High-Throughput Reclassification of SCN5A Variants

Abstract

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 (VUSs). The designation of VUS is a barrier to the use of sequence data in clinical care. We selected 83 variants: 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 individuals 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. The ten previously studied controls had functional properties concordant with published manual patch clamp data. All 10 suspected benign variants had wild-type-like function. 22 suspected BrS variants had loss of channel function (<10% normalized peak current) and 22 variants had partial loss of function (10%-50% normalized peak current). The previously unstudied variants were initially classified as likely benign (n ¼ 2), likely pathogenic (n ¼ 10), or VUSs (n ¼ 61). After the patch clamp studies, 16 variants were benign/likely benign, 45 were pathogenic/likely pathogenic, and only 12 were still VUSs. Structural modeling identified likely mechanisms for loss of function including altered thermostability and disruptions to alpha helices, disulfide bonds, or the permeation pore. High-throughput patch clamping enabled reclassification of the majority of tested VUSs in SCN5A.

Phenotypic and biochemical characteristics and molecular basis in 36 Chinese patients with androgen receptor variants

“…According to the guideline of ACMG, well-established in vitro functional studies (PS3 and BS3) may reclassify the variants. In a previous study, 61 SCN5A variants initially predicted as VUS by in-silico analyses were reclassified as benign/likely benign (n = 14), likely pathogenic (n = 35) and still VUS (n = 12) via high-throughput automated patch clamping [ 27 ]. In our study, it was found that all the novel AR variants, which were initially predicted to be VUS, significantly impaired the transcriptional activity of AR and were reclassified as pathogenic/likely pathogenic, except p.L295P (Table 1 and Fig.…”

A Bayesian method to estimate variant-induced disease penetrance

“…clinically phenotyping individuals heterozygous for these variants. The interchangeability of this information was additionally demonstrated recently by taking the reverse approach: functionally characterizing variants with different estimates of BrS1 penetrance [21]. In these experiments, Glazer et al found that variants with a higher estimated BrS1 penetrance had a higher probability of producing a variant-induced loss-of-function protein phenotype (Fig 3A in reference 21).…”

Enhancing rare variant interpretation in inherited arrhythmias through quantitative analysis of consortium disease cohorts and population controls

“…Second, loss-offunction evidence (PVS1) was implemented according to recommendations from the ClinGen Sequence Variant Interpretation Workgroup. 14 Third, functional evidence rules (PS3/ BS3) for BrS/SCN5A were applied using published data curated by Denham et al 6 and a high-throughput functional study by Glazer et al 15 Note that evidence relating to cosegregation (PP1/ BS4), de novo inheritance (PS2/PM6), and functional data for LQTS (PS3/BS3) could not be applied due to the lack of curated resources. While this will affect the yield of (likely) pathogenic variants, cosegregation evidence is not expected to be particularly informative for BrS as family pedigrees tend to be of limited sizes with numerous examples of non-Mendelian segregation.…”