A novel mutation in the connexin 29 gene may contribute to nonsyndromic hearing loss

Abstract

Connexins (Cxs) are homologous four-transmembrane domain proteins and constitute the major components of gap junctions. Among a cohort of patients with nonsyndromic hearing loss, we recently identified a novel missense mutation, E269D, in the GJC3 gene encoding connexin 29 (Cx29), as being causally related to hearing loss. The functional alteration of Cx29 caused by the mutant GJC3 gene, however, remains unknown. This study compared the intracellular distribution and assembly of mutant Cx29 (Cx29E269D) with that of the wild-type Cx29 (Cx29WT) in HeLa cells and the effect the mutant protein had on those cells. Cx29TW showed continuous staining along apposed cell membranes in the fluorescent localization assay. In contrast, the p.E269D missense mutation resulted in accumulation of the Cx29 mutant protein in the endoplasmic reticulum (ER) rather than in the cytoplasmic 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. Based on these findings, we suggest that Cx29E269D has a dominant negative effect on the formation and function of the gap junction. These results provide a novel molecular explanation for the role Cx29 plays in the development of hearing loss.

Prospective variants screening of connexin genes in children with hearing impairment: genotype/phenotype correlation

“…In addition, our previous study found that p.E269 is only moderately conserved (Conseq score = 3-4) in the C-terminal domain of CX29, suggesting p.E269 being none critical in the function of the CX29 protein. However, our results showed that the p.E269D mutation leads to loss of function of the CX29 protein (Hong et al 2010). Based on these results, we conjecture that those variants in this study may be causative for HL.…”

Human Connexin30.2/31.3 (GJC3) does not Form Functional Gap Junction Channels but Causes Enhanced ATP Release in HeLa Cells

“…Our earlier study established that mutations of GJC3 are associated with nonsyndromic hearing loss [17,18]. Moreover, our data demonstrate that the p.E269D missense mutation of CX30.2/CX31.3 results in the accumulation of the CX30.2/CX31.3 mutant protein in the endoplasmic reticulum, rather than in the cytoplasmic membrane [19].…”

“…The ORFs of GJC3 were obtained from the pcDNA3.1 clone [19] after digestion with KpnI and SacII, and then subcloned into the KpnI and SacII restriction sites in vectors p-eGFPN1 (Clontech, Palo Alto, CA, USA). The dideoxy DNA sequencing method, using a DNA Sequencing Kit (Applied Biosystems Corporation, Foster city, CA), an ABI Prism 3730 Genetic Analyzer (Applied Biosystems Corporation, Foster city, CA), and restriction digests were used to confirm the DNA sequence of all constructs.…”

Identification of novel variants in the TMIE gene of patients with nonsyndromic hearing loss

“…Then, we compared the intracellular distribution and assembly of mutant Cx29 (Cx29E269D) with those of the wild-type Cx29 (Cx29WT) in HeLa cells and the effect of the mutant protein had on the cells. Our results showed that Cx29E269D had a dominant negative effect on the formation and function of the gap junction [23]. Further studies on the protein structure and functional effect of sequence variant in the TMIE will provide important insight into the mechanisms that lead to hearing loss.…”