Next generation sequencing for the molecular analysis of sarcomeric and non-sarcomeric genes in patients with hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is a common genetic heart disorder characterized by unexplained left ventricle hypertrophy associated with non-dilated ventricular chambers. Several genes encoding heart sarcomeric proteins have been associated to HCM, but a small proportion of HCM patients harbor alterations in other non-sarcomeric loci. The variable expression of HCM seems influenced by genetic modifier factors and new sequencing technologies are redefining the understanding of genotype-phenotype relationships, even if the interpretations of the numerous identified variants pose several challenges.We investigated 62 sarcomeric and non-sarcomeric genes in 41 HCM cases and in 3 HCM-related disorders patients. We found that 82% of the patients harbored at least one rare nsSNV: 11% of the patients showed only sarcomere nsSNVs, 20% of cases harbored at least one sarcomeric nsSNV with at least a desmosomal one and 14% displayed at least one desmosomal nsSNV but no other sarcomere change. We reported an association between desmosomal variations and the pathogenesis of HCM that has not been described to date. We employed an integrated approach that combines multiple tools for the prediction, annotation and visualization of functional variants. Several different methods were employed to predict the functional consequences of alleles that result in amino acid substitutions, to study the effect of some variants over the splicing process and to investigate the impact of these changes respect to the evolutionary conservation. Genotype-phenotype correlations were carried out for inspecting the involvement of each gene in age onset and clinical variability of HCM. Statistical analyses revealed an inverse correlation between the number of nsSNVs and age at onset, and a relationship between the clinical variability and number and type of variants. Then, we describe the clinical, pathological, and molecular features of the novel LAMP2 c.453delT mutation in one of our HCM-related disorders patients affected by Danon disease characterized by severe hypertrophic cardiomyopathy, mild intellectual impairment and rapid progression to heart failure, requiring heart transplant. Immunohistochemical analysis of LAMP2 in the explanted heart revealed a mosaic pattern of distribution, with discrete clusters of either stained or unstained cardiac myocytes, the latter being more frequent in the septum. Interestingly, multiple foci of microscarring were found on histology in the Left Ventricle (LV) free wall and septum. Our findings suggest that several features may contribute to the early and severe cardiac phenotype in female patients affected by Danon disease. In conclusion, this work aims to extend the mutational spectrum of HCM and to contribute in defining the molecular pathogenesis and inheritance pattern(s) of this condition. Besides, we delineate a specific procedure for the identification of the most likely pathogenetic variants for a next generation sequencing approach embodied in a clinical context