A fast and cost-effective molecular diagnostic tool for genetic diseases involved in sudden cardiac death.

BACKGROUND: Cardiomyopathies and arrhythmia syndromes are common genetic cardiac diseases that account for a significant number of sudden cardiac death (SCD) cases. METHODS: NGS workflow based on a panel of 95 genes was developed on Illumina NextSeq500™ sequencer for sequencing prevalent SCD-causing genes. A cohort of 90 patients (56 genotype-positive, 27 genotype-negative and 7 new cases) was screened to evaluate this strategy in terms of sensitivity, specificity, practicability and cost. In silico analysis were performed using a pipeline based on NextGENe® software and a personalized Sophia Genetics pipeline. RESULTS: Using our panel custom, 100% of targeted sequences were efficiently covered and all previously identified genetic variants were readily detected. Applied to 27 genotype-negative patients, this molecular strategy allowed the identification of pathogenic or likely pathogenic variants into 12 cases. It confirmed the involvement of HCN4 mutations in the combined bradycardia­myocardial non-compaction phenotype, and also suggested, for the first time, the involvement of PKP2, usually associated with arrhythmogenic right ventricular dysplasia, in ventricular non-compaction. CONCLUSION: This NGS approach is a fast, cheap, sensitive and high-throughput mutation detection method that is ready to be deployed in clinical laboratories and would provide new insights on physiopathology of SCD, more particularly of cardiomyopathies and arrhythmia syndromes.

Development of a high resolution melting method for the detection of genetic variations in Long QT Syndrome.

BACKGROUND: Inherited Long QT Syndrome (LQTS) is a cardiac channelopathy associated with a high risk of sudden death. The prevalence has been estimated at close to 1:2000. Due to large cohorts to investigate, the size of the 3 prevalent mutated genes, and the presence of a large spectrum of private mutations, mutational screening requires an extremely sensitive and specific scanning method. METHODS: Efficiency of high resolution melting (HRM) analysis was evaluated for the most prevalent LQTS-causing genes (KCNQ1, KCNH2) using control DNAs and DNAs carrying previously identified gene variants. A cohort of 34 patients with a suspicion of LQTS was further blindly screened. To evaluate HRM sensitivity, this cohort was also screened using an optimized DHPLC strategy. RESULTS: HRM analysis was successfully optimized for KCNQ1 but optimisation of KCNH2 was more laborious as only 3 KCNH2 exons could be finally optimized. Remaining KCNH2 exons were analysed by direct sequencing. This molecular approach, which combined HRM and direct sequencing, was applied on the cohort of 34 cases and 9 putative mutations were identified. Using this approach, molecular investigation was completed faster and cheaper than using DHPLC strategy. CONCLUSIONS: This HRM/sequencing procedure represents an inexpensive, highly sensitive and high-throughput method to allow identification of mutations in the coding sequences of prevalent LQTS genes.