Functional study of GJB2 in hereditary hearing loss

Objectives/Hypothesis: The gene GJB2 of the gap junction protein connexin 26 (Cx26) was found to be the main causative gene of autosomal recessive nonsyndromic hearing loss (DFNB1). Although 35delG has been known as the major mutation in Western countries, 235delC was reported to be a specific form of mutation in Asian populations. The objective of the study was to identify how 235delC and E114G changes found in the Korean population affected the function of GJB2 using molecular biological techniques. Methods. Genes containing 235delC and E114G were cloned into the pcDNA3 vector, and HeLa cells were transfected with the recombinant DNA samples by the liposome complex method. The expression and subcellular localization of Cx26 were determined, using antibodies against amino acid sequences in the intracellular loop (IL) and N-terminal (NT) portions of Cx26. To analyze functions of the GJB2 as a gap junction channel, we examined Lucifer yellow dye transfer between cells with a scrape-loaded technique. Wild-type GJB2 (WT) with normal hearing was used as a positive control, and mock transfected cells were used as a negative control. Results: Immunocytochemical analysis showed that cells transfected with E114G and WT gave characteristic punctate patterns of reaction in the cell membrane with both antibodies. However, 235delC cells were not stained with anti-IL antibody but stained slightly just around the nucleus only with anti-NT antibody. In a functional study of GJB2, transfer of Lucifer yellow into contiguous cells was detected in both WT and E114G, but no transfer activity was observed in 235delC. Conclusions: The 235delC mutation showed a loss of targeting activity to the cell membrane and severe deterioration of gap junction activity. For the E114G, we did not find any difference from WT transfected cells. Key Words: Hereditary hearing loss, gap junction, connexin 26, functional study.