Background and objective: 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 1:2,000. Due to large cohorts to investigate and high rate of private mutations, mutational screening must be performed using an extremely sensitive and specific detection method. Mutational screening is crucial as this may have implications for therapy and management of LQTS patients.
Methods: Next-generation sequencing (NGS) workflow based on a custom AmpliSeq™ panel was designed for sequencing the five most prevalent cardiomyopathy-causing genes (KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2) on Ion PGM™ Sequencer. A cohort of 30 previously studied patients was screened to evaluate this strategy in terms of sensitivity, specificity, practicability, and cost. In silico analysis was performed using NextGENe(®) software.
Results: Our AmpliSeq™ custom panel allowed us to explore 86 % of targeted sequences efficiently. Using adjusted alignment settings, all genetic variants (40 substitutions, 17 indels) present in covered regions and previously detected by high-resolution melt (HRM)/sequencing were readily identified. Uncovered targeted regions, which were mainly located in KCNH2, were further analyzed by HRM/sequencing strategy. Complete molecular investigation was performed faster and cheaper than with previously used mutation detection methods.
Conclusion: Finally, these results suggested that our new NGS approach based on AmpliSeq™ libraries and Ion PGM™ sequencing is a highly efficient, fast, and cheap high-throughput mutation detection method that is ready to be deployed in clinical laboratories. This method will allow fast identification of LQTS mutations that will have further implications for therapeutics.