A novel compound heterozygous mutation in the GJB2 gene causing non-syndromic hearing loss in a family

Abstract

Mutations in the GJB2 gene are responsible for up to 50% of cases of non-syndromic recessive hearing loss, with c.35delG, c.167delT and c.235delC being the predominant mutations in many world populations. However, a large number of rare mutations in this gene may also contribute to hearing loss. The aim of the present study was to conduct a clinical and molecular characterization of a Chinese family with non-syndromic hearing loss. Sequence analysis of the GJB2 gene led to the identification of a novel compound heterozygous mutation c.257C>G (p.T86R)/c.605ins46 in two profoundly deaf siblings whose hearing parents were each heterozygous, either for the c.257C>G (paternal) or for the c.605ins46 (maternal) mutations. Both c.257C>G and c.605ins46 are rare GJB2 mutations that have previously been reported to segregate with autosomal recessive hearing loss exclusively in East Asian populations. To study the pathogenic effect of the compound heterozygous mutation, a three-dimensional model was constructed and Anolea mean force potential energy was predicted for a bioinformatic structural analysis. HEK293 cells were used to study the pathogenic effect of mutant connexin 26 proteins. The results suggested that the c.257C>G (p.T86R)/c.605ins46 mutations in the GJB2 gene provides a novel molecular explanation for the role of the GJB2 gene in hearing loss.

Figure 1

Pedigree and genotypes of the family showing the novel compound heterozygous GJB2 c.257C>G (p.T86R) and c.605ins46 mutations.

Figure 2

Bioinformatic estimation of the 3D structures of the wild-type (WT) and c.257C>G Cx26 proteins. The secondary structure of Cx26 is shown by solid purple cylinders (α-helices), yellow arrows (β-sheet) and white loops (turn). (A). 3D structures of the Cx26-WT and Cx26-c.257C>G proteins (blue, amino acid residue position before mutation; red, position after mutation). Local 3D structures of (a) the WT protein, (c) the Cx26-c.257C>G protein and (b) their overlapping structures are shown (blue and red indicate amino acid residues of the WT and Cx26-c.257C>G proteins, respectively). (B). The graphs show computer modeling from the SWISS-MODEL workspace. An evaluation was performed using the Anolea mean force potential to assess the quality of the model and the stability of the folding of protein chains. The Anolea mean force potential changed the WT (top) and c.257C>G (bottom) versions of the Cx26 protein. The arrows and boxes indicate amino acid residue 86, located in the TM2 region of the human Cx26 protein.

Figure 3

Bioinformatic estimation of the 3D structures of the wild-type (WT) and c.605ins46 Cx26 proteins. (A). 3D structures of the Cx26-WT and Cx26-c.605ins46 proteins (blue, amino acid residue position before mutation; red, position after mutation). The local 3D structures of the (a) WT and (b) Cx26-c.605ins46 proteins are shown. (B) The graphs show computer modeling from the SWISS-MODEL workspace. An evaluation was performed using the Anolea mean force potential to assess the quality of the model and the stability of the folding of protein chains. Anolea mean force potential changes of the WT (top) and c.605ins46 (bottom) versions of the Cx26 protein. The arrows and boxes indicate amino acid residues 105–125, located in the cytoplastic loop (CL) region, and residues 202–217, located in the TM4 region of the human Cx26 protein.

Figure 4

Immunocytochemical analysis of wild-type (WT) and mutant Cx26 protein expression in transfected HEK293 cells. (A). The cellular localization of wild-type and mutant Cx26 proteins. The Cx26-WT protein localized to the membrane and formed gap junction plaques between adjacent cells, unlike the mutant Cx26 proteins. (B). Co-expression of Cx26-WT-mCherry and Cx26-c.257C>G-EGFP. The gap junction is visible between cell pairs. (C) mCherry-tagged Cx26-WT co-expressed with EGFP-tagged Cx26-605ins46 also formed gap junctions in cell pairs. (D) Co-expressed Cx26-c.257C>G-EGFP and Cx26-605ins46-mCherry did not form gap junctions but were expressed in the cytoplasm.