Two novel mutations of connexin genes in Chinese families with autosomal dominant congenital nuclear cataract
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《英国眼科学杂志》
1 Eye Center of Tianjin Medical University, Tianjin, China
2 National Center of Human Genome Research (Beijing), Beijing, China
Keywords: cataract; connexin genes; Chinese families
Congenital or childhood cataract is a clinically and genetically highly heterogeneous lens disorder, with autosomal dominant inheritance being most common. Non-syndromic congenital cataracts have an estimated frequency of 1–6 per 10 000 live births,1 with one third of cases familial. Underlying mutations have identified 14 genes involved in the pathogenesis of isolated inherited cataract, including seven genes coding for crystallins (CRYAA, CRYAB, CRYBA1/A3, CRYBB1, CRYBB2, CRYGC, CRYGD), two for gap junctional channel protein (GJA3 and GJA8), two for lens membrane protein (LIM2 and MIP), one for beaded filament structural protein 2 (BFSP2), and one for glucosaminyl (N-acetyl) transferase 2 (GCNT2), one for heat shock transcription factor (HSF4). Here we report two novel heterozygous mutations in the GJA8 and GJA3 genes, in two Chinese families affected by autosomal dominant congenital nuclear cataracts.
Case report
We studied two Chinese three generation nuclear cataract families with a dominant pattern of inheritance. Clinical information and blood specimens were obtained from 16 members of family A (seven affected and nine unaffected), and 13 members of family B (nine affected and four unaffected). All participants had a full ocular assessment to document the phenotype. The phenotype of two families was characterised by bilateral nuclear cataract that was present at birth or developed during infancy. There was no evidence of other systemic or ocular defects.
After obtaining informed consent, we used a panel of 46 microsatellite markers to study 13 loci for known candidate genes of autosomal dominant congenital cataract susceptibility. The markers’ order and position were obtained from the Marshfield Genetic Database (www.marshfield.org/genetics/maps). Genotyping and data collection were conducted by ABI Prism GeneMapper v 3.0 software. We carried out two point linkage analysis using the MLINK program from the Linkage v.5.10 software package. It suggested positive linkage on chromosome 1q21.1 (lod score was 2.44 for marker D1S1167) in family A and chromosome 13q11–12 (lod score was 1.63 for marker D13S1326) in family B (tables 1 and 2).
Table 1 Two point LOD scores for linkage between the cataract locus and 1q markers in family A
Table 2 Two point LOD scores for linkage between the cataract locus and 13q markers in family B
There are two strong candidate genes in these regions, GJA8 encoding connexin 50 (Cx50) and GJA3 encoding connexin 46 (Cx46). We screened the mutation of candidate genes by bidirectional sequencing polymerase chain reaction products (300–700 bp). Sequence analysis of the entire coding region and immediate flanking regions detected a heterozygous 191 T G (AF217524) transition in exon 2 of GJA8, resulting in a Val Gly substitution at codon 64 (fig 1B). Sequence analysis of GJA3 detected a heterozygous 134 G C (AF075290) transition, resulting in a Trp(TGG) Ser (TCG) substitution at codon 45 (fig 2B). We examined all unaffected members of two families and 200 unrelated normal controls for GJA3 and GJA8 gene mutations but failed to detect these sequence variations.
Figure 1 (A) Pedigree and haplotype analysis of family A showing segregating nine microsatellite markers on chromosome 1, listed in descending order from the centromere. Squares and circles symbolise males and females, respectively. Solid and open symbols denote affected and unaffected individuals, respectively. IV:2 is the proband. (B) Sequence chromatograms showing the heterozygous 191 T G transition that converts a Val residue (GTC) to a Gly residue (GGC) at codon 64. (C) Sequence chromatograms of wild type allele. (D) Schematic diagram of the predicted Cx50 polypeptide and location of V64G and known mutations. M1–M4, transmembrane domains 1–4; E1 and E2, extracellular domains 1 and 2, respectively. (E) Cx50 multiple protein sequence alignment in different specices. Reference sequence numbers of protein are human (NP_005258), mouse (NP_032149), and chicken (NP_990328). The arrow directed the mutant amino acid residue.
Figure 2 (A) Pedigree and haplotype analysis of family B showing segregation of four microsatellite markers on chromosome 13q. Squares and circles symbolise males and females, respectively. Solid and open symbols denote affected and unaffected individuals, respectively. IV:5 is the proband. (B) Sequence chromatograms showing the heterozygous 134 G C transition resulting in a Trp(TGG) Ser (TCG) substitution at codon 45. (C) Sequence chromatograms of wild type allele. (D) Exon organisation and mutation profile of GJA3. Cx46 has nine structural domains including a cytoplasmic amino-terminus (NT), four transmembrane domains (M1–M4), two extracellular loops (E1–E2), a cytoplasmic loop (CL), and a cytoplasmic carboxy-terminus (CT). The relative locations of the W45S mutation and other mutations associated with dominant cataracts in humans are indicated. (E) Cx50 multiple protein sequence alignment in different species. Reference sequence numbers of protein are human (NP_068773), mouse (NP_058671), rat (Rattus norvegicus) (NP_077352), and zebrafish (Donio rerio) (NP_997525). The arrow directed the mutant amino acid residue.
Comment
Three connexins are expressed in the lens: connexin 43, connexin 46, and connexin 50. Gap junction intercellular communication is an essential part of the cell–cell communication system, which facilitates the exchange of ions, metabolites, signalling molecules, and other molecules with a molecular weight up to 1 kDa.2
Each gap junction channel is composed of two hemi-channels, or connexons, which dock in the extracellular space between adjacent cells, and each connexon comprised six integral transmembrane protein subunits known as connexins. All connexins have four transmembrane domains and two extracellular loops with cytoplasmic N and C termini.
To date, four heterozygous missense Cx50 mutations (P88S, E48K, R23T, and I247M) have been described, causing a nuclear or zonular nuclear pulverulent cataract.3–6 Six mutations of Cx46 have been associated with ADCC, including five missense mutations (F32L, P59L, N63S, P187L, and N188T) and one insertion mutation (1137 insC), which resulted in a frame shift at codon 380 (S380fs).7,8,9,10,11,12
Currently, two mutations occurred: Cx50 (G22R and D47A) results in cataracts in the mouse,13,14 but no dominant spontaneous or mutagen induced cataracts have been associated with the murine gene for GJA3 (Gja3).
V64G and W45S substitutions in two Chinese families occurred within evolutionarily conserved residues across species for Cx50 and Cx46 (figs 1E and 2E). These two mutant amino acid residue locate at the phylogenetically conserved extracellular loop 1 (E1). The two extracellular loops mediate docking between connexons and the E1 loop has also been shown to be important for determinant of the transjunctional voltage required for closure of gap junction pores.15 The mutant proteins may disrupt normal interactions between the two connexons, which may reduce resistance of the intercellular channel to the leakage of small ions.
In conclusion, two novel heterozygous mutations, V64G in Cx50 and W45S in Cx46, were identified in two Chinese families. These further expand the genetic and phenotypic heterogeneity of cataract.
References
Lambert SL, Drack AV. Infantile cataracts. Surv Ophthalmol 1996;40:427–58.
White TW. Unique and redundant connexin contributions to lens development. Science 2002;295:319–20.
Shiels A, Mackay D, Ionides A, et al. A missense mutation in the human connexin 50 gene (GJA8) underlies autosomal dominant "zonular pulverulent" cataract on chromosome 1q. Am J Hum Genet 1998;62:526–32.
Berry V, Mackay D, Khaliq S, et al. Connexin 50 mutation in a family with congenital "zonular nuclear" pulverulent cataract of Pakistani origin. Hum Genet 1999;105:168–70.
Polyakov AV, Shagina IA, Khlebnikova OV, et al. Mutation in the connexin 50 gene (GJA8) in a Russian family with zonular pulverulent cataract. Clin Genet 2001;60:476–8.
lloughby CE, Arab S, Gandhi R, et al. A novel GJA8 mutation in an Iranian family with progressive autosomal dominant congenital nuclear cataract. J Med Genet 2003;40:e124.
Mackay D, Ionides A, Kibar Z, et al. Connexin46 mutations in autosomal dominant congenital cataract. Am J Hum Genet 1999;64:1357–64.
Gerido DA, White TW. Connexin disorders of the ear, skin, and lens. Biochim Biophys Acta 2004;1662:159–70.
Rees MI, Watts P, Fenton I, et al. Further evidence of autosomal dominant congenital zonular pulverulent cataracts linked to 13q11 (CZP3) and a novel mutation in connexin 46 (GJA3). Hum Genet 2000;106:206–9.
Jiang H, Jin Y, Bu L, et al. A novel mutation in GJA3 (connexin46) for autosomal dominant congenital nuclear pulverulent cataract. Mol Vis 2003;9:579–83.
Bennett TM, Mackay DS, Knopf HL, et al. A novel missense mutation in the gene for gap-junction protein alpha3 (GJA3) associated with autosomal dominant "nuclear punctate" cataracts linked to chromosome 13q. Mol Vis 2004;10:376–82.
Li Y, Wang J, Dong B, et al. A novel connexin46 (GJA3) mutation in autosomal dominant congenital nuclear pulverulent cataract. Mol Vis 2004;10:668–71.
Tusnady GE, Simon I. The HMMTOP transmembrane topology prediction server. Bioinformatics 2001;17:849–50.
Rong P, Wang X, Niesman I, et al. Disruption of Gja8 (a8 connexin) in mice leads to microphthalmia associated with retardation of lens growth and lens fiber maturation. Development 2002;129:167–74.
Rubin JB, Verselis V, Bennett MVL, et al. domain substitution procedure and its use to analyze voltage dependence of homotypic gap junctions formed by connexins 26 and 32. Proc Natl Acad Sci USA 1992;89:3820–4.(Z Ma1,2, J Zheng1, F Yang)
2 National Center of Human Genome Research (Beijing), Beijing, China
Keywords: cataract; connexin genes; Chinese families
Congenital or childhood cataract is a clinically and genetically highly heterogeneous lens disorder, with autosomal dominant inheritance being most common. Non-syndromic congenital cataracts have an estimated frequency of 1–6 per 10 000 live births,1 with one third of cases familial. Underlying mutations have identified 14 genes involved in the pathogenesis of isolated inherited cataract, including seven genes coding for crystallins (CRYAA, CRYAB, CRYBA1/A3, CRYBB1, CRYBB2, CRYGC, CRYGD), two for gap junctional channel protein (GJA3 and GJA8), two for lens membrane protein (LIM2 and MIP), one for beaded filament structural protein 2 (BFSP2), and one for glucosaminyl (N-acetyl) transferase 2 (GCNT2), one for heat shock transcription factor (HSF4). Here we report two novel heterozygous mutations in the GJA8 and GJA3 genes, in two Chinese families affected by autosomal dominant congenital nuclear cataracts.
Case report
We studied two Chinese three generation nuclear cataract families with a dominant pattern of inheritance. Clinical information and blood specimens were obtained from 16 members of family A (seven affected and nine unaffected), and 13 members of family B (nine affected and four unaffected). All participants had a full ocular assessment to document the phenotype. The phenotype of two families was characterised by bilateral nuclear cataract that was present at birth or developed during infancy. There was no evidence of other systemic or ocular defects.
After obtaining informed consent, we used a panel of 46 microsatellite markers to study 13 loci for known candidate genes of autosomal dominant congenital cataract susceptibility. The markers’ order and position were obtained from the Marshfield Genetic Database (www.marshfield.org/genetics/maps). Genotyping and data collection were conducted by ABI Prism GeneMapper v 3.0 software. We carried out two point linkage analysis using the MLINK program from the Linkage v.5.10 software package. It suggested positive linkage on chromosome 1q21.1 (lod score was 2.44 for marker D1S1167) in family A and chromosome 13q11–12 (lod score was 1.63 for marker D13S1326) in family B (tables 1 and 2).
Table 1 Two point LOD scores for linkage between the cataract locus and 1q markers in family A
Table 2 Two point LOD scores for linkage between the cataract locus and 13q markers in family B
There are two strong candidate genes in these regions, GJA8 encoding connexin 50 (Cx50) and GJA3 encoding connexin 46 (Cx46). We screened the mutation of candidate genes by bidirectional sequencing polymerase chain reaction products (300–700 bp). Sequence analysis of the entire coding region and immediate flanking regions detected a heterozygous 191 T G (AF217524) transition in exon 2 of GJA8, resulting in a Val Gly substitution at codon 64 (fig 1B). Sequence analysis of GJA3 detected a heterozygous 134 G C (AF075290) transition, resulting in a Trp(TGG) Ser (TCG) substitution at codon 45 (fig 2B). We examined all unaffected members of two families and 200 unrelated normal controls for GJA3 and GJA8 gene mutations but failed to detect these sequence variations.
Figure 1 (A) Pedigree and haplotype analysis of family A showing segregating nine microsatellite markers on chromosome 1, listed in descending order from the centromere. Squares and circles symbolise males and females, respectively. Solid and open symbols denote affected and unaffected individuals, respectively. IV:2 is the proband. (B) Sequence chromatograms showing the heterozygous 191 T G transition that converts a Val residue (GTC) to a Gly residue (GGC) at codon 64. (C) Sequence chromatograms of wild type allele. (D) Schematic diagram of the predicted Cx50 polypeptide and location of V64G and known mutations. M1–M4, transmembrane domains 1–4; E1 and E2, extracellular domains 1 and 2, respectively. (E) Cx50 multiple protein sequence alignment in different specices. Reference sequence numbers of protein are human (NP_005258), mouse (NP_032149), and chicken (NP_990328). The arrow directed the mutant amino acid residue.
Figure 2 (A) Pedigree and haplotype analysis of family B showing segregation of four microsatellite markers on chromosome 13q. Squares and circles symbolise males and females, respectively. Solid and open symbols denote affected and unaffected individuals, respectively. IV:5 is the proband. (B) Sequence chromatograms showing the heterozygous 134 G C transition resulting in a Trp(TGG) Ser (TCG) substitution at codon 45. (C) Sequence chromatograms of wild type allele. (D) Exon organisation and mutation profile of GJA3. Cx46 has nine structural domains including a cytoplasmic amino-terminus (NT), four transmembrane domains (M1–M4), two extracellular loops (E1–E2), a cytoplasmic loop (CL), and a cytoplasmic carboxy-terminus (CT). The relative locations of the W45S mutation and other mutations associated with dominant cataracts in humans are indicated. (E) Cx50 multiple protein sequence alignment in different species. Reference sequence numbers of protein are human (NP_068773), mouse (NP_058671), rat (Rattus norvegicus) (NP_077352), and zebrafish (Donio rerio) (NP_997525). The arrow directed the mutant amino acid residue.
Comment
Three connexins are expressed in the lens: connexin 43, connexin 46, and connexin 50. Gap junction intercellular communication is an essential part of the cell–cell communication system, which facilitates the exchange of ions, metabolites, signalling molecules, and other molecules with a molecular weight up to 1 kDa.2
Each gap junction channel is composed of two hemi-channels, or connexons, which dock in the extracellular space between adjacent cells, and each connexon comprised six integral transmembrane protein subunits known as connexins. All connexins have four transmembrane domains and two extracellular loops with cytoplasmic N and C termini.
To date, four heterozygous missense Cx50 mutations (P88S, E48K, R23T, and I247M) have been described, causing a nuclear or zonular nuclear pulverulent cataract.3–6 Six mutations of Cx46 have been associated with ADCC, including five missense mutations (F32L, P59L, N63S, P187L, and N188T) and one insertion mutation (1137 insC), which resulted in a frame shift at codon 380 (S380fs).7,8,9,10,11,12
Currently, two mutations occurred: Cx50 (G22R and D47A) results in cataracts in the mouse,13,14 but no dominant spontaneous or mutagen induced cataracts have been associated with the murine gene for GJA3 (Gja3).
V64G and W45S substitutions in two Chinese families occurred within evolutionarily conserved residues across species for Cx50 and Cx46 (figs 1E and 2E). These two mutant amino acid residue locate at the phylogenetically conserved extracellular loop 1 (E1). The two extracellular loops mediate docking between connexons and the E1 loop has also been shown to be important for determinant of the transjunctional voltage required for closure of gap junction pores.15 The mutant proteins may disrupt normal interactions between the two connexons, which may reduce resistance of the intercellular channel to the leakage of small ions.
In conclusion, two novel heterozygous mutations, V64G in Cx50 and W45S in Cx46, were identified in two Chinese families. These further expand the genetic and phenotypic heterogeneity of cataract.
References
Lambert SL, Drack AV. Infantile cataracts. Surv Ophthalmol 1996;40:427–58.
White TW. Unique and redundant connexin contributions to lens development. Science 2002;295:319–20.
Shiels A, Mackay D, Ionides A, et al. A missense mutation in the human connexin 50 gene (GJA8) underlies autosomal dominant "zonular pulverulent" cataract on chromosome 1q. Am J Hum Genet 1998;62:526–32.
Berry V, Mackay D, Khaliq S, et al. Connexin 50 mutation in a family with congenital "zonular nuclear" pulverulent cataract of Pakistani origin. Hum Genet 1999;105:168–70.
Polyakov AV, Shagina IA, Khlebnikova OV, et al. Mutation in the connexin 50 gene (GJA8) in a Russian family with zonular pulverulent cataract. Clin Genet 2001;60:476–8.
lloughby CE, Arab S, Gandhi R, et al. A novel GJA8 mutation in an Iranian family with progressive autosomal dominant congenital nuclear cataract. J Med Genet 2003;40:e124.
Mackay D, Ionides A, Kibar Z, et al. Connexin46 mutations in autosomal dominant congenital cataract. Am J Hum Genet 1999;64:1357–64.
Gerido DA, White TW. Connexin disorders of the ear, skin, and lens. Biochim Biophys Acta 2004;1662:159–70.
Rees MI, Watts P, Fenton I, et al. Further evidence of autosomal dominant congenital zonular pulverulent cataracts linked to 13q11 (CZP3) and a novel mutation in connexin 46 (GJA3). Hum Genet 2000;106:206–9.
Jiang H, Jin Y, Bu L, et al. A novel mutation in GJA3 (connexin46) for autosomal dominant congenital nuclear pulverulent cataract. Mol Vis 2003;9:579–83.
Bennett TM, Mackay DS, Knopf HL, et al. A novel missense mutation in the gene for gap-junction protein alpha3 (GJA3) associated with autosomal dominant "nuclear punctate" cataracts linked to chromosome 13q. Mol Vis 2004;10:376–82.
Li Y, Wang J, Dong B, et al. A novel connexin46 (GJA3) mutation in autosomal dominant congenital nuclear pulverulent cataract. Mol Vis 2004;10:668–71.
Tusnady GE, Simon I. The HMMTOP transmembrane topology prediction server. Bioinformatics 2001;17:849–50.
Rong P, Wang X, Niesman I, et al. Disruption of Gja8 (a8 connexin) in mice leads to microphthalmia associated with retardation of lens growth and lens fiber maturation. Development 2002;129:167–74.
Rubin JB, Verselis V, Bennett MVL, et al. domain substitution procedure and its use to analyze voltage dependence of homotypic gap junctions formed by connexins 26 and 32. Proc Natl Acad Sci USA 1992;89:3820–4.(Z Ma1,2, J Zheng1, F Yang)