Beta Connexin Mutations and Epidermal Dysplasia

Abstract

Connexins (Cx) are the basic unit of gap junction channels and consist of highly conserved amino acid sequences that identify subtypes. The genes are scattered throughout the chromosome based on the subtype and classified into two groups; a and p. Gap junction (GJ) proteins are expressed in different tissues and particularly in epidermal tissue. Autosomal dominant mutations in a and P subgroups are associated with a range of dominant epidermal dysplasias. These mutations are divided into three main classes; Class 1: 'loss of function' mutations that form deficient Cx26 channels at the plasma membrane; Class 2: that are not trafficked to the plasma membrane and not forming functional gap junctions; Class 3 'gain of function' mutations form leaky hemi-channels that are associated with inflammatory skin disorders. Several mutations were discovered that are scattered throughout the Cx26 coding region associated with syndromic hyper-proliferative skin disorders and deafness. These include a recently reviewed Cx26F142Lmutation, discovered in a 2 year old Caucasian female with psoriasiform mucocutaneous involvement. In addition, Erythrokeratodermia variabilis (EKV) is an autosomal dominant genodermatosis caused by several mutations in Cx31 that are associated with the EKV syndrome. In this study, the pathological mechanisms that underlie the syndromic phenotypes were investigated. The methodology was divided into two sections; in vitro and in vivo. In the first section, HaCat cells and HeLa Ohio cells were transfected with different plasmids expressing wild type P JGs (WT) or mutations. All examined wild-type Cxs form punctate staining at the cell membrane and clear GJ plaques. Cx26F142L, Cx26D66H and Cx31G45E mutations were retained at the nuclear area and co-localised with the wild type Cxs such asCx26 and Cx43. The absence of the ER stress in our HeLa Ohio cells transfected with the mutation of interest was investigated. A transdominant effect of the mutations on wild-type Cxs that was analysed by colocalisation and co-immunopercipitation assays. Mutations associated with Keratitis-ichthyosis-deafness (KID), EKV and Mucositis-deafness syndrome when expressed in HeLa Ohio cells altered nuclear morphology resulting in cellapoptosis. In in vivo study, KID and non-KID syndrome mice model (G12RIXand D66H) were employed to investigate the pathology mechanisms that causing the syndromes. Histological analysis of the epidermal layer of theCx26G12R/D66H transgenic mice compared to WT mice revealed significant differences and hyperproliferation. Following exposure of mouse skin to proinflammatory mediators mice expressing the KID mutations G12Rresponded with an enhanced proinflammatroy status, proliferation and Cxexpression compared to those expressing the non-functional non-KID D66Hmutation. In this study, we proposed to classify the new mutationsCx26F142L and Cx31G45E into a separate class (class 4) as they exhibiting unique features that lead to skin hyperplasia by a unique pathological mechanism. The spatial localisation and inflammatory gene expression differences between the transgenic and WT mice were observed aftertreatment with inflammation inducers. We conclude that both loss and gain of function are underlying the pathology mechanisms of the clinical phenotype outcome as all the mutations employed in our project are dominant and impacted the biological function of the Cx negatively. We suggest a unique therapy including hemichannel blockers for KID patients and regulated cellular function via paclitaxel drug that recovered the cell death phenotype.

Date of Award	2019
Original language	English
Awarding Institution	Glasgow Caledonian University
Supervisor	Patricia Martin (Supervisor), Susan Lang (Supervisor) & Janice Spencer (Supervisor)