Comparative functional characterization of novel non-syndromicGJB2gene variant p.Gly45Arg and lethal syndromic variant p.Gly45Glu

Abstract

We characterized a novel GJB2 missense variant, c.133G>A, p.Gly45Arg, and compared it with the only other variant at the same amino acid position of the connexin 26 protein (Cx26) reported to date: c.134G>A, p.Gly45Glu. Whereas both variants are associated with hearing loss and are dominantly inherited, p.Gly45Glu has been implicated in the rare fatal keratitis-ichthyosis-deafness (KID) syndrome, which results in cutaneous infections and septicemia with premature demise in the first year of life. In contrast, p.Gly45Arg appears to be non-syndromic. Subcellular localization experiments in transiently co-transfected HeLa cells demonstrated that Cx26-WT (wild-type) and p.Gly45Arg form gap junctions, whereas Cx26-WT with p.Gly45Glu protein does not. The substitution of a nonpolar amino acid glycine in wildtype Cx26 at position 45 with a negatively charged glutamic acid (acidic) has previously been shown to interfere with Ca2+ regulation of hemichannel gating and to inhibit the formation of gap junctions, resulting in cell death. The novel variant p.Gly45Arg, however, changes this glycine to a positively charged arginine (basic), resulting in the formation of dysfunctional gap junctions that selectively affect the permeation of negatively charged inositol 1,4,5-trisphosphate (IP3) and contribute to hearing loss. Cx26 p.Gly45Arg transfected cells, unlike cells transfected with p.Gly45Glu, thrived at physiologic Ca2+ concentrations, suggesting that Ca2+ regulation of hemichannel gating is unaffected in Cx26 p.Gly45Arg transfected cells. Thus, the two oppositely charged amino acids that replace the highly conserved uncharged glycine in p.Gly45Glu and p.Gly45Arg, respectively, produce strikingly different effects on the structure and function of the Cx26 protein.

Molecular Mechanisms and Clinical Phenotypes of GJB2 Missense Variants

“…Dominant variants cause mainly postlingual progressive hearing loss. Dominant variants, such as G45R, may cause milder HL because the mutant can traffic to the cell membrane with only partially impaired hemichannel or GJC function [ 54 ]. On the other hand, the M163L variant cannot traffic itself, but when co-expressed with the wildtype Cx26, it can traffic to the cell membrane, thus leading to a milder phenotype [ 67 ].…”

“…Another distinctive feature of variants causing syndromic HL is a dominant-negative or trans-dominant effect on wildtype Cx26 or other Cxs. For example, HeLa cells cotransfected with G45R and wildtype Cx26 could form GJs, whereas G45E (causing nonsyndromic HL) and wildtype Cx26 did not [ 54 ]. The N54K mutant protein was retained intracellularly and displayed a dominant or transdominant effect on wildtype Cx26 and coexpressed Cx30 and Cx43.…”

Connexins and the Epithelial Tissue Barrier: A Focus on Connexin 26

“…The molecular mechanisms underlying the condition likely relate to “leaky” hemichannels, with differing sensitivities to pro-inflammatory mediators, and ionic sensitivity including calcium and zinc levels thereby altering channel function [ 66 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 ]. Several mutations induce lethal phenotypes and are associated with loss of cell viability [ 83 , 84 , 85 ]. Recently, we proposed a further Class 4 mutation group associated with hyperkeratosis, mucositis and deafness, where cell model studies revealed cell death and a collapse of the microtubule network, but limited connexin channel function (e.g., F142L, CX31G45E) [ 86 , 87 ].…”

A heterozygous mutation in GJB2 (Cx26F142L) associated with deafness and recurrent skin rashes results in connexin assembly deficiencies

“…[8,9,16,[30][31][32] Each KID mutation studied to date has unique characteristics rendering the channels more sensitive to changes in calcium, zinc and other ions and are influenced by pro-inflammatory mediators such as peptidoglycan a key component of gram-positive bacterial cell wall, [8,9,16,31,32] with several mutations resulting in lethal phenotypes. [10,[33][34][35] Overexpression of CX26 (for example in psoriasis) or these leaky KID hemichannels release ATP from the cells feeding into purinergic signalling pathways and associated pro-inflammatory signalling cascades leading to hyperproliferation loss of epidermal integrity and inflammation. [4,21,36] Cx channel blockers or inhibition of the overexpression of CX26…”