GJB2 (Connexin 26) Gene Mutations In 140 Patients Of Hunan Province With NSHL And Intracellular Distribution Of Two Cx26 Mutants In HeLa Cells

Objective:To identify genetic characteristics in patients of Hunan province with nonsydromic hearing loss (NSHL), determine the prevalence and spectrum of mutations in GJB2(Cx26) gene, explore the pathogenic molecular mechanism.Methods:140 sporadic cases of NSHL without genetic relationship were collected after enquiring history and clinical examinations. Molecular studies were performed by amplifing the coding region of Cx26 gene, purifing the PCR products, then bidirectional sequencing directly.Sequences were analysed by DNAStar software, determine the prevalence and spectrum of mutations in Cx26 gene. Cx26 gene mutants, which primers were designed to amplify,were selected, then constructed into pEGFP vector,transfected into Hela cells after identification by bacteria PCR, double restriction enzyme analysis and sequence. Western blot was performed. Expression of Cx26-EGFP protein were observed in fluorescence microscope after fluorescent staining,compare the differences of fluorescent localization between the wild type and the mutants.Account the mumber of cells that formed gap junction channels, statistic analysis were used to define whether there were differences.Results:Among the 140 NSHL patients without genetic relationship,56 patients were detected Cx26 gene mutation, the relevance ratio was 40%(56/140). Both of the two Cx26 alleles were mutated in 29 patients and one Cx26 allele in 27 patients, the rate of allele mutation was 30.4%(85/280).10 types of variations were detected, containing 7 types of pathogenic mutations and 3 types of polymorphisms. The 7 types of pathogenic mutations were nonsense mutation c.139G>T, frameshift mutation c.235delC and c.176-191del16, and missense mutation c.109G>A,c.344T>G,c.550C>T and c.571T>C. c.344T>G is a newly reported missense mutation. The c.235delC mutation which was detected in 27 patients was the most prevalent mutation, the relevance ratio was 19.3%(27/140), including homozygous mutation in 20 patients, heterozygous in 5 and compound heterozygous in 2, accounting for 55.3%(47/85)of the pathogenic alleles. The c.109G>A mutation which was detected in 25 patients was just next to c.235delC, the rate was 17.9%(25/140), including homozygous mutation in 7 patients and heterozygous in 18, accounting for 37.6%(32/85) of the pathogenic alleles. pEGFP-Cx26 wt, pEGFP-Cx26 V37I and pEGFP-Cx26 F115C eukaryotic expression vectors were constructed, after transfected into Hela cell,Cx26 wt and two mutants'Cx26 and GFP fusion protein were expressed successfully. The fluorescent localization and distribution assay of the two mutants showed no differences with the wildtype, as well as the numbers of cells that formed gap junction channels, indicating that they can not impair the conformation of the gap junctions. mutation is the most hot-spot in patients of Hunan province. The two mutants of Cx26,c.V371 and c.F115C,do not affect the conformation of gap junction after transfected into Hela cell,but whether the gap junction exist normal function need to do further research.