Genotype/phenotype analysis and pathophysiology of Desmoglein-2 propeptide cleavage-site mutations in Arrhythmogenic right ventricular cardiomyopathy

Abstract

Purpose: Desmoglein-2 (DSG2) mutations are common causes of Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC). A hot spot of missense mutations targets the consensus cleavage-site of DSG2 pro-peptide (Arg-X-Arg/Lys-Arg) by Kex2-like proprotein-convertases (PCs). Through mutational screening in 200 independent ARVC probands we identified heterozygous missense DSG2 mutations of the cleavage site (p.Arg46Trp, p.Arg46Gln, p.Arg49His and p.Lys48Asn) in ten probands and seven additional relatives. In the present work we analysed the genotype/phenotype correlations associated with these specific mutations and experimentally explored their molecular consequences. Methods and results: The pro-DSG2 cleavage site mutations were associated with a severe phenotype in 53% of cases with diffuse right ventricular (RV) dilatation, severe RV dysfunction and frequent left ventricular involvement leading to end-stage heart failure in 3 out of 17 mutation-carriers. To understand molecular dysfunctions associated with the observed severe phenotype, we performed ex vivo analysis on heart samples obtained from two mutation carriers and in vitro analysis by expressing a wild-type (WT) or mutant DSG2-GFP fusion protein in cellular models. First, we demonstrated, by using an antibody specifically directed against the propeptide, that all mutations prevented efficient propeptide cleavage. However pro-DSG2 mutants were correctly addressed to the intercellular junctions (cells) or at the intercalated disk (human tissue). Although co-immunoprecipitation analysis and extracellular crosslinking studies showed the persistence of homophilic interactions of mutant pro-DSG2, the presence of propeptide led to the abolition of interactions between the EC1 (N-terminal) domains of the cadherins. This was accompanied with a mis-incorporation of pro-DSG2 into desmosomes as revealed by immunofluorescent labelling at low calcium concentration compared to WT DSG2, due to an EGFR-dependant internalization of mutant pro-DSG2 and partners (Plakophillin-2 and Plakoglobin). Finally, we showed by western-blot an increase in the soluble pool of mutant pro-DSG2 compared to the DSG2 WT when cells were submitted to mechanical stress. Conclusion: DSG2 propeptide cleavage-site mutations are associated with a severe phenotype and an increased risk of heart failure. Our experimental results indicated a loss of DSG2 adhesiveness properties and a decrease of the incorporation of mutant DSG2 into desmosomes due to propeptide cleavage abolition, that could play an important role in ARVC pathophysiology.