Identification of a novel GJA8 (Cx50) point mutation causes human dominant congenital cataracts

Abstract

Hereditary cataracts are clinically and genetically heterogeneous lens diseases that cause a significant proportion of visual impairment and blindness in children. Human cataracts have been linked with mutations in two genes, GJA3 and GJA8, respectively. To identify the causative mutation in a family with hereditary cataracts, family members were screened for mutations by PCR for both genes. Sequencing the coding regions of GJA8, coding for connexin 50, revealed a C > A transversion at nucleotide 264, which caused p.P88T mutation. To dissect the molecular consequences of this mutation, plasmids carrying wild-type and mutant mouse ORFs of Gja8 were generated and ectopically expressed in HEK293 cells and human lens epithelial cells, respectively. The recombinant proteins were assessed by confocal microscopy and Western blotting. The results demonstrate that the molecular consequences of the p.P88T mutation in GJA8 include changes in connexin 50 protein localization patterns, accumulation of mutant protein, and increased cell growth.

Figure 1. Cataracts pedigree and phenotype.

(A). Cataracts pedigree. Squares and circles symbolize males and females, respectively. Black and white lines denote affected status and unaffected status, respectively. Arrow indicates proband. Individuals underlined in blue represent those enrolled in the study. + represents wild-type GJA8 allele, − represents allele with mutation. (B). Photographs of affected individuals of this family. The phenotype of the proband (IV: 3) is bilateral complete opacification of the fetal nucleus and the cortex; its phenotype is total cataract.

Figure 2. Mutation analysis.

(A). DNA sequence chromatogram analysis. DNA sequence chromatograms of the unaffected members (top) and affected members (bottom) in a family with autosomal dominant total cataracts. A single base alteration of C > A transversion in exon 2 causes a conservative substitution of Pro to Thr at codon 88 (p.P88T). (B). Multiple protein sequence alignments. Multiple-sequence alignment of Cx50 from different species and Cx family members (Cx43, Cx40) from human revealed that codon 88, where the mutation (p.P88T) occurred, was located within a highly conserved region. The “mut.” sequence indicates the sequence with the mutation detected in this family.

Figure 3. Schematic diagram of Cx50 reported mutations.

Schematic diagram of Cx50 protein containing all reported human mutations associated with cataracts. The identified mutation in this study is marked with red arrow.

Figure 4. Distinct protein localization patterns of p.P88T mutation.

(A, B). Immunofluorescent imaging of Cx50-Flag and EGFP (control) showing transiently transfected 293 cells (A) and HLE cells (B) with Cx50-WT, p.P88T mutant, and pSin EGFP backbone plasmids (control). Cells were immunostained with anti-Flag monoclonal antibody. DAPI shows nuclear DNA staining. Arrows mark the accumulation of the mutant protein in the cytoplasm and plasma membrane. (C, D). Immunofluorescent imaging of EGFP fusion proteins showing transiently transfected 293 cells (C) and HLE cells (D) with Cx50-WT p.P88T mutant, and pEGFPc1 backbone plasmids (control). DAPI shows nuclear DNA staining.

Figure 5. Much higher expression level of p.P88T mutant protein.

Western blotting for Cx50 and EGFP (control). The blots were probed with the anti-Flag antibody. Only two groups of cells produced a protein band, which includes about 60 kDa, 50 kDa, 43 kDa. No band was detected in the EGFP control.

Figure 6. A positive effect of p.P88T mutant protein on cell growth.

(A, B). Colony size of 293 cells with stable ectopic overexpression of Cx50. Stable ectopic overexpression of p.P88T mutated Cx50 led to a much larger cell colony when compared with wild-type control.