Localization And Function Detection Of The Disease-associated Gene Of A Congenital Nuclear Pulverulent And Posterior Polar Cataract Family

Purpose:Congenital cataract is the leading cause of visual disability in children worldwide. Approximately 1/3 of congenital cataracts are inherited, with the most common being the autosomal dominant form.Up to now, researchers in our courtury have done so much work on the localization of disease-associated genes of congenital cataract families. Functional detection of mutant genes is needed. Here, we studied on a family affected with congenital nuclear pulverulent and posterior polar cataract by localizaing and functional detecting of the disease- associated gene. Genomic DNA was extracted from the blood after the analysis of inheritance mode. Mutation was detected by gene cloning and DNA sequencing.Plasmid with the mutant gene was constructed, Molecular mechanisms by which this mutation caused congenital cataract were detected by gene transfection into cells. Method:The family with congenital cataracts was ascertained by ophthalmologic examination. Inherit mode was analyzed through the pedigree. Lens material was aspirated by irrigation and aspiration during cataract surgery from the proband. and was examined using transmission electron microscopy. Genomic DNA was extracted from the blood. Nuclear cataract-associated gene was amplified and sequenced directly.the mutation was verified by denaturing high-performance liquid chromatography (DHPLC). Wild type genes were PCR amplified from genomic DNA, then the amplifications were subcloned into pEGFP-N1.Quick change site directed mutagenesis was performed to construct the mutant plasmid. Behavior and cellular distribution of connexins were examined by expression in human lens epithelial cells(HLEB-3) and HeLa cells:protein distribution was observed by confocal fluorescence microscopy; distribution and function of Cx43 were detected by immunofiuorescene; Stably transfected clones were selected by their resistance to G418;cell apoptosis were detected by flow cytometry analysis and Western-blot.Results:We identified a four generation family with autosomal dominant congenital cataracts. The phenotype of the affected individuals'cataract was unique:not only were they nuclear pulverulent, but also exhibited posterior polar opacity. There were foci of dense, globular intracellular deposits in the proband's lens fiber cells. Direct sequencing of the GJA3 gene revealed a novel heterozygous transition G→A at position 5(c.5G>A) in the affected individuals, resulting in the replacement of highly conserved Glycine by Aspaingine (p.G2N).DHPLC analysis confirmed this mutation. We successfully constructed plasmids with wt Cx46 and Cx46G2N. After transfection into HeLa and HLEB-3 cells, we detected protein aggregation, which inhibited the formation of gap junction plaques, and disrupted the activity of Cx43. After selected by G418 for about 1 week, all of the cells transfected with Cx46G2N died.This indicated that the mutation lead to cell apotosis. Flow cytometry analysis showed increased cell apoptosis. Immunoblotting showed a decreased level of caspase3 and increased level of cleaved caspase3 in the Cx46G2N group compared to wt Cx46. indicating the activity of caspase3.Conclusion:We identified a novel congenital cataract associated mutation, G2N. in the N terminal of the GJA3 gene. This mutation may cause protein aggregation, reduce gap junction plaques and influence intercellular communication, thus leading to cell apoptosis. Our study widened the mutation spectrum of congenital cataract, represented a novel mechanism by which mutant connexin can cause cataracts. It also suggested a potential contribution of the Cx46 NH2- terminal in maintaining lens homeostasis, provided more theoretical basis for the prevention and gene therapy of congenital cataracts.