Federal Research Center Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia.
Novosibirsk State University, Novosibirsk 630090, Russia.
Biomolecules. 2021 Jan 5;11(1):61. doi: 10.3390/biom11010061.
Mutations in the 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 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 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 knockout HeLa cell line has been generated using CRISPR/Cas9 genome editing tool. Subsequently, the HeLa transgenic cell lines stably expressing different 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.
基因编码跨膜蛋白连接蛋白 26（Cx26）的突变是全球范围内导致听力损失的最常见原因。Cx26 在内耳的离子和代谢稳态中发挥着至关重要的作用，是正常听力过程所必需的。基因中的不同致病突变会影响 Cx26 生命周期的所有阶段，并导致常染色体隐性（DFNB1）或显性（DFNA3）耳聋以及与听力损失相关的皮肤疾病综合征。本研究旨在阐明在来自南西伯利亚（俄罗斯）土著人群的聋病患者中高频发现的罕见变异 c.516G>C（p.Trp172Cys）的功能后果。该替换导致在 Cx26 蛋白的第二细胞外环中保守的第 172 位色氨酸被半胱氨酸（p.Trp172Cys）取代。我们通过免疫细胞化学分析了突变体 Cx26-p.Trp172Cys 蛋白的亚细胞定位，并通过染料加载测定分析了半通道通透性。使用 CRISPR/Cas9 基因组编辑工具生成了 Cx26 基因敲除的 HeLa 细胞系。随后，基于敲除细胞，建立了稳定表达不同变体（野生型和与听力损失相关的突变）的 HeLa 转基因细胞系，并用于比较功能分析。突变体 Cx26-p.Trp172Cys 蛋白向质膜的运输受损和半通道通透性降低支持了 c.516G>C（p.Trp172Cys）变体的致病性及其与非综合征性听力损失的关联。我们的数据有助于更好地理解 Cx26 蛋白第二细胞外环突变在耳聋发病机制中的作用。
