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

Abstract

Mutations in GJB2 encoding Connexin 26 (CX26) are associated with hearing loss and hyperproliferative skin disorders of differing severity including keratitis‐ichthyosis‐deafness (KID) and Vohwinkel syndrome. A 6‐year‐old Caucasian girl who presented with recurrent skin rashes and sensorineural hearing loss harboured a heterozygous point mutation in GJB2 (c.424T > C; p.F142L). To characterize the impact of CX26F142L on cellular events. Plasmids CX26WT, CX26F142L, CX26G12R (KID) or CX26D66H (Vohwinkel) were transfected into HeLa cells expressing Cx26 or Cx43 or into HaCaT cells, a model keratinocyte cell line. Confocal microscopy determined protein localization. MTT assays assessed cell viability in the presence or absence of carbenoxolone, a connexin‐channel blocker. Co‐immunoprecipitation/Western blot analysis determined Cx43:Cx26 interactions. Quantitative real‐time polymerase chain reaction assessed changes in gene expression of ER stress markers. Dye uptake assays determined Connexin‐channel functionality. F142L and G12R were restricted to perinuclear areas. Collapse of the microtubule network, rescued by co‐treatment with paclitaxel, occurred. ER stress was not involved. Cell viability was reduced in cells expressing F142L and G12R but not D66H. Unlike G12R that forms “leaky” hemichannels, F142L had restricted permeability. Cell viability of F142L and G12R transfected cells was greater in HeLa cells expressing Cx43 than in native Cx‐free HeLa cells. Co‐immunoprecipitation suggested a possible interaction between Cx43 and the three mutations. Expression of CX26F142L and G12R results in microtubule collapse, rescued by interaction with Cx43. The GJB2 mutations interacted with Cx43 suggesting that unique Cx43:Cx26 channels are central to the diverse phenotype of CX26 skin‐related channelopathies.

Molecular Mechanisms and Clinical Phenotypes of GJB2 Missense Variants

“…Dye uptake assays showed that cells expressing the N54K mutant demonstrated a highly variable incidence of PI uptake, and cells expressing the S183F mutant displayed no increase in PI uptake, indicating nonfunctional hemichannels [ 24 ]. Marziano et al [ 52 ] and Albuloushi et al [ 87 ] obtained similar results for the hemichannel activity of the D66H and F142L mutants. Both variants resulted in syndromic HL and had reduced hemichannel activity.…”

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

“…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 ].…”

“…As such, CX26 and CX43, critical connexins in epithelial tissue are unable to form heterotypic structures [ 88 , 89 ]. Recent studies report changes in CX26 mutation oligomerization compatibility, allowing aberrant interactions with CX43 with exacerbated hemichannel activity but non-functional gap junction channels [ 78 , 86 , 90 ], with each mutation uniquely altering the 3D structure in terms of charge, pore size, hydrophobicity, etc. It is also conceivable that altered CX43:CX26 heteromeric channels influence unique metabolic exchange and influence asymmetric cell division required for the stratification of the epidermis, thereby contributing to the hyperproliferative status of the skin [ 91 ].…”

“…Mutations in CX26 affect hemichannel permeability. As discussed, CX26 KID syndrome mutations provoke ‘leaky’ hemichannels and allow the aberrant formation of CX43-CX26 hetero-hemichannels presenting abnormal permeability [ 66 , 78 , 86 , 145 , 146 , 147 ]. Some of these mutations also alter CX26 PCO 2 gating sensitivity, which may contribute to CO 2 -dependent regulation of breathing in mammals [ 148 , 149 , 150 , 151 ].…”

4‐octyl itaconate improves the viability of D66H cells by regulating the KEAP1‐NRF2‐GCLC/HO‐1 pathway

“…This disease is usually accompanied by many other medical disorders, for example sensorineural deafness, ichthyosis, vitiligo and mental retardation, severely affecting the patients' physical and mental health 2 . However, the effective therapeutic treatment of VS is still lacking in the clinical settings 3 …”