DNA sequence capture and high-throughput sequencing technology: a novel approach to identify a large number of hypertrophic cardiomyopathy-causing genes

Hypertrophic cardiomyopathy (HCM) is the most frequent genetic cardiovascular disease worldwide and is an important cause of heart failure-related disability in young people. To date, more than 20 different genes have been identified and the number is increasing. We evaluted a novel approach to identify causative mutations in a large number of HCM-related and candidate genes. Four HCM patients previously analysed by DHPLC/Sanger sequencing for causative mutations in 8 sarcomeric genes were enrolled in this study. Overall, 234 genes were selected for array on the chip, and a custom sequence capture array was designed for target enrichment of all coding regions. The size of our target was 3,908,196 bp. Each DNA sample was enriched using one custom array, and then sequenced in two runs by the GS FLX System. We obtained an average of 164 Mb/sample, which is equivalent to 503,775 sequencing reads/sample with an average read length of 325.6 bp. Sequence and data analysis were performed using the Roche/454 gsMapper software. High confidence variants were blasted against the SNP database to distinguish between known and unknown variants. We found 7864 different variants, of which 6725 were intronic, 424 intergenic and 715 exonic. About 31% of these variants were novel and 56 novel variants were in 35 HCM related genes. In all patients, we confirmed the mutations and polymorphisms previously identified in them with the DHPLC/Sanger approach. The simultaneous analysis of a vocabulary of genes so to increase sensitivity for the molecular diagnosis may in turn increase the information for those patients in whom traditional screening was not adequate. With this new technology it may be possible to identify mutations in genes that, also acting as phenotype modifiers, could be responsible for clinical variability thereby explaining the pathogenetic mechanism underlying HCM development. Consequently, by reducing time and costs and increasing the sensitivity of molecular testing, routine HCM molecular diagnostics could be implemented also to obtain a model readily applicable to other genetic diseases