Functional assessment of new MYBPC3 variants associated with Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is the most common genetic disease of the myocardium. In ~60% of the cases HCM is caused by mutations in sarcomeric proteins, such as cardiac Myosin Binding Protein C (cMyBPC), which are responsible for generating the molecular force of myocyte contraction. A cohort of HCM patients have been screened for mutations in sarcomeric genes, and some new variants of cMyBPC of uncertain significance (VUS) were found. These new variants include two intronic variants (MYBPC3-c.506-2 A>C and MYBPC3-c.2308+3 G>C) and one missense variant (cMyBPC I603M), which were selected for functional study to determine pathogenicity. The MYBPC3-c.506-2 A>C mutation was analysed in mRNA extracted from peripheral blood of the patient. The analysis revealed the loss of the canonical splice site and the utilization of an alternative splicing site, causing the loss of the first 7 nucleotides of exon 5. For the other variant, minigene constructs were generated to transfect HEK-293 cells. The minigene assay showed that mutation MYBPC3-c.2308+3 G>C also produces altered pre-mRNA processing, resulting in the skipping of the exon 23. The mutation I603M localizes to domain C4 of cMyBPC. Using bioinformatics sequence analyses, a deleterious effect for I603M was predicted, but mRNA studies do not show any alteration of the splicing mechanism. At the protein level, homology modelling of domain C4 shows I603 to be buried in the protein structure, suggesting a potential destabilizing role of the I603M mutant. Indeed, circular dichroism spectroscopy and differential scanning calorimetry show a ~15oC lower melting temperature for the mutant C4 domain. Finally, results obtained by single-molecule atomic force microscopy do not show a mechanical fingerprint for C4 indicating a very low mechanical stability of this domain. Taken our results together, we propose that mutations c.506-2 A>C, c.2308+3 G>C and I603M lead to haploinsufficiency and cMyBPC protein destabilization, respectively causing the development of HCM. In conclusion, the study of the functional consequences of mutations leads to assignment of pathogenicity of variants of uncertain significance.