Characterization of a Novel Gja8 (Cx50) Mutation in a New Cataract Rat Model

Abstract

Purpose To describe a novel spontaneous cataract inbred strain isolated from large-scale breeding SD rats, identify the responsible gene mutation, and understand how this mutation affects lens function. Methods Exome sequencing of 12 cataract-associated genes was performed in the affected and healthy relatives. Sequences of rat wild-type or mutant gap junction protein alpha 8 gene (Gja8) were transfected into cells. The expression level of protein was assayed by Western blot analysis. Subcellular localization of connexin 50 (Cx50) was analyzed in confocal fluorescent images. Wound-healing, 5-ethynyl-2ʹ-deoxyuridine incorporation, and attachment assay were performed to characterize the cell migration, proliferation and adhesion. Results The abnormality was found to be inheritable in an autosomal semi-dominant pattern through different mating patterns. We found a G to T transversion at codon 655 in Gja8, leading to a substitution of valine by phenylalanine (p.V219F). Gja8 V219F/+ heterozygotes expressed nuclear cataract while Gja8 V219F/V219F homozygotes manifested microphthalmia in addition to cataract. Histology revealed fiber disorders and loss of organelle-free zone in the mutant lens. Cx50 V219F altered its location in HeLa cells and inhibited the proliferation, migration and adhesion abilities of HLEB3 cells. The mutation also reduced the expression of focal adhesion kinase and its phosphorylation. Conclusions The c.655G>T mutation (p.V219F) is a novel mutation in Gja8, inducing semi-dominant nuclear cataracts in a new spontaneous cataract rat model. The p.V219F mutation altered Cx50 distribution, inhibited lens epithelial cell proliferation, migration, and adhesion, and disrupted fiber cell differentiation. As a consequence, the nuclear cataract and small lens formed.