Molecular Vision 2011; 17:1343-1349
Received 1 March 2011 | Accepted 13 May 2011 | Published 20 May 2011
The first two authors contributed equally to this work
1Department of Ophthalmology, The Second Affiliated Hospital of Harbin Medical University, Harbin, China; 2Department of Genetics, National Research Institute for Family Planning, Beijing, China
Correspondence to: Dr. Yanhua Qi, 1Department of Ophthalmology, The Second Affiliated Hospital of Harbin Medical University, Harbin, China; Phone: 86-451-86605851; FAX: 86-451-86605116; email: email@example.com
Purpose: To identify the potential pathogenic mutation in a three-generation Chinese family with congenital nuclear pulverulent cataracts.
Methods: A three-generation pedigree was recruited for our study. Three patients and four healthy members of the family underwent a comprehensive clinical examination. Genomic DNA extracted from peripheral blood was amplified using the polymerase chain reaction (PCR) method and the exons of all candidate genes were sequenced.
Results: When sequencing the encoding regions of the candidate genes, a novel mutation (c.559C>T) was identified in the gap junction protein alpha 3 (GJA3) gene, which resulted in the substitution of highly conserved proline by serine at codon 187 (P187S). There was no noticeable nucleotide polymorphism in other candidate genes. The mutation co-segregated with all patients, but was absent in the healthy members and 100 normal individuals.
Conclusions: The present study identified a novel mutation (c.559C>T) in the GJA3 gene associated with autosomal dominant pulverulent cataracts in a Chinese family. As the first report to relate p.P187S mutation in GJA3, it expands the mutation spectrum of GJA3 in association with congenital cataracts.
Cataracts are the most common cause of visual impairment in the world. They are especially evident in blindness in infants, as it is reported that approximately 10% of all childhood blindness worldwide is due to congenital cataracts . Congenital cataracts have been reported primarily as an autosomal dominant disorder and rarely as an autosomal recessive or X-linked pattern. To date, the genes that have been identified in congenital cataracts include ten crystalline genes (αA-crystallin [CRYAA] , αB-crystallin [CRYAB] , βA1-crystallin [CRYBA1] , βA4-crystallin [CRYBA4] , βB1-crystallin [CRYBB1] , βB2-crystallin [CRYBB2] , βB3-crystallin [CRYBB3] , γC-crystallin [CRYGC] , γD-crystallin [CRYGD] , and γS-crystallin [CRYGS] ), two cytoskeletal protein genes (beaded filament structural protein 2, phakinin [BFSP2] , beaded filament structural protein 1, filensin [BFSP1] ), three transcription factors genes (heat shock transcription factor 4 [HSF4] , Maf-like protein [MAF]  and paired-like homeodomain 3 [PITX3] ), Glucosaminyl (N-acetyl) transferase 2 (GCNT2) , chromatin-modifying protein-4B (CHMP4B) , transmembrane protein 114 (TMEM114) , and four membrane transport protein genes (major intrinsic protein of lens fiber [MIP] , lens intrinsic membrane protein 2 gene [LIM2] , gap junction protein [alpha 8, GJA8]  and gap junction protein [alpha 3, GJA3] ).
To date, several mutations in genes have been reported as being associated with congenital nuclear pulverulent cataracts such as MIP ,CRYBB1 , CRYBA1 , GJA8 , and GJA3 . These mutations had all been identified as a pathogenic mutation. The gap junction protein alpha 3 (GJA3) gene consists of a single exon encoding a 435 amino acid protein in humans and is predominantly expressed in lens fiber cells. In the human eyes, three members of the connexin family, including connexin 43 (Cx43), connexin 46 (Cx46), and connexin 50 (Cx50) are expressed. Connexins are the critical function groups on the gap junction channels that play an irreplaceable role in the transportation of metabolites between cells , such as ions, metabolites, signaling molecules, and other molecules with a molecular weight up to 1 kDa.
In this study, we detected a novel mutation (P187S) in GJA3 associated with autosomal dominant nuclear pulverulent cataracts in a Chinese family. The mutation was co-segregated with all patients, but was absent in the healthy members and 100 normal individuals. This mutation has not been reported previously in terms of congenital cataracts, thus knowledge of the mutation will help in understanding the mechanisms of cataractogenesis.
A three-generation Chinese Han family (Figure 1) with autosomal dominant pulverulent cataracts from Heilongjiang province was identified. Seven members of the pedigree were involved in this study. The research was approved by the Institutional Review Board of Harbin Medical University. The affected and unaffected individuals in this family were accepted full ophthalmic and clinical examinations. In addition, 100 healthy normal were recruited as the normal controls. Photographs of significant findings were taken. After informed consent, venous blood (5 ml) was collected for genomic DNA extraction. Briefly, 5 ml of venous blood from family members and controls was collected in a BD Vacutainer (BD, San Jose, CA) containing EDTA. Genomic DNA was extracted by QIAamp DNA Blood Mini Kits (QIAGEN Science, Germantown, MD).
The coding exons and the flanking regions of all the known candidate genes associated with autosomal dominant congenital nuclear pulverulent cataracts, such as CRYAA, CRYAB, CRYBA1, CRYBB2, CRYGC, CRYGD, CRYGS, GJA3, and GJA8, were amplified by PCR with primers listed in Table 1. Through sequencing both directions with an ABI 3130XL Genetic Analyzer (Applied Biosystems, Foster City, CA), the results were analyzed using Chromas (version 1.0) software and compared with the reference sequences in the NCBI gene bank.
The wild-type and mutant Cx46 protein sequences were analyzed with PolyPhen to predict whether the amino acid substitution affects the structure and function of proteins.
There were four affected individuals in this Chinese family, and three of them (II:2, II:4, and III:2) participated in this study (Figure 1). A cataract characterized as a central nuclear opacity with punctate opacities (Figure 2) was exhibited fully in the affected patients. There were no other ocular or systemic abnormalities. To date, all of the affected individuals have had cataract surgery.
The candidate genes were sequenced in three affected individuals and four healthy members. A heterozygous missense mutation, C>T at position 559 in exon 2 of GJA3, was identified (Figure 3). The mutation led to the substitution of proline by serine, which is highly conserved across various species, as determined from NCBI (Figure 4). There was no noticeable nucleotide polymorphism in the other candidate genes. This change was co-segregated with the affected individuals of the family, but was not detected in either the unaffected family individuals or the 100 normal controls.
PolyPhen analysis demonstrated that the substitution in GJA3 at position 187 from P to S scored 1.753, which meant that “this variant is predicted to be possibly damaging.”
The GJA3 gene, which encodes a 435-amino acid protein, is located on chromosome 13q11 and was first reported by Willecke et al. , in 1990. Our study identifies a new mutation (c.559C>T) in GJA3. This substitution resulted in the replacement of proline by serine at amino acid 187 (P187S). The variant is likely to be disease causative, as it co-segregates with the phenotype of ADCC in all affected individuals in this family, but not with the unaffected individuals and 100 normal controls. As an avascular organ, the eye lens has developed an extensive cell to cell communication system via gap junction channels belonging to connexins that allow the transport of small metabolites between cells. In humans, more than 20 genes that code connexins have been identified. Three of these (connexin 43, connexin 46 and connexin 50) are expressed in the lens. Cx43 is expressed mainly in the lens epithelial cells; Cx46 and Cx50 are expressed in lens fiber cells. Both Cx46 and Cx50 knockout mice develop nuclear cataracts, while deletion of Cx50 also is associated with a significant ocular growth defect . Mutations in either connexin 46 or connexin 50 have been linked with congenital cataracts in humans [32,33].
Like other connexins, GJA3 has four transmembrane domains (M1, M2, M3, and M4), two extracellular loops (E1 and E2), an intracellular loop (CL), and intracellular NH2 and COOH termini. Our finding is the third mutation detected in E2 of connexin 46 [34,35]. Sequence comparison of connexin 46 showed that the proline at position 187 is phylogenetically conserved in the second transmembrane domain in different species (Figure 4). Extracellular domains of connexins (E1 and E2) play an important part in both mediating hemichannel docking and regulating voltage gating of the channel [36-38]. They provide the strong interaction between the two hemichannels, ensure the formation of an intercellular channel with no leakage of current and molecules to the extracellular environment . The process of the formation of intercellular channels is dependent on the connexins expressed, and the second extracellular domain plays a decisive heterotypic compatibility between connexins . In our study, Polyphen data shows that the mutation in GJA3 is likely to be damaged. Although the activity of the P187S mutation identified in our study on the connexin 46 has not been immediately certificated, we presume that this mutation would result in an incorrect association of connexins and thus change the function of endogenous wild-type connexins in the affected individuals. Similarly, the mutation (P187L) in connexin 46 reported by Rees et al. , in 2000, which also localizes to the extracellular domain (E2) of the connexin 46 protein, strongly indicates an association of C560T with congenital zonular pulverulent cataracts.
The cataract phenotypes of GJA3 mutations share genotype-phenotype similarities to some extent. Most of these were described as nuclear or zonular pulverulent types. Thus far, 16 mutations in GJA3 have been reported to be associated with congenital cataracts in humans (Table 2). The mutation (P187L) in GJA3 described a Caucasian family with congenital cataracts, presenting with zonular pulverulent cataracts phenotype . In our study, nuclear pulverulent cataracts (Figure 2) were fully exhibited in all the affected individuals in this Chinese family.
In conclusion, we identified a novel mutation (P187S) in GJA3 associated with autosomal dominant nuclear pulverulent cataracts in a Chinese family. As the first report to relate the P187S mutation in GJA3, it expands the mutation spectrum of GJA3 in terms of congenital cataracts.
The authors are grateful to all patients, the family, and normal volunteers for their participation in this research. This study was supported by the National Science & Technology Pillar Program of China (No.2008BAH24B05), the Science and Technology Department of Heilongjiang Province Projects of International Cooperation (WB09C104) and the Natural Science Foundation of China (Grant No. 30801274). Professors Xu Ma (firstname.lastname@example.org) and Yanhua Qi contributed equally to the research project and can be considered co-corresponding authors.