PMC

Linkage analysis of autosomal dominant cataract segregating in a 5-generation Caucasian American pedigree (family Sh). A: Pedigree and haplotype analysis showing segregation of 3 microsatellite markers on chromosome 13q listed in descending order from the centromere (13p-tel). Squares and circles denote males and females respectively. Filled symbols denote affected status. B: Ideogram of chromosome 13 showing the cytogenetic location of the cataract locus.

Mutation analysis of GJA3 in family Sh. A: Sequence profile of the wild-type allele showing translation of valine (V) at codon 44 (GTG) in exon 2. B: Sequence trace of the mutant allele showing the heterozygous c.130G>A transition (denoted R by the International Union of Pure and Applied Chemistry [IUPAC] code) at the first base of codon 44 (ATG) that is predicted to result in a missense substitution of methionine (M) for valine (p.V44M). C: Restriction fragment length analysis on agarose gels showing gain of an Hsp92 II site (5′CATG↓) that co-segregated with affected individuals heterozygous for the mutant A-allele (167/174 bp) but not with unaffected individuals homozygous for the wild-type G-allele (341 bp).

Schematic showing gene structure and protein domains of GJA3. A: Exon organization and mutation profile of GJA3. The entire coding region (435 amino-acids) is located in exon-2. Based on hydrophobicity analysis [], GJA3 has nine structural domains including: a cytoplasmic N-terminus (NT), 4 transmembrane domains (TM-1 – TM-4); 2 extracellular loops (EC1, EC2), a cytoplasmic loop (CL), and a cytoplasmic C-terminus (CT). The relative locations, with respect to the translation start codon, of the p.V44M mutation and 19 other mutations associated with autosomal dominant cataract in humans are indicated. The rat p.E42K mutation associated with autosomal recessive cataract is also indicated. B: Amino-acid sequence alignment of the first extracellular (EC-1) domain (amino-acids 42–71) from human GJA3 and homologs from other species. Dots denote identical amino-acids. Cysteine residues involved in hemi-channel docking are underlined. Missense substitutions are shown in red.