Ebrahim, H.Y.; (2008) Genetic and functional studies of mutations affecting cell adhesion proteins in arrhythmogenic right ventricular cardiomyopathy (ARVC). Doctoral thesis , University of London.

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Abstract

ARVC is a cardiac disease associated with ventricular cardiomyocyte fibro-fatty replacement and sudden death. It presents with incomplete penetrance and variable clinical expression. Desmoplakin (DP) and plakoglobin (PG) gene mutations were previously identified. This study aimed: to identify desmosomal (DS) gene mutations in an ARVC cohort by DNA sequencing study the gene transmission and disease expression in affected families and determine functional implications of three identified mutations (A733fsX740PKP-2, S140FPKP-2 and Q273fsX288DP) using wild-type and mutant cDNA plasmid cloning and cell line protein expressions. Eight PKP-2 mutations were identified of which four were novel: frame-shifts disrupting Armadillo domains (ARM) 4, 5 and 8, and a non-sense disrupting ARM 2. One missense and two frame-shift novel DP mutations occurred. Pedigree analysis showed incomplete gene penetrance and variable ARVC expression. Stable cells over-expressing A733fsX740PKP-2 showed increased cellular adhesion and apoptosis following mechanical stretch recovery, and absence of cell junction Connexin-43 (Cx43) protein without significant change in cell-input resistance. Desmosomal lengths were statistically unaltered, but intermittent pale DS coupling occurred. Truncated PKP-2 showed reduced PG interaction. Stable cells over-expressing S140FPKP-2 showed no differences in cell proliferation, adhesion, apoptosis following mechanical stretch recovery, and in cell junction Cx43 protein localization. DS lengths, however, were significantly increased, and missense PKP-2 showed reduced p-catenin interaction. Stable cells over-expressing Q273fsX288 DP showed reduced DS widths, vimentin filament retraction, and lower monolayer adhesion. PG and PKP-2 interacted normally with truncated DP. Altered DS morphologies featured prominently in all three mutant protein expressions. Reduced inter-protein interactions of truncated and missense PKP-2 proteins with PG and p-catenin respectively suggests PG and p-catenin signalling may be affected. Q273fsX288DP expression lacking DP distal domains correlated with loss of DS filament association. These data suggests that expression of mutant desmosomal proteins leads to abnormal DS formation, thus providing the substrate for arrhythmia and cardiomyopathy.

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