👤 Giulia Frisso

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease. Variants in MYBPC3, the gene encoding cardiac myosin-binding protein C (cMyBP-C), are the leading cause of HCM. However, the pathogenicity status of hundreds of MYBPC3 variants found in patients remains unknown, as a consequence of our incomplete understanding of the pathomechanisms triggered by HCM-causing variants. Here, we examined 44 nontruncating MYBPC3 variants that we classified as HCM-linked or nonpathogenic according to cosegregation and population genetics criteria. We found that around half of the HCM-linked variants showed alterations in RNA splicing or protein stability, both of which can lead to cMyBP-C haploinsufficiency. These protein haploinsufficiency drivers associated with HCM pathogenicity with 100% and 94% specificity, respectively. Furthermore, we uncovered that 11% of nontruncating MYBPC3 variants currently classified as of uncertain significance in ClinVar induced one of these molecular phenotypes. Our strategy, which can be applied to other conditions induced by protein loss of function, supports the idea that cMyBP-C haploinsufficiency is a fundamental pathomechanism in HCM. Show less

In the era of next generation sequencing (NGS), genetic testing for inherited disorders identifies an ever-increasing number of variants whose pathogenicity remains unclear. These variants of uncertain significance (VUS) limit the reach of genetic testing in clinical practice. The VUS for hypertrophic cardiomyopathy (HCM), the most common familial heart disease, constitute over 60% of entries for missense variants shown in ClinVar database. We have studied a novel VUS (c.1809T>G-p.I603M) in the most frequently mutated gene in HCM, MYBPC3, which codes for cardiac myosin-binding protein C (cMyBPC). Our determinations of pathogenicity integrate bioinformatics evaluation and functional studies of RNA splicing and protein thermodynamic stability. In silico prediction and mRNA analysis indicated no alteration of RNA splicing induced by the variant. At the protein level, the p.I603M mutation maps to the C4 domain of cMyBPC. Although the mutation does not perturb much the overall structure of the C4 domain, the stability of C4 I603M is severely compromised as detected by circular dichroism and differential scanning calorimetry experiments. Taking into account the highly destabilizing effect of the mutation in the structure of C4, we propose reclassification of variant p.I603M as likely pathogenic. Looking into the future, the workflow described here can be used to refine the assignment of pathogenicity of variants of uncertain significance in MYBPC3. Show less

Point mutations are the most common cause of inherited diseases. Bioinformatics tools can help to predict the pathogenicity of mutations found during genetic screening, but they may work less well in determining the effect of point mutations in non-coding regions. In silico analysis of intronic variants can reveal their impact on the splicing process, but the consequence of a given substitution is generally not predictable. The aim of this study was to functionally test five intronic variants ( Show less

End-stage hypertrophic cardiomyopathy (ES-HC) has an ominous prognosis. Whether genotype can influence ES-HC occurrence is unresolved. We assessed the spectrum and clinical correlates of HC-associated mutations in a large multicenter cohort with end-stage ES-HC. Sequencing analysis of 8 sarcomere genes (MYH7, MYBPC3, TNNI3, TNNT2, TPM1, MYL2, MYL3, and ACTC1) and 2 metabolic genes (PRKAG2 and LAMP2) was performed in 156 ES-HC patients with left ventricular (LV) ejection fraction (EF) <50%. A comparison among mutated and negative ES-HC patients and a reference cohort of 181 HC patients with preserved LVEF was performed. Overall, 131 mutations (36 novel) were identified in 104 ES-HC patients (67%) predominantly affecting MYH7 and MYBPC3 (80%). Complex genotypes with double or triple mutations were present in 13% compared with 5% of the reference cohort (p = 0.013). The distribution of mutations was otherwise indistinguishable in the 2 groups. Among ES-HC patients, those presenting at first evaluation before the age of 20 had a 30% prevalence of complex genotypes compared with 19% and 21% in the subgroups aged 20 to 59 and ≥60 years (p = 0.003). MYBPC3 mutation carriers with ES-HC were older than patients with MYH7, other single mutations, or multiple mutations (median 41 vs 16, 26, and 28 years, p ≤0.001). Outcome of ES-HC patients was severe irrespective of genotype. In conclusion, the ES phase of HC is associated with a variable genetic substrate, not distinguishable from that of patients with HC and preserved EF, except for a higher frequency of complex genotypes with double or triple mutations of sarcomere genes. Show less

Hypertrophic cardiomyopathy is a relatively frequent disease with a prevalence of 0.2% worldwide and a remarkable genetic heterogeneity, with more than 30 causative genes reported to date. Current PCR-based strategies are inadequate for genomic investigations involving many candidate genes. Here, we report a next-generation sequencing procedure associated with DNA sequence capture that is able to sequence 202 cardiomyopathy-related genes simultaneously. We developed a complementary data analysis pipeline to select and prioritize genetic variants. The overall procedure can screen a large number of target genes simultaneously, thereby potentially revealing new disease-causing and modifier genes. By using this procedure, we analyzed hypertrophic cardiomyopathy patients in a shorter time and at a lower cost than with current procedures. The specificity of the next-generation sequencing-based procedure is at least as good as other techniques routinely used for mutation searching, and the sensitivity is much better. Analysis of the results showed some novel variants potentially involved in the pathogenesis of hypertrophic cardiomyopathy: a missense mutation in MYH7 and a nonsense variant in INS-IGF2 (patient 1), a splicing variant in MYBPC3 and an indel/frameshift variant in KCNQ1 (patient 2), and two concomitant variations in CACNA1C (patient 3). Sequencing of DNA from the three patients within a pool allowed detection of most variants identified in each individual patient, indicating that this approach is a feasible and cost-effective procedure. Show less