Automated patch clamp data improve variant classification and penetrance stratification for SCN5A –Brugada syndrome

Figure 1. Schematic of project background and experimental design. (A) The automated patch-clamp (APC) assay measures electrophysiological properties of SCN5A variants that are stably expressed in HEK293 cell lines. Variant peak current densities are normalized to WT measurements made on the same plate. (B) For implementation into the ACMG/AMP framework, we report variant function as a Z-score, which is calculated as multiples of the standard deviation from the mean of 25 B/LB control variants. Datapoints indicate the median of each benign and pathogenic variant control, and the lines represent the mean and interquartile range of each group. (C) Here, we studied 252 missense and in-frame insertion/deletion variants observed among 458 patients undergoing evaluation for BrS. The APC uncovered a spectrum of effects among the studied channels. Datapoints indicate the median of each cohort variant (grey), and lines represent the mean and interquartile range of the cohort

Figure 2. Functional scores of SCN5A-BrS variants by protein domain. (A) Z-scores of variants in the cohort plotted against position in the protein. (B) Two-dimensional topological view of NaV1.5. (C) Three-dimensional structure of hNaV1.5 (PDB: 8VYJ24). Many N-term, IDL, and C-term variants are not present in the structure and are omitted from this plot. (D) Counts of functional criteria by location in protein. A–D. Variants are colour-coded by ACMG/AMP classification level as defined in the legend in the lower right. Most variants found in the BrS cases are in the transmembrane and pore loop regions, and these regions are also enriched for loss-of-function variants. N-term indicates N-terminus; TM, transmembrane; P, pore; IDL, interdomain linker; C-term, C-terminus; pCD, peak current density

Figure 3. Proportion of individuals that have ultra-rare or rare SCN5A variants by protein domains. (A) Ultra-rare variants (gnomAD v4.1 FAF <0.00001) are located mostly in the transmembrane (TM) domains and are functionally abnormal except for the few in the Inter-domain Linker (IDL) region. (B) Rare variants with a gnomAD v4.1 FAF between 0.00001 and 0.001 are mostly functionally normal in all domains

Figure 4. Impact of functional data (PS3/BS3) on variant classifications. Variant classifications for 252 SCN5A variants from the BrS cohort were pre- and post-applying functional APC evidence (PS3_strong to BS3_moderate). Variants were classified using population frequency (BS1), variant rarity (PM2_supporting), hotspot evidence (PM1 at a maximum of moderate), in-frame indel (PM4), and case enrichment (PS4). A through D, Calibrated in silico predictions (PP3/BP4) from REVEL (A, B) or AlphaMissense (C, D) were applied, at a maximum level of 4 points when combined with PM1. Sankey plots show flow between functional evidence and variant classifications for calibrated REVEL (B) and calibrated AlphaMissense (D). A full list of criteria is provided in Table 1

Figure 5. BrS Penetrance and odds ratio based on classifications or variant features. Variants were stratified by hotspot evidence (A), calibrated REVEL (B), calibrated AlphaMissense (C), functional evidence (using ACMG/AMP evidence categories) (D), final classification (integrating all criteria) (E) or functional evidence derived from pCD Z-score with brackets inclusive and parentheses exclusive of respective values (F). Data shown represent mean and 95% CI (see Supplementary data online, Table S9)