High-throughput reclassification ofSCN5Avariants

Abstract

Rationale: Partial or complete loss of function variants in SCN5A are the most common genetic cause of the arrhythmia disorder Brugada Syndrome (BrS1). However, the pathogenicity of SCN5A variants is often unknown or disputed; 80% of the 1,390 SCN5A missense variants observed in at least one individual to date are variants of uncertain significance (VUS). The designation of VUS is a barrier to the use of sequence data in clinical care. Objective:We selected 83 variants for study: 10 previously studied control variants, 10 suspected benign variants, and 63 suspected Brugada Syndrome-associated variants, selected on the basis of their frequency in the general population and in patients with Brugada Syndrome. We used high-throughput automated patch clamping to study the function of the 83 variants, with the goal of reclassifying variants with functional data.Methods and Results: Ten previously studied variants had functional properties concordant with published manual patch clamp data. All 10 suspected benign variants had wildtype-like function. 22 suspected BrS variants had loss of channel function (<10% normalized peak current) and 23 variants had partial loss of function (10-50% normalized peak current). The 73 previously unstudied variants were initially classified as likely benign (n=2), likely pathogenic (n=11), or VUS (n=60). After the patch clamp studies, 16 variants were benign/likely benign, 47 were pathogenic/likely pathogenic, and only 10 were still VUS. 8/22 loss of function variants were partially rescuable by incubation at lower temperature or pretreatment with a sodium channel blocker. Structural modeling identified likely mechanisms for loss of function including altered thermostability, and disruptions to alpha helices, disulfide bonds, or the permeation pore. Conclusions:High-throughput automated patch clamping enabled the reclassification of the majority of tested VUS's in SCN5A.

Brugada syndrome masked by complete left bundle branch block: A clinical and functional study of its association with the p.1449Y>H SCN5A variant

“…Nowadays, functional evidence is considered the major score driver for pathogenicity assumption for missense variants. According to a recent re‐evaluation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG‐AMP) rules for assessment of pathogenicity, 2,9 the SCN5A variant identified in this report can be classified as pathogenic, since it fulfills the following criteria: (i) the p.1449Y>H variant is a missense variant that leads to the replacement of tyrosine by histidine at position 1449, in the transmembrane S6 region of DIII, which is part of the pore region of the Na v 1.5 channel and, according to the SCN5A variant browser, is a hotspot region for BrS1 (ACMG category PM1, moderate evidence); 8 (ii) other variants producing amino acid changes in the same residue has been previously established as pathogenic and associated to clinical BrS (p.1449Y>S) and also to conduction disease with partial loss of function in in vitro studies (p.1449Y>C; PS1, strong evidence) 9–12 . These results indicate that the conserved Y1449 is crucial for the proper functioning of the Na v 1.5 channel, and that its alteration induces dramatic changes in channel activity; and (iii) p.1449Y>H mutant encodes a not fully functional channel that generates extremely small (2% as compared to the WT) sodium currents, and a decrease more than 50% in the peak current is significantly associated with BrS1 penetrance (PS3, strong evidence) 2,9 …”

Dominant negative effects ofSCN5Amissense variants

“…Variants for this study were selected from previously published functionally characterized variants (6, 14). Variants with peak currents <10% compared to WT were considered LoF, and variants with peak currents between 10-50% compared to WT were considered partial LoF.…”

“…The SCN5A variant plasmids were mutagenized using a previously described “zone” system (14). Briefly, SCN5A individual zones on small plasmids were mutagenized using the QuikChange Lightning Multi kit (Agilent) with primers designed using the online QuikChange Primer Design tool.…”

“…Primers used in this study are listed in Supplementary Table 2. The variant-containing zone was then subcloned by restriction digestion into a plasmid containing an AttB:SCN5A:IRES:mCherry-blasticidinR plasmid (14–16). The entire sequence of the zone containing the variant was confirmed by Sanger sequencing.…”

Incessant atrial and ventricular tachycardias associated with an SCN5A mutation

“…The L939P mutation was introduced into an SCN5A expression plasmid, 9 and wildtype and L939P plasmids were transiently transfected into Chinese Hamster Ovary (CHO) cells and studied by manual whole cell patch clamping, as previously described. 10 Compared to wildtype, L939P showed a ~75% decrease in peak current density at -30 mV (119.5±16.7 pA/pF for WT vs. 30.3±4.2 pA/pF for L939P, p<0.01, Figure 3).…”