A Novel Connexin46 (GJA3) Mutation in Autosomal Dominant Con- Genital Nuclear Pulverulent Cataract

A novel connexin46 (GJA3) mutation in autosomal dominant congenital nuclear pulverulent cataract

Yang Li, Jun Wang, Bing Dong, Hong Man

Beijing Institute of Ophthalmology, Beijing Tongren Hospital, Capital University of Medical Science, Beijing, 100730, China

(The first two authors contributed equally to this work.)

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 this family for sequence variants by direct sequencing of the blindness in children [1,2]. Approximately one third of con- GJA3 gene. A novel missense mutation in GJA3 was detected genital cataracts are hereditary and most often in a in this family. nonsyndromic autosomal dominant fashion [1,2]. Congenital cataract exhibits high clinical and genetic heterogeneity. To METHODS date at least 17 independent loci for autosomal dominant con- Clinical data and sample collection: This study was granted genital cataract (ADCC) have been mapped on human chro- approval from the Beijing Tongren Hospital Joint Committee mosomes 1p36, 1q21-25, 2p12, 2q33-36, 3q21-35, 10q24-25, on Clinical Investigation. A five generation Chinese family 11q22, 12q12, 13q11, 15q21-22, 16q22, 17p12-13, 17q11-12, was referred to the Beijing Tongren Hospital. After informed 17q24, 20p12-q12, 21q22.3, and 22q11-12 and thirteen genes consents were obtained, all participants underwent full oph- have been implicated in human cataractogenesis [3,4]. The thalmologic examination including visual function, slit lamp, genes that have been identified or cloned in ADCC include and fundus examination with the dilated pupil. Blood samples seven crystalline genes (CRYAA at 21q22 [5], CRYAB at 11q22 were obtained by venipuncture, and genomic DNA was ex- [6], CRYBA1 at 17q11 [7,8], CRYBB1 at 22q11 [9], CRYBB2 tracted using the QIAmp Blood kit. at 22q11 [10], CRYGC and CRYGD at 2q33 [11,12]), two tran- Genotyping and linkage analysis: Some of the known scription factors genes (HSF4 at 16q22 [13] and PITX3 at ADCC loci screening was performed using 31 microsatellite 10q24 [14]), one cytoskeletal protein gene (BFSP22 at 3q21- markers from autosomes. The fine mapping primer sequences 22 [15]), and three membrane transport protein genes (MIP at were obtained from the Genome Database. Genotyping and 12q12 [16], GJA8 at 1q21 [17] and GJA3 at 13q11 [18]). The linkage analysis were carried out as described elsewhere ADCC gene on chromosome 13q11 was identified as the [23,24]. Lod scores were calculated for each marker by two Connexin46 (GJA3) gene, that encodes a 435 amino acid pro- point linkage analysis using the linkage package 5.2 [25]. Pedi- tein belonging to the connexin protein family and is predomi- gree and haplotype were constructed using Cyrillic V. 2.0 soft- nantly expressed in the lens [18]. To date there are five muta- ware. tions of GJA3 that have been reported to cause ADCC [18- Mutation analysis: The entire coding region of GJA3 was 22]. In this study, we mapped a Chinese family with congeni- sequenced with five pairs of primers from both directions us- tal nuclear pulverulent cataract to locus 13q11 with polymor- ing ABI3100 sequencer. Four pairs of primers were the same phic markers around the known ADCC loci. Then we screened as was used by Jiang et al. [21] 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 Correspondence to: Yang Li, MD, Beijing institute of Ophthalmol- ogy, Beijing Tongren Hospital, Hougou Lane 17, Chong Nei Street, AG-3') were used. All affected and unaffected members of Beijing, 100730, China; Phone: 8610-65288426; FAX: 8610- this family and 100 unrelated normal controls were examined 65288561 or 65130796; email: [email protected] for GJA3 gene mutations. 668 Molecular Vision 2004; 10:668-71 ©2004 Molecular Vision

RESULTS cDNA, replacing asparagine with threonine at amonino acid Clinical findings: We have identified a large Chinese family 188 (Figure 3A). This missense mutation creates a novel AleI with clear diagnosis of congenital cataract. The inheritance restriction site that segregated with all affected members in pattern in this family appears to be autosomal dominant (Fig- this Chinese family, but was not detected in 100 unrelated ure 1). After careful ophthalmologic examination, 12 individu- normal controls and unaffected pedigree members (Figure 3B). als presented with bilateral congenital cataracts. Among them 10 had had a cataract extraction prior the examination. In two DISCUSSION patients without cataract extraction, a cataract was bilateral We report the identification of a novel mutation (A563C) of and consisted of a central polverulent opacity affecting the GJA3. We provide two lines of evidence that strongly sug- 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. Linkage analysis: 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. [26], so our genetic analysis of this kindred was then shifted to mutation analysis of GJA3. Figure 2. Slit lamp photograph of individual V:1 of the cataractous Mutation analysis: By direct sequencing of GJA3, we Chinese family. A slit lamp photograph showing punctate opacities found a novel base change (A->C) at position 563 of GJA3 located in the central zone (nuclear) of the lens.

Figure 1. Family structure and haplotype analysis of the cataractous Chinese family. Pedigree and haplotype analysis of the large Chinese family with ADCC showed segregation of four microsatellite markers on chromosome 13, listed in descending order from the centromeric end. In the pedigree, square symbols indicate males, circular symbols indicate females, a slash through the symbol indicates the person is deceased, solid symbols indicate affected persons, and open symbols indicate unaffected persons. 669 Molecular Vision 2004; 10:668-71 ©2004 Molecular Vision gests that this mutation is causal. First, this mutation co-seg- 380 (S380fs) [18-22]. The mutation N188T reported here is regates with the phenotype of ADCC in all affected members located in the second extracellular loop (E2) of connexin46 in this kindred, but not with unaffected family members and just next to the P187L substitution reported by Rees et al. [20]. 100 normal controls. Second, this sequence variant replaces Similarly, both the N63S substitution reported by Mackay et asparagine with threonine at amonino acid 188. The connexin al. [18] and the N59L substitute reported by Bennett et al. [22] gene family encodes gap junction proteins that form channels are located in the first extracellular loop (E1) domain. Extra- to allow passage of ions and small biomolecules (<1 kDa) cellular domains of connexins, containing two extracellular including metabolites and second messengers between adja- loops (E1 and E2), play a key role in both mediating cent cells [27,28]. In humans, at least 20 connexin genes have hemichannel docking [27,28] and regulating of voltage gat- been identified and mutations of specific connexin genes have ing of the channel [29]. White et al. [30] has found that the been associated with several disease including genetic deaf- specificity of heterotypic interactions between hemichannels ness, skin disease, peripheral neuropathies, heart defects and composed of different connexins appears to be largely dic- cataracts [19]. The len expresses three distinct connexins Cx43, tated by the primary sequence of the second extracellular loop. Cx46, and Cx50, all of which appear to have different func- Our finding is the second mutation detected in E2 of tions in maintaining lens homeostasis [19]. Both Cx46 and connexin46. These missense mutations, which change the pri- Cx50 knockout mice develop nuclear cataracts, however de- mary sequence of E2, may induce a defect in the E2 second- letion of Cx50 also is associated with a significant ocular ary structure that impairs Cx46 mediated coupling of lens fi- growth defect [19]. In humans, all the families which have ber cells. A mouse model for dominant congenital cataract Cx46 or Cx50 mutations have had the same nuclear pulveru- (No2) also has an E1 missense mutation in the connexin50 lent phenotype [3], consistent with the clinical finding in this gene [3,4]. The E1/E2 mutations detected in human or murine Chinese family. probably share a similar mechanism in compromising In our study, a novel mutation (N188T) was detected in connexion binding, however the precise way in which this GJA3 in a large Chinese family. Sequence comparison of GJA3 kind of mutations of connexins causing lens opacity repre- from various species showed that asparagine is relatively consents the next challenge in understanding the basis of connexin served in Rattus Norvegicus (Norway rat), chicken, and also mediated cataractogenesis. connexin50 in human. To date five mutations of GJA3 have In summary, our study further expanded the mutation spec- been associated with ADCC, including four misense muta- trum of GJA3 and confirmed that GJA3 is important in the tions (F32L, P59L, N63S, and P187L) and one insertion mu- maintenance of optical clarity. tation (1137 insC), which resulted in a frame shift at codon ACKNOWLEDGEMENTS We thank the patients and their families for participation in this study.

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Figure 3. DNA sequence chromatograms and co-segregation analysis of the N188T mutation with the disease phenotype. A: Heterozygote sequence (sense strand) showing an A->C transition (arrow) in codon 188 that changed asparagine (AAC) to threonine (ACC). B: Restriction fragment length analysis showing that a gain of the novel AleI site co-segregated with affected individuals heterozygous for the A->C transition (237, 128, and 109 bp) but not with unaffected individuals and spouses (237 bp only). 670 Molecular Vision 2004; 10:668-71 ©2004 Molecular Vision

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The print version of this article was created on 14 Sep 2004. This reflects all typographical corrections and errata to the article through that date. Details of any changes may be found in the online version of the article. 671