Localization And Function Characterization Of The Disease-associated Gene Of Autosomal Dominant Congenital Perinuclear Cataract Families

Purpose:Congenital cataract is the leading cause of visual disability in children worldwide.Among all the causes, genetic mutation is the most common one. To date, more than37cataract-related genes have been characterized.We studied on two Chinese autosomal dominant congenital perinuclear cataract(CPNC) families by localizing and functional detecting of the disease-associated gene.Methods:Two families affected with bilateral congenital cataracts were recruited from the Eye Center of Affiliated Second Hospital, College of Medicine, Zhejiang University, Hangzhou, China. Affected individuals was determined by history records and ophthalmologic examination, which including slit-lamp examination under dilated pupils, visual acuity testing and fudus examination. Genomic DNA samples were extracted from peripheral blood of the pedigree members. All the exons and flanking intronic sequences of candidate gene were amplified by polymerase chain reaction (PCR) and screened for mutation by direct bidirectional DNA sequencing. Sequencing results were analyzed by NCBI Blast.Computational algorithms,including SIFT,PolyPhen are used to predict whether a specific amino acid substitution of a protein sequence is deleterious or neutral to the function of the protein. Additionally, we used Kyte-Doolittle hydropathy plots for hydropathy analysis.CX46gene was acquired from human lens cDNA library,Cx46mutant was generated by PCR site-directed mutagenesis. Cx46wt-EGFP, Cx46H95Y-EGFP, Cx46R76H-EGFP, pEGFP-N1was transfected into Hek293cell,and selected by G418.Stable experssed Cx46was localized by laser scanning.Hemichannel function was analyzed by dye uptake in external free Ca2+HBSS,HBSS containing1.8mM Ca2+or HBSS containing10μM FFA.Results:The two families’phenotype was characterized as perinuclear, inherited model was identified as autosomal dominant, according to the genealogy. Sequencing of the candidate genes detected a novel heterozygous c.283C>T change in the coding region of the Connexin46gene (CX46) in FAMILY I, resulting in the substitution of a highly conserved histidine to tyrosine (p.H95Y).In FAMILY II, a heterozygous c.227G>A change of CX46lead to the substitution of a highly conserved arginine to histidine (p. R76H). These changes were co-segregated with the affected individuals in two families, but were not observed in unaffected family members or in the100unrelated normal controls. All results of SIFT, PolyPhen and Kyte-Doolittle algorithm for hydrophobicity analysis indicated the Cx46-H95Y, R76H substitution was likely deleterious and possibly contributed to the disease.In stable transfectants,Cx46-wild type and Cx46-H95Y protein fluorescene were all detected mainly at cell membranes,while Cx46-R76H primarily at cytoplasm.Though gap junction plaques between cells could be observed in all three groups, plaques in Cx46-H95Y group was much less than in Cx46-wt group, and there is very little gap junction plaques expressing in Cx46-R76H group.Besides, we dected cytopalsmic protein aggregation both in Cx46-H95Y and Cx46-R76H group.After hemichannel was opened in the absence of external calcium for30min with PI and10min with DAPI,Hek293cells expressing Cx46-wt took up both dyes,while cells expressing Cx46-H95Y,Cx46-R76H,or GFP took up no dyes.Dye loading could be blocked by1.8mM Ca2+and10μM FFA.Conclusions:This study has identified two Cx46gene mutations associated with congenital perinuclear cataracta novel one,c.283C>T,and a recurrent one, c.227G>A. Cx46-H95Y and Cx46-R76H mutations may result in protein aggregation,reduce gap junction plaques and affect hemichannel function.Our work widened the mutation spectrum of congenital cataract,represent the mechanism of connexin mutation cause cataract,privied more theoretical basis for the prevention and gene therapy of congenital cataracts.