Arrhythmogenic right ventricular cardiomyopathy: mutation screening of candidate genes and in vitro functional studies

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetically determined heart muscle disorder that presents clinically with ventricular arrhythmias, heart failure, and sudden death. The pathological process consists of progressive loss of ventricular myocardium with fibro-fatty replacement. Right ventricle is mostly involved, but presentation of the disease with predominantly left ventricular involvement has been reported. ARVC is typically inherited as a dominant disease, although recessive variants exist and the involvement of family members often can only be detected by molecular genetic analysis (low penetrance of mutations). Genetic studies over the last few years have offered insight into the potential causes of ARVC. Early works demonstrated substantial genetic heterogeneity, and at least 12 independent loci and 7 disease-genes have now been identified. These findings also implicated desmosomal proteins or proteins involved in desmosomal function as candidate causes of the disorder. In the present study Perp was investigated as a candidate gene for ARVC because of its possible role in cell-cell adhesion, as a structural constituent of desmosomes or as a protein playing an yet unknown role in desmosome assembly. After PERP human cardiac expression was tested and confirmed, 90 ARVC index cases were screened for PERP mutations by DHPLC analysis and direct sequencing. Two variations G59R in exon1 and c.1091C>T in 3'UTR were detected in two patients, in whom a mutation in a known ARVC gene was previously identified. The missense variation G59R was detected in 1 control out of 250 screened and the variation c.1091C>T was identified in 2 controls out of 192 screened. Moreover these two novel variants involved respectively a highly conserved amino acid and a highly conserved nucleotide. Interestingly index cases, carrying two mutations (one in PERP gene and one in a known ARVC gene), showed a more severe phenotype than family members carriers for only one of these variations. It is impossible to establish whether these single PERP mutations might lead to ARVC determination, but on the other hands, in patients carrying a pathogenic mutation in a different gene involved in ARVC, PERP mutations might worsen the clinical phenotype. The idea that ARVC is due to desmosomal dysfunction was strengthened by two recent studies that reported mutations in the desmosomal desmocollin-2 (DSC2) gene as the cause of ARVC. During the present study six different DSC2 mutations were identified in seven out of sixty-four ARVC unrelated Italian index cases. Two nucleotide substitutions (c.-92G>T and c.3241A>T) in 5' and 3' UTR regions were detected in two different index cases. Neither of the nucleotide changes were found in 300 chromosomes from the same population, but to exclude that these mutations could correspond to rare polymorphisms the size of the control group should be increased to 500. Moreover in order to test whether this UTR mutations could affect the expression levels of DSC2 gene, specific in vitro functional studies are needed. Another nucleotide substitution (c.348A>G) absent among 500 control chromosomes, was detected in exon 3; although this mutation corresponds to a synonymous variation (Q116Q), it has been demonstrated that it creates a cryptic splice site, leading to a deletion of 9 nucleotides. Although skipping of 9bp in the mutant transcript doesn't alter the reading frame of DNA sequence, at protein level it leads to loss of three amino acids very conserved among species. This mutation mapped on a region important for maturation of the protein. Two heterozygous point substitutions c.304G>A and c.1034T>C were detected in other two patients. Both nucleotide changes was never found in 250 unrelated controls (500 control chromosomes). Variations c.304G>A in exon 3 and c.1034T>C in exon 8 result in predicted p.E102K and p.I345T amino acid substitutions. The mutated amino acids had completely different physico-chemical properties when compared to the wild type. Both these changes occurred in a residue highly conserved among species and are located in protein regions involved on DSC2 adhesion function. The sixth mutation c.2687_2688insGA in exon 17 was detected in two different patients and in six control subjects, suggesting the possibility of a polymorphism. This mutation would affect the C terminus of DSC2a, precisely the ICS domain, by altering 4 aa residues before a termination codon is prematurely introduced. The change occurred in the last five aa residues of the protein, which are non conserved among mammals, in contrast with the high conservation of the upstream region. The final part of this thesis work was focused on the analysis of potential pathogenic effects of the last three DSC2 mutations described above in cultured cardiomyocytes. Once human cDNAs coding for wild type, two polymorphic variants and mutant proteins were obtained, constructs containing also GFP protein were expressed by transient transfection of HL-1 cell line. In transfected HL-1 cells, wild type protein and the two polymorphic variants were detected in the cell membrane, into cell-cell contact regions since co-localised with the endogenous desmoglein which was marked with a monoclonal dsg antibody. In contrast the three mutant proteins were almost exclusively distributed throughout the cytoplasm with very scarce cell membrane localisation, affecting the normal localisation of DSC2 and suggesting the potential pathogenic effect of the mutations.