Functional evaluation of gene mutations in Long QT Syndrome: strength of evidence from in vitro assays for deciphering variants of uncertain significance

Abstract

Abstract Background Genetic screening is now commonplace for patients suspected of having inherited cardiac conditions. Variants of uncertain significance (VUS) in disease-associated genes pose problems for the diagnostician and reliable methods for evaluating VUS function are required. Although function is difficult to interrogate for some genes, heritable channelopathies have established mechanisms that should be amenable to well-validated evaluation techniques. The cellular electrophysiology techniques of ‘voltage-’ and ‘patch-’ clamp have a long history of successful use and have been central to identifying both the roles of genes involved in different forms of congenital Long QT Syndrome (LQTS) and the mechanisms by which mutations lead to aberrant ion channel function underlying clinical phenotypes. This is particularly evident for KCNQ1, KCNH2 and SCN5A, mutations in which underlie > 90% of genotyped LQTS cases (the LQT1-LQT3 subtypes). Recent studies utilizing high throughput (HT) planar patch-clamp recording have shown it to discriminate effectively between rare benign and pathological variants, studied through heterologous expression of recombinant channels. In combination with biochemical methods for evaluating channel trafficking and supported by biophysical modelling, patch clamp also provides detailed mechanistic insight into the functional consequences of identified mutations. Whilst potentially powerful, patient-specific stem-cell derived cardiomyocytes and genetically modified animal models are currently not well-suited to high throughput VUS study. Conclusion The widely adopted 2015 American College of Medical Genetics (ACMG) and Association for Molecular Pathology (AMP) guidelines for the interpretation of sequence variants include the PS3 criterion for consideration of evidence from well-established in vitro or in vivo assays. The wealth of information on underlying mechanisms of LQT1-LQT3 and recent HT patch clamp data support consideration of patch clamp data together (for LQT1 and LQT2) with information from biochemical trafficking assays as meeting the PS3 criterion of well established assays, able to provide ‘strong’ evidence for functional pathogenicity of identified VUS.