Functional Evaluation of a Rare Variant c.516G>C (p.Trp172Cys) in the GJB2 (Connexin 26) Gene Associated with Nonsyndromic Hearing Loss

Abstract

Mutations in the GJB2 gene encoding transmembrane protein connexin 26 (Cx26) are the most common cause for hearing loss worldwide. Cx26 plays a crucial role in the ionic and metabolic homeostasis in the inner ear, indispensable for normal hearing process. Different pathogenic mutations in the GJB2 gene can affect all stages of the Cx26 life cycle and result in nonsyndromic autosomal recessive (DFNB1) or dominant (DFNA3) deafness and syndromes associating hearing loss with skin disorders. This study aims to elucidate the functional consequences of a rare GJB2 variant c.516G>C (p.Trp172Cys) found with high frequency in deaf patients from indigenous populations of Southern Siberia (Russia). The substitution c.516G>C leads to the replacement of tryptophan at a conserved amino acid position 172 with cysteine (p.Trp172Cys) in the second extracellular loop of Cx26 protein. We analyzed the subcellular localization of mutant Cx26-p.Trp172Cys protein by immunocytochemistry and the hemichannels permeability by dye loading assay. The GJB2 knockout HeLa cell line has been generated using CRISPR/Cas9 genome editing tool. Subsequently, the HeLa transgenic cell lines stably expressing different GJB2 variants (wild type and mutations associated with hearing loss) were established based on knockout cells and used for comparative functional analysis. The impaired trafficking of mutant Cx26-p.Trp172Cys protein to the plasma membrane and reduced hemichannels permeability support the pathogenic effect of the c.516G>C (p.Trp172Cys) variant and its association with nonsyndromic hearing loss. Our data contribute to a better understanding of the role of mutations in the second extracellular loop of Cx26 protein in pathogenesis of deafness.

Keywords: C (p.Trp172Cys); Connexin 26; GJB2; functional assay; gap junction channels; hearing loss; transgenic HeLa cell lines; variant c.516G>.

Figure 1

Localization of Cx26 in several newly established HeLa cell lines (confocal micrographs). The panels of Cx26 variants are designated (top down) as KO, WT, W172C, R75Q, and 35delG for cell lines HeLa Cx26-KO, HeLa-Cx26wt, HeLa-p.W172C, HeLa-p.R75Q, and HeLa-c.35delG, respectively. Nuclei were visualized with DAPI (blue), transgenic HeLa cells were visualized with Green Fluorescent Protein (GFP) and Cx26 was immunostained with antibodies against C-terminus of Cx26 (red). Scale bar = 20 µm.

Figure 2

A fraction of HeLa-p.W172C cells with reduced number of protein granules (confocal micrographs). Blue color indicates DAPI nuclei staining, green color corresponds to GFP signal, red color corresponds to Cx26 signal. Scale bar = 10 μm.

Figure 3

Analysis of the Cx26 protein expression in transgenic HeLa cell lines. The total lysates of cells were subjected to Western blot analysis with antibodies against Cx26 and the loading control, α-tubulin. The panels of Cx26 variants are designated as KO, WT, W172C, R75Q, and 35delG for cell lines HeLa Cx26-KO, HeLa-Cx26wt, HeLa-p.W172C, HeLa-p.R75Q, and HeLa-c.35delG, respectively.

Figure 4

Percentage of cells loaded with propidium iodide (PI) through hemichannels. (A) Proportion of PI-positive cells estimated by analysis of fluorescent microscopy images (the data were obtained for cells in Ca²⁺-free HBSS). Bars represent the mean (± SEM) for each of analyzed cell lines. * denotes significant differences at p < 0.05. (B) Percentage of cells loaded with PI estimated by flow cytometry in Ca²⁺-containing (left) and Ca²⁺-free (right) extracellular conditions. KO, WT, W172C, R75Q, and 35delG denote lines HeLa Cx26-KO, HeLa-Cx26wt, HeLa-p.W172C, HeLa-p.R75Q, and HeLa-c.35delG, respectively.

Figure 5

(A) Topological structure of Connexin 26. TM1-4—four transmembrane domains, E1 and E2—extracellular loops, CL—cytoplasmic loop, NT—N-terminal segment, CT—C-terminal segment. The variants p.Trp172Cys (W172C), p.Arg75Gln (R75Q), p.Gly12Valfs*2 (35delG) examined in this study are shown in red. Amino acid positions for ”pathogenic” and ”likely pathogenic” GJB2 variants located in E2 previously subjected to functional analysis [19,25,27,28,38,40,41,43,46,48,69,70] are marked in black. The mutations linked to recessive deafness are marked in blue; the mutations linked to dominant hearing loss (nonsyndromic and syndromic) are marked in green; ambiguously defined (recessive or dominant) mutation p.M163V is shown in grey. (B) Wild (Trp172) and mutant (Cys172) types of Cx26 (modified Figure 1 from [50]).