Analysis of and expression of connexin 26 mutations

Abstract

Problem: Mutations in the gap junction beta-2 (GJB2) gene, which encodes the connexin 26 protein, account for 50% of all the patients with autosomal recessive nonsyndromic hearing loss. Connexins are proteins that oligomerize to form a gap junction channel. Gap junctions enable intercellular communication between cell membranes of 2 neighboring cells. Mutations in these connexins lead to absent or malformed gap junctions and congenital deafness. The expression of different connexin mutations’ molecules has not been fully evaluated. Methods: Based on their prevalence and inheritance, 4 specific mutations were chosen (35delG, R184P, R184Q, and C202F). Primers were designed based on specific mutations and site-directed PCR mutagenesis was performed using Stratagene’s Quikchange site-directed mutagenesis kit. Mutations were confirmed via DNA sequencing. The mutated GJB2 genes and wild-type GJB2 were inserted into the EcoRI site of the pNUT vector and transfected into baby-kidney-cells (BHK) using Lipofectamine. Expression of mutated genes was analyzed using immunofluorescence. Results: Mutations (35delG, R184P, R184Q, C202F) were successfully created using site-directed PCR mutagenesis. DNA sequencing confirmed desired mutations. Mutations and wild-type GJB2 genes were successfully expressed in BHK cells and analyzed using immunofluorescence. Specific results and difference will be presented. Conclusion: Specific GJB2 mutations (35delG, R184P, 184Q, C202F) were successfully created and expressed. Immunofluorescence results confirm specific difference between wild-type GJB2 and GJB2 mutations. This study was the first to successfully express and analyze mutations R184Q and C202F and to express mutations 35delG and R184P in a mouse in vitro system. Significance: As noted above, mutations in the gap junction beta-2 (GJB2) gene, which encodes the connexin 26 protein, account for 50% of all the patients with autosomal recessive nonsyndromic hearing loss. Expression and analysis of these mutations are crucial to our understanding of this disease and to our ability to provide potential therapy via targeted gene therapy in the future. Support: None reported.