語言學習前非症候群感因神經聽障基因CONNEXIN29,CONNEXIN43和Pseudo CONNEXIN43基因突變之功能研究

Abstract

Hearing loss, caused by gene mutations and environmental factors, is a common sensory disorder in the human population. In the developed countries, the incidence of congenital hearing loss is estimated at 1 in 1000 births, of which approximately 60% cases are attributed to genetic factors. To date, 59 auditory genes have been identified. Connexin (Cx) belongs to a large gene family, and the products of Cx gene family constitute a gap junction channel responsible for regulation of the physiological and developmental process, involving the exchange of ions and small molecules. Every six Cxs compose a hemichannel (so called as connexon), and then two hemichannel to form an integral gap junction. This study applied cell model to determine the Cx29 and Cx43 gene mutations in the pathogenic mechanisms causing hearing loss. To confirm the localization patterns seen in the immunolabeling assay, HeLa cells were transfected with Cx29 constructs that were directly ‘tagged’ with GFP or DsRed at the C-terminal end of the protein. The results reveal the Cx29WT-EGFP was expressed along apposed cell membranes in the fluorescent localization assay with a continuous staining. In contrast, the p.E269D- EGFP (missense mutation) resulted in the accumulation of the Cx29 mutant protein in the endoplasmic reticulum (ER) rather than in the plasma membrane. Co-expression of Cx29WT and Cx29E269D proteins by a bi-directional tet-on expression system demonstrated that the heteromeric connexon accumulated in the cytoplasm, thereby impairing the formation of the gap junction. Assuming these findings, we suggest that Cx29E269D has a dominant negative effect on the formation of the gap junction. To determine the crucial relationship between wild type Cx43 and three of mutant Cx43 genes( S69P、T326Iand 932 del C) were cloned and their functions were analysed in HeLa cells. Localization assay of WT Cx43 reveals a typical punctuate fluorescence pattern of a gap junction channel between neighboring expression cells. Additionally, immunoblotting analysis of the transfectants confirms the production of mutant proteins, in which their distributions along appositional membranes are determined using immunofluorescent staining procedures. Furthermore, dye transfer assay results demonstrate that gap junctional intercellular communication (GJIC) is less in HeLa cells carrying mutant GJA1 orψGJA1 gene than in WT-expressing cell. In summary, we use molecular biology methods to create the normal and mutant constructs containing Cx29 or Cx43 gene and determine the pathogenic effects of normal and mutant Cx29or Cx43 gene in transfected HeLa cells. Our data reveal the Cx29 and Cx43 gene mutations play causal roles in the pathogenesis of hearing loss.