B-AB13-05 HIGH-THROUGHPUT PATCH CLAMP OF INDEL AND MISSENSE VARIANTS IN SCN5A

Abstract

Variants in SCN5A are associated with congenital arrhythmias including Brugada Syndrome (BrS; decreased channel function) and type 3 Long QT Syndrome (LQT3; increased channel function). A majority of SCN5A variants are currently classified as variants of uncertain significance (VUS). Thus, clinicians are unable to make genotype-guided medical decisions for carriers of these variants. We aimed to clinically (re)classify and identify the mechanisms underlying SCN5A insertion/deletion (indel) and missense variants reported in cases of BrS and/or LQT3. Candidate BrS/LQT3-associated indel (n=23) and LQT3-associated missense (n=64) variants were selected based on prevalence in reported cases and their low frequency in the population database gnomAD. Suspected-benign variants (n=25) were selected which were present at least 15 times in gnomAD but absent from published cases. We created stable HEK293T lines expressing these variants, validated their SCN5A expression by flow cytometry, and studied them with automated patch clamping. For each variant, 6 parameters were measured, including peak and late current, inactivation time, and recovery from inactivation, in at least 20 cells. 10/23 in-frame indel variants had partial or complete loss of function (LoF, <50% peak current), 6 of which have previously been found in at least 1 case of BrS. 4/23 indel variants had at least one gain of function (GoF) feature, including elevated peak or late current, slower inactivation time and faster recovery from inactivation; all 4 have been found in at least 1 case of LQT3. 22/64 suspected LQT3-associated missense variants had at least one GoF feature. In contrast, only 2/25 suspected benign variants had GoF features. This result indicates that GoF features are readily captured by automated patch-clamp and are specific for LQT3-associated variants. Using the missense and indel data, 33 variants were reclassified from VUS to likely pathogenic. High-throughput patch clamp can identify loss and gain of function variants in SCN5A. In-frame indels are a substantial contributor to SCN5A-mediated arrhythmia risk. Missense variants in SCN5A associated with LQT3 can display multiple electrophysiological defects.