Genetic complexity in hypertrophic cardiomyopathy revealed by high-throughput sequencing

Abstract

Background: Clinical interpretation of the large number of rare variants identified by high throughput sequencing (HTS) technologies is challenging. The aim of this study was to explore the clinical implications of a HTS strategy for patients with hypertrophic cardiomyopathy (HCM) using a targeted HTS methodology and workflow developed for patients with a range of inherited cardiovascular diseases. By comparing the sequencing results with published findings and with sequence data from a large-scale exome sequencing screen of UK individuals, we sought to quantify the strength of the evidence supporting causality for detected candidate variants.

Methods and results: 223 unrelated patients with HCM (46±15 years at diagnosis, 74% males) were studied. In order to analyse coding, intronic and regulatory regions of 41 cardiovascular genes, we used solution-based sequence capture followed by massive parallel resequencing on Illumina GAIIx. Average read-depth in the 2.1 Mb target region was 120. Rare (frequency<0.5%) non-synonymous, loss-of-function and splice-site variants were defined as candidates. Excluding titin, we identified 152 distinct candidate variants in sarcomeric or associated genes (89 novel) in 143 patients (64%). Four sarcomeric genes (MYH7, MYBPC3, TNNI3, TNNT2) showed an excess of rare single non-synonymous single-nucleotide polymorphisms (nsSNPs) in cases compared to controls. The estimated probability that a nsSNP in these genes is pathogenic varied between 57% and near certainty depending on the location. We detected an additional 94 candidate variants (73 novel) in desmosomal, and ion-channel genes in 96 patients (43%).

Conclusions: This study provides the first large-scale quantitative analysis of the prevalence of sarcomere protein gene variants in patients with HCM using HTS technology. Inclusion of other genes implicated in inherited cardiac disease identifies a large number of non-synonymous rare variants of unknown clinical significance.

Figure 1

Box-and-whisker plot, showing the read-depths across the targeted genes.

Figure 2

Number of patients with variants in each of the sarcomeric genes.

Figure 3

Percentage of patients with rare variants in hypertrophic cardiomyopathy/dilated cardiomyopathy associated genes. This figure is only reproduced in colour in the online version.

Figure 4

Rare nsSNPs frequency comparison between our sequencing results and a set of 1287 UK controls with exome sequence data generated by the UK10K project (http://www.uk10k.org) for the eight sarcomere genes most commonly associated with hypertrophic cardiomyopathy. nsSNP: non-synonymous single nucleotide polymorphism. The frequency of MYH7, MYBPC3, TNNT2 and TNNI3 candidate nsSNPs is significantly higher in our cohort, as also shown in table 5. This figure is only reproduced in colour in the online version.