligner, and single nucleotide polymorphisms (SNPs) were called using SOAPsnp set with the default parameters after the duplicated reads (produced mainly in the polymerase chain reaction [PCR] step) were deleted.8Y10 Short insertions or deletions (indels) altering coding sequence or splicing sites were also identified by GATK.11,12 We filtered candidate SNPs with the following criteria: SNP quality greater than or equal to 20, sequencing depth greater than or equal to 4, estimated copy number less than or equal to 2, and distance between two SNPs greater than 5 (the quality score is a Phred score, generated by the program SOAPsnp1.03, quality score 20 represents 99% accuracy of a base call). Based on the assumption that the variant of interest is rare within the normal population, all variants were filtered against the SNP database (dbSNP132, http://www.ncbi.nlm.nih.gov/projects/SNP/snp_summary.cgi), 1000 genomes data (1000 genomes release_20100804, http:// www.1000genomes.org/), HapMap (2010-08_phase II + III, http://hapmap.ncbi.nlm.nih.gov/), YanHuang1 (YH1) project, as well as synonymous substitutions, and the variants were kept as candidates with the frequency less than 0.50%.4,7Y9 Variants that were not annotated in the above public databases were further
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Novel GJA3 Mutation in Congenital Nuclear CataractVYuan et al.
filtered by in-house database from BGI-Shenzhen with 2375 ethnically matched controls. Sorting intolerant from tolerant (SIFT) prediction was performed to evaluate whether the amino acid substitutions affect protein function.13
Mutation Validation Locus-specific PCR amplification primers were designed. Sanger sequencing was then performed in all available family members and 100 ethnically matched normal controls to validate the presence and identity of potential disease-causing variants. Sequences of primers used for the causative variation were as follows: 5¶-GGAGGAGCAGCTGAAGAGAG-3¶ and 5¶-ATGAAGCAGTCCACCGTGTT-3¶. Polymerase chain reaction amplification was conducted as described previously,14 and PCR products were sequenced using ABI3500 genetic analyzer (Applied Biosystems, Foster City, CA) following the standard procedures.15
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regions or the splicing sites. A prioritization scheme was applied to identify the pathogenic mutation in the patient, similar to recent studies.4,11,18,21,22 We excluded known variants identified in dbSNP132, 1000 genomes project, HapMap, and YH1. After this, we reduced the number of candidate genes by more than 90.33%. Sequence variants that were not annotated in any of the above public databases were further filtered by in-house database from BGI-Shenzhen, and only 56 novel variants predicted to affect protein function were identified. Except for the GJA3 p.G143E mutation, other known disease-causing gene mutations for congenital cataract were excluded. After validation by Sanger sequencing, a c.428G>A variant (p.G143E) in the GJA3 gene was identified in the patient, and no other known disease-causing gene mutations for congenital cataract were found (Fig. 2B). The same heterozygous variant was subsequently identified in both her affected father and daughter. The variant cosegregated with disease in the family and was unidentified in three unaffected family members and in the 100 ethnically matched unrelated controls.
Bioinformatics Analysis of the Mutation Multiple sequence alignment was performed using the Basic Local Alignment Search Tool (http://blast.st-va.ncbi.nlm.nih.gov/ Blast.cgi). Online tools including Polymorphism Phenotyping version 2 (PolyPhen-2, http://genetics.bwh.harvard.edu/pph2/), SIFT (http://sift.jcvi.org/, scores less than 0.05 are deleterious), and MutationTaster (http://www.mutationtaster.org/) were used to evaluate the possible effects of amino acid substitution on protein structure and function in terms of sequence conservation, chemical change, and likelihood of pathogenicity.13,16Y18 A threedimension model of the protein was created by PyMOL 1.7 based on the CPHmodels-3.2.19,20
RESULTS Clinical Findings According to the medical records, three patients (I:1, II:1, and III:1, Fig. 1) were diagnosed as having bilateral nuclear cataract by slit lamp examination. All patients presented with cataract at birth. The proband (II:1) also presented with bilateral myopia and had phacoemulsification and intraocular lens implantation performed at 21 years. Her father (I:1) had cataract extraction performed but had no medical records available. Her daughter (III:1) had mild nuclear opacities. Both her mother (I:2) and brother (II:3) were normal based on ophthalmic evaluation.
Mutation Screening We sequenced the exome of the proband (II:1) of the Chinese Han family with congenital nuclear cataract and attained an average of 8.14 billion bases of 90-bp paired-end read sequence. Among the 8.14 billion bases, 7.88 billion (96.81%) passed the quality assessment, 7.37 billion (93.53%) were aligned to the human reference sequence, and 3.60 billion bases (48.85%) were mapped to the targeted regions with an average coverage of 81.65-fold.8 A total of 105,963 genetic variants, including 14,723 nonsynonymous variants, were identified in either the coding
Bioinformatics Analysis of the Mutation The glycine at position 143 is phylogenetically conserved among various species (Fig. 2C) and human gap junctions (Fig. 2D) by multiple sequence alignment.23 PolyPhen-2 analysis produced a score of 0.98 on the HumVar database (sensitivity, 0.55; specificity, 0.94) (Fig. 2E), which is predicted to be probably damaging. The SIFT prediction revealed a score of 0.00, indicating that the substitution is predicted to affect protein function. MutationTaster predicted that the alteration was disease causing with a probability value close to 1, which indicates the high security of prediction. Cartoon representation of the model structure of the protein was shown in Fig. 3. Computer-based protein analysis of the variant indicated that the p.G143E variant in the GJA3 gene was likely deleterious and the disease-causing mutation for congenital cataract in our family.
DISCUSSION The GJA3 gene, located on 13q12.11, consists of two exons, one of which encodes a 435Yamino acid protein. The GJA3 gene belongs to the connexin gene family. GJA3 protein, also known as connexin 46, is predominantly expressed in the lens fiber cells and functions in gap junction communication. GJA3 is essential for maintaining lens transparency.1 Each connexin includes cytoplasmic amino and carboxyl termini as well as four transmembrane domains linked by a single cytoplasmic loop (CL) and two extracellular domains.23 GJA3, a structurally related transmembrane protein, functions as a gap junction allowing intercellular passage of lowYmolecular weight metabolites, ions, and second messengers between adjacent cells.4,20 Diverse gap junction channels formed by GJA3 and GJA8 subunits are important for differentiation, elongation, and maturation of lens fiber cells.24 Gap junction coupling largely contributes to the avascular lens homeostasis and the maintenance of transparency.23 Mutations in the GJA3 gene have been considered to lead to nuclear cataract in both humans and mice.4
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340 Novel GJA3 Mutation in Congenital Nuclear CataractVYuan et al.
FIGURE 2. Mutation analysis of c.428G>A (p.G143E) mutation in the GJA3 gene. (A) Unaffected member (II:3) of the family. (B) Heterozygous p.G143E mutation patient (II:1). (C, D) Glycine at position 143 is highly conserved in different animal species and human gap junction proteins (>-connexins). (E) The p.G143E is predicted to be probably damaging by PolyPhen-2, with a score of 0.98. A color version of this figure is available online at www.optvissci.com.
FIGURE 3. Cartoon representation of the model structure of the GJA3 protein. (A) Overall structure of the protein is displayed in three dimensions. E1 and E2 stand for two extracellular domains, and M1, M2, M3, and M4 mean four transmembrane domains of the protein. (B, C) Amino acids at position 143, the glycine (B) and the mutated glumatic acid (C), are shown as sticks. A color version of this figure is available online at www.optvissci.com. Optometry and Vision Science, Vol. 92, No. 3, March 2015
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Novel GJA3 Mutation in Congenital Nuclear CataractVYuan et al.
Gja3j/j mice developed nuclear cataract associated with the proteolysis of crystallins,25 and the Gja3 gene maintained cellular differentiation.26 Genetic background influenced the cataract phenotype of Gja3j/j mice, consistent with clinical findings and genetic studies in humans.4,27 The missense p.E42K mutation in the Gja3 gene was found to be responsible for a rat model of autosomal recessive congenital nuclear cataract.28 Knocked-in Gja3 replacing the endogenous wild-type Gja8 gene could suppress nuclear cataract caused by Crygb p.S11R mutation, suggesting that elevated Gja3 can prevent or delay nuclear cataract formation.29 To our knowledge, at least 29 mutations, including nucleotide substitution, deletion, and insertion mutations involving different domains, have been described to contribute to assorted phenotypic cataracts in humans (the Human Gene Mutation Database, http://www.hgmd.cf.ac.uk/ac/).4,20,30Y32 Several mutations (p.L11S, p.V44M, p.D47N, p.P59L, and p.R76H) were reported in at least two families,4 and founder effect or mutation hotspot should be considered. Most of these mutations occur in extracellular loop and transmembrane domains, which are associated with gap junction formation and pore structure/gating, respectively.23,33 Mutations in the GJA3 gene tend to produce phenotypically similar autosomal dominant nuclear and especially zonular pulverulent cataract,2 although there are some interfamilial and intrafamilial differences regarding the appearance and location of opacities within the lens.4,34 Radically different GJA3-related cataract morphologies and severities were reported in different families or even in the same family, which may be explained by the interaction of modifier genes or background environmental factors affecting the expression of the gene and hence resulting in various phenotypes.2,34 In 2011, the GJA3 p.V139M variant, the first report about an amino acid change in the CL domain, was found in a 76-year-old sporadic Chinese patient with cortical cataract, and the variant was predicted to be benign, making it difficult to assess the real role of the p.V139M variant in the development of the disease.35 However, another GJA3 mutation, p.G143R, in the CL domain was identified in a five-generation Chinese pedigree with congenital Coppock-like cataract,23 and the mutation enhanced its interaction with the carboxyl terminus, resulting in reduced gap junction channel function, increased hemichannel function, and cell death, a possible underlying mechanism for cataract formation in a dominant negative manner.33 Intriguingly, a different amino acid substitution, p.G143E, at the same position, potentially changing the normal protein function, was identified in our family with congenital nuclear cataract. The variant cosegregated with patients with congenital nuclear cataract in our family and was unidentified in unaffected family members and in unrelated ethnically matched controls. The c.428G>A transition led to the replacement of a neutral amino acid residue, glycine, by a negatively charged residue, glutamic acid. A multiple sequence alignment of the amino acid sequence of GJA3 showed that glycine at position 143 in the CL domain is phylogenetically conserved in various animal species and also in different human gap junctions, although the CL and carboxylterminal domains are highly variable between gap junction family members.23 The recurring variant at position p.G143 further supported the pathogenicity of this missense variant and the essential
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role of the CL domain in the GJA3 gene function.23,33 PolyPhen-2, SIFT, and MutationTaster analyses predicted the variant to be ‘‘damaging’’ or intolerant, further supporting the variant to be the disease-causing mutation in this family. Interestingly, in our family, the paternal allele carries the cataract-related GJA3 gene mutation, and the maternal allele harbors the COL4A5 gene mutation, c.499delC (p.P167Qfs*36), responsible for X-linked Alport syndrome in the proband.36 Both mutations were found in the proband’s daughter. Alport syndrome is associated with typical ocular abnormalities, including dot-and-fleck retinopathy, anterior lenticonus, and posterior polymorphous corneal dystrophy, and other rare changes, including cataract, can be observed.37 However, we identified that the congenital nuclear cataract in these three patients was caused by the GJA3 p.G143E mutation instead of being a symptom of Alport syndrome. The genetic basis of cataract and glomerulopathy in the proband and her daughter appears to be independent. In summary, our data support that the novel missense mutation, c.428G>A (p.G143E), in the GJA3 gene is probably the genetic cause of congenital nuclear cataract in this family. Our finding extends the spectrum of GJA3 mutations. Our strategy of using exome sequencing for genetic diagnosis is highly efficient and cost-effective for disorders with high genetic and clinical heterogeneity and even in small pedigrees.18,36 Further functional studies in the GJA3 gene mutations may help uncover pathogenic mechanisms of congenital cataract and therefore provide a possible genetic therapy for this disorder in the future.
ACKNOWLEDGMENTS We thank the participating patients and investigators for their cooperation and their efforts in collecting the genetic information and DNA specimens. This work was supported by grants from the National Natural Science Foundation of China (81271921, 81101339, and 81441033); the Sheng Hua Scholars Program of Central South University, China (HD); the Research Fund for the Doctoral Program of Higher Education of China (20110162110026); the Natural Science Foundation of Hunan Province, China (10JJ5029); the Construction Fund for Key Subjects of the Third Xiangya Hospital, Central South University, China (HD); the Hunan Provincial Innovation Foundation for Postgraduate, China (7138000008); the Students Innovative Pilot Scheme of Central South University, China (YC12417); and the Top-notch Innovative Doctoral Scholarship of Central South University, China (2014bjjxj039). Received August 25, 2014; accepted October 16, 2014.
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Hao Deng Center for Experimental Medicine The Third Xiangya Hospital, Central South University Tongzipo Rd 138 Hunan Changsha 410013 P.R. China e-mail: [email protected]
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