Additional value of screening for minor genes and copy number variants in hypertrophic cardiomyopathy

Abstract

Introduction: Hypertrophic cardiomyopathy (HCM) is the most prevalent inherited heart disease. Next-generation sequencing (NGS) is the preferred genetic test, but the diagnostic value of screening for minor and candidate genes, and the role of copy number variants (CNVs) deserves further evaluation.

Methods: Three hundred and eighty-seven consecutive unrelated patients with HCM were screened for genetic variants in the 5 most frequent genes (MYBPC3, MYH7, TNNT2, TNNI3 and TPM1) using Sanger sequencing (N = 84) or NGS (N = 303). In the NGS cohort we analyzed 20 additional minor or candidate genes, and applied a proprietary bioinformatics algorithm for detecting CNVs. Additionally, the rate and classification of TTN variants in HCM were compared with 427 patients without structural heart disease.

Results: The percentage of patients with pathogenic/likely pathogenic (P/LP) variants in the main genes was 33.3%, without significant differences between the Sanger sequencing and NGS cohorts. The screening for 20 additional genes revealed LP variants in ACTC1, MYL2, MYL3, TNNC1, GLA and PRKAG2 in 12 patients. This approach resulted in more inconclusive tests (36.0% vs. 9.6%, p<0.001), mostly due to variants of unknown significance (VUS) in TTN. The detection rate of rare variants in TTN was not significantly different to that found in the group of patients without structural heart disease. In the NGS cohort, 4 patients (1.3%) had pathogenic CNVs: 2 deletions in MYBPC3 and 2 deletions involving the complete coding region of PLN.

Conclusions: A small percentage of HCM cases without point mutations in the 5 main genes are explained by P/LP variants in minor or candidate genes and CNVs. Screening for variants in TTN in HCM patients drastically increases the number of inconclusive tests, and shows a rate of VUS that is similar to patients without structural heart disease, suggesting that this gene should not be analyzed for clinical purposes in HCM.

Fig 1. Classification of rare variants in MYBPC3, MYH7, TNNI3, TNNT2 and TPM1 (pooled data from Sanger sequencing and NGS cohorts).

Fig 2. Classification of the rare variants found in the 25 genes screened in the NGS cohort.

Fig 3. Classification of the novel variants identified in the NGS cohort.

Fig 4. Distribution of rare variants according to gene-level supporting evidence, ACMG clinical classification and minor allele frequency filtering.

Fig 5. Cases with confirmed CNVs.

NGS results, schematic representation of the breakpoints and precise characterization by Sanger sequencing of (A) the deletion of exon 27 of MYBPC3 (P168, brown sample in the graph), (B) the deletion spanning from exon 4 to exon 12 of MYBPC3 (P259, turquoise sample in the graph), and (C) the well-characterized PLN deletion (blue sample in the graph). (D) NGS results are shown for the non-characterized PLN deletion (orange sample in the graph).