Mutation Analysis of the Main Hypertrophic Cardiomyopathy Genes Using Multiplex Amplification and Semiconductor Next-Generation Sequencing

Background: Mutations in at least 30 genes have been linked to hypertrophic cardiomyopathy (HCM). Due to the large size of the main HCM genes, Sanger sequencing is labor intensive and expensive. The purpose was to develop a next-generation sequencing (NGS) procedure for the main HCM genes. Methods and Results: Multiplex amplification of the coding exons of MYH7, MYBPC3, TNNT2, TNNI3, ACTC1, TNNC1, MYL2, MYL3, and TPM1 was designated, followed by NGS with the Ion Torrent PGM (Life Technologies). A total of 8 pools containing DNA from HCM patients were sequenced in a 2-step approach. First, a total of 60 patients (validation cohort) underwent both PGM and Sanger sequencing for the 9 genes. No false-negative variants were found on NGS (100% sensitivity), and a specificity of 97% and 80% was achieved for single-nucleotide and insertion/deletion variants, respectively. Second, the PGM was used to search for mutations in a total of 76 cases not previously studied (discovery cohort). A total of 19 putative mutations were identified in the discovery pools, which were confirmed and assigned to specific patients on Sanger sequencing. Conclusions: An NGS procedure has been developed for the main sarcomeric genes that would facilitate the screening of large cohorts of patients. In addition, this procedure would facilitate the uncovering of rare gene variants on a population scale.