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.

Molecular Mechanisms and Clinical Phenotypes of GJB2 Missense Variants

“…The life cycle of Cx26 includes protein translation, post-translational modifications, degradation of Cx26 molecules, assembly into hemichannel, intracellular trafficking of connexons to the plasma membrane, docking of the connexons from neighboring cells to form GJs, and interactions between other coexpressed connexins ( Figure 2 ) [ 19 , 20 , 21 ]. The most common variant type for the GJB2 gene is frameshift, such as c.35delG in Europeans and c.235delC in East Asians.…”

“…As the most abundant variant type for the GJB2 gene, different missense variants can disrupt intercellular communication by affecting all of the Cx26 life cycle stages [ 20 , 22 ]. A recent review categorized GJB2 missense variants based on whether the affected residues were involved in maintaining the structural stability or impaired the function of Cx26 [ 22 ].…”

“…These in vitro functional studies were performed using model cell lines, including transfected HeLa, HEK-293, NEB1 cells or the cochlear-relevant HEI-OC1 cells from mice, and the paired Xenopus oocytes expression system. The models were used to investigate the cellular localization and membrane trafficking of the mutant proteins, their ability to form functional gap junctions, and their relationship with the Cx26 wild type and other expressed connexins caused by missense variants [ 20 ].…”

Analysis of Serum Inflammatory Markers in Infants Under 6 Months of Age with Non-Syndromic Moderate and Severe Hearing Loss Associated with GJB2 Gene Mutations

“…Mutations in the GJB2 gene are classified as truncating and nontruncating [42]. Truncating mutations, also known as inactivating mutations or nonsense mutations, inhibit protein synthesis and lead to severe hearing loss.…”

Connexins, Innexins, and Pannexins: From Biology to Clinical Targets

“…Apart from clinical mutational screening, considerable effort is being expended in understanding the pathogenicity of different mutations. In this SI, Maslova et al [8] report how they used CRISPR/Cas9 GJB2 knockout cells for functional analysis of the rare GJB2 mutation c.516 G > C (p.Trp172Cys) to show that this W172C mutation-localized to the second extracellular loop of Cx26-impairs Cx26 trafficking to the membrane and thus hemichannel permeability.…”