|Molecular Vision 2004;
Received 5 July 2004 | Accepted 14 September 2004 | Published 14 September 2004
A novel connexin46 (GJA3) mutation in autosomal dominant congenital nuclear pulverulent cataract
Bing Dong, Hong Man
Beijing Institute of Ophthalmology, Beijing Tongren
Hospital, Capital University of Medical Science, Beijing, 100730,
(The first two authors contributed equally to this work.)
Correspondence to: Yang Li, MD, Beijing institute of Ophthalmology, Beijing Tongren Hospital, Hougou Lane 17, Chong Nei Street, Beijing, 100730, China; Phone: 8610-65288426; FAX: 8610-65288561 or 65130796; email: email@example.com
Purpose: To report the identification of a novel mutation of connexin46 in a large Chinese family with autosomal dominant congenital nuclear pulverulent cataract.
Methods: Genetic linkage analysis was performed on the known genetic loci for autosomal dominant congenital nuclear pulverulent cataract with a panel of polymorphic markers and mutations were screened by direct sequencing.
Results: Significant two point lod score was generated at marker D13S175 (Zmax=3.61, θ=0), further linkage and haplotype studies confined the disease locus to 13q11-13. Mutation screening of connexin46 in this family revealed an A->C transition at position 563 (N188T) of the cDNA sequence, creating a novel AleI restriction site that co-segregated with affected members of the pedigree, but was not present in unaffected relatives or 100 normal individuals.
Conclusions: Our finding expands the spectrum of connexin46 mutations causing autosomal dominant congenital nuclear pulverulent cataract, and confirms the role of connexin46 in the pathogenesis of autosomal dominant congenital nuclear pulverulent cataract.
Congenital or infantile cataract is an important cause of blindness in children [1,2]. Approximately one third of congenital cataracts are hereditary and most often in a nonsyndromic autosomal dominant fashion [1,2]. Congenital cataract exhibits high clinical and genetic heterogeneity. To date at least 17 independent loci for autosomal dominant congenital cataract (ADCC) have been mapped on human chromosomes 1p36, 1q21-25, 2p12, 2q33-36, 3q21-35, 10q24-25, 11q22, 12q12, 13q11, 15q21-22, 16q22, 17p12-13, 17q11-12, 17q24, 20p12-q12, 21q22.3, and 22q11-12 and thirteen genes have been implicated in human cataractogenesis [3,4]. The genes that have been identified or cloned in ADCC include seven crystalline genes (CRYAA at 21q22 , CRYAB at 11q22 , CRYBA1 at 17q11 [7,8], CRYBB1 at 22q11 , CRYBB2 at 22q11 , CRYGC and CRYGD at 2q33 [11,12]), two transcription factors genes (HSF4 at 16q22  and PITX3 at 10q24 ), one cytoskeletal protein gene (BFSP22 at 3q21-22 ), and three membrane transport protein genes (MIP at 12q12 , GJA8 at 1q21  and GJA3 at 13q11 ). The ADCC gene on chromosome 13q11 was identified as the Connexin46 (GJA3) gene, that encodes a 435 amino acid protein belonging to the connexin protein family and is predominantly expressed in the lens . To date there are five mutations of GJA3 that have been reported to cause ADCC [18-22]. In this study, we mapped a Chinese family with congenital nuclear pulverulent cataract to locus 13q11 with polymorphic markers around the known ADCC loci. Then we screened this family for sequence variants by direct sequencing of the GJA3 gene. A novel missense mutation in GJA3 was detected in this family.
Clinical data and sample collection
This study was granted approval from the Beijing Tongren Hospital Joint Committee on Clinical Investigation. A five generation Chinese family was referred to the Beijing Tongren Hospital. After informed consents were obtained, all participants underwent full ophthalmologic examination including visual function, slit lamp, and fundus examination with the dilated pupil. Blood samples were obtained by venipuncture, and genomic DNA was extracted using the QIAmp Blood kit.
Genotyping and linkage analysis
Some of the known ADCC loci screening was performed using 31 microsatellite markers from autosomes. The fine mapping primer sequences were obtained from the Genome Database. Genotyping and linkage analysis were carried out as described elsewhere [23,24]. Lod scores were calculated for each marker by two point linkage analysis using the linkage package 5.2 . Pedigree and haplotype were constructed using Cyrillic V. 2.0 software.
The entire coding region of GJA3 was sequenced with five pairs of primers from both directions using ABI3100 sequencer. Four pairs of primers were the same as was used by Jiang et al.  except for the middle coding region, where primers 455F (5'-ATC ATC TTC AAG ACG CTG TTC G-3') and 697R (5'-CCT GCT TGA GCT TCT TCC AG-3') were used. All affected and unaffected members of this family and 100 unrelated normal controls were examined for GJA3 gene mutations.
We have identified a large Chinese family with clear diagnosis of congenital cataract. The inheritance pattern in this family appears to be autosomal dominant (Figure 1). After careful ophthalmologic examination, 12 individuals presented with bilateral congenital cataracts. Among them 10 had had a cataract extraction prior the examination. In two patients without cataract extraction, a cataract was bilateral and consisted of a central polverulent opacity affecting the embryonal, fetal, and infantile nucleus of the lens (Figure 2). From the hospital records, bilateral cataract was present at birth or developed during infancy, the best corrected visual acuity of the affected eye varied from 0.1 to 0.5 before cataract extraction.
Since many genetic loci have been identified in ADCC, our initial genetic study of this family was focused on linkage analysis with markers linked to some of the known genetic loci for ADCC (Table 1). Two point lod scores varied from negative to 0.000018 with all markers tested except D13S175, which yielded a positive lod score of 3.61. Two point linkage and haplotype analysis of additional 13q markers confined the minimal disease haplotype within 13q11-q13 interval between D13S1316 (Zmax of 0.22), D13S175 (Zmax of 3.61), D13S292 (Zmax of 3.73), and D13S1243 (Zmax of 4.26, Figure 1). This result was consistent with the findings of Mackay et al. , so our genetic analysis of this kindred was then shifted to mutation analysis of GJA3.
By direct sequencing of GJA3, we found a novel base change (A->C) at position 563 of GJA3 cDNA, replacing asparagine with threonine at amonino acid 188 (Figure 3A). This missense mutation creates a novel AleI restriction site that segregated with all affected members in this Chinese family, but was not detected in 100 unrelated normal controls and unaffected pedigree members (Figure 3B).
We report the identification of a novel mutation (A563C) of GJA3. We provide two lines of evidence that strongly suggests that this mutation is causal. First, this mutation co-segregates with the phenotype of ADCC in all affected members in this kindred, but not with unaffected family members and 100 normal controls. Second, this sequence variant replaces asparagine with threonine at amonino acid 188. The connexin gene family encodes gap junction proteins that form channels to allow passage of ions and small biomolecules (<1 kDa) including metabolites and second messengers between adjacent cells [27,28]. In humans, at least 20 connexin genes have been identified and mutations of specific connexin genes have been associated with several disease including genetic deafness, skin disease, peripheral neuropathies, heart defects and cataracts . The len expresses three distinct connexins Cx43, Cx46, and Cx50, all of which appear to have different functions in maintaining lens homeostasis . Both Cx46 and Cx50 knockout mice develop nuclear cataracts, however deletion of Cx50 also is associated with a significant ocular growth defect . In humans, all the families which have Cx46 or Cx50 mutations have had the same nuclear pulverulent phenotype , consistent with the clinical finding in this Chinese family.
In our study, a novel mutation (N188T) was detected in GJA3 in a large Chinese family. Sequence comparison of GJA3 from various species showed that asparagine is relatively conserved in Rattus Norvegicus (Norway rat), chicken, and also connexin50 in human. To date five mutations of GJA3 have been associated with ADCC, including four misense mutations (F32L, P59L, N63S, and P187L) and one insertion mutation (1137 insC), which resulted in a frame shift at codon 380 (S380fs) [18-22]. The mutation N188T reported here is located in the second extracellular loop (E2) of connexin46 just next to the P187L substitution reported by Rees et al. . Similarly, both the N63S substitution reported by Mackay et al.  and the N59L substitute reported by Bennett et al.  are located in the first extracellular loop (E1) domain. Extracellular domains of connexins, containing two extracellular loops (E1 and E2), play a key role in both mediating hemichannel docking [27,28] and regulating of voltage gating of the channel . White et al.  has found that the specificity of heterotypic interactions between hemichannels composed of different connexins appears to be largely dictated by the primary sequence of the second extracellular loop. Our finding is the second mutation detected in E2 of connexin46. These missense mutations, which change the primary sequence of E2, may induce a defect in the E2 secondary structure that impairs Cx46 mediated coupling of lens fiber cells. A mouse model for dominant congenital cataract (No2) also has an E1 missense mutation in the connexin50 gene [3,4]. The E1/E2 mutations detected in human or murine probably share a similar mechanism in compromising connexion binding, however the precise way in which this kind of mutations of connexins causing lens opacity represents the next challenge in understanding the basis of connexin mediated cataractogenesis.
In summary, our study further expanded the mutation spectrum of GJA3 and confirmed that GJA3 is important in the maintenance of optical clarity.
We thank the patients and their families for participation in this study.
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