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 = 2), likely pathogenic (n = 10), or VUSs (n = 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.

Keywords: Brugada syndrome; Na(V)1.5; SCN5A; high-throughput; patch clamp; variant of uncertain significance.

Figure 1

Molecular and Functional Expression of SCN5A (A) Flow cytometry plot of representative wild-type stable cell line. Histogram of mCherry signal is shown. 89.8% of cells expressed a high-level of mCherry, indicating successful plasmid integration. (B) Boxplot of percentage of wild-type wells with Na_V1.5-like current across eight independent transfections. Only wells with a cell passing seal resistance and capacitance criteria (see Material and Methods) were considered. (C) Wild-type current density-voltage plot, averaged across 405 wild-type cells passing quality control (seal resistance, capacitance, in voltage control, and minimum peak current criteria, see Material and Methods). The predicted reversal potential given the internal and external solutions used in this study is 45.3 mV. Error bars indicate SEM. (D) Example SyncroPatch experiment. A typical experiment studied 5 variants and wild-type on a single 384-well chip. In this experiment all 5 variants had wild-type-like currents. Green wells indicate a cell with seal resistance >0.5 GΩ, blue wells indicate a cell with seal resistance 0.2–0.5 GΩ (not included in analysis), and gray wells indicate no cell present or a cell with seal resistance <0.2 GΩ (not included in analysis). Randomly selected cells with seals >0.5GΩ are highlighted with a black square and displayed on the right.

Figure 2

Suspected Brugada Syndrome Variants Have Reduced Peak Current (A) Peak current density of ten variants previously studied by manual patch clamp. Mean ± standard errors. Left: literature values. For some variants, standard errors were not reported. Right: automated patch clamp values (this study). (B) Selection of suspected benign and suspected BrS-associated variants. Each data point is a variant. Points were “jittered” in the x and y axes with a small random number so that points with identical values would be visible. The x axis is the minor allele frequency in the gnomAD database. The y axis indicates the number of individuals with Brugada syndrome in the literature. Values are taken from a recent collation of SCN5A data from the literature. p.Arg1958Ter was classified as a suspected BrS variant because it is a nonsense variant, even though it has not been observed in any cases of BrS. (C) Current-voltage curves for selected variants, showing an example of a normal variant (p.Val924Ile), a partial loss-of-function variant (p.Val396Leu), and a loss-of-function variant (p.Glu901Lys). Error bars indicate standard error of the mean. (D and E) Violin plots comparing peak current density (D) or voltage of 1/2 activation (E) for suspected Brugada syndrome versus suspected benign variants. Wild-type values are indicated with a dashed black line, and cutoffs for deleteriousness are indicated with a red line (50% peak current or 10 mV rightward shift in V_1/2 activation). p values are from a two-tailed Mann-Whitney U test. For a complete list of measured parameters, see Table S7 and Data S1. (F) Peak current density (normalized to wild-type) for all previously unstudied variants. Error bar indicates standard error of the mean.

Figure 3

Reclassifications with Patch Clamp Data (A) Variants were classified according to the American College of Medical Genetics and Genomics criteria. Above: classifications before the patch clamp data in this study. Below: classifications with the patch clamp data in this study. All classifications indicate Brugada syndrome, except for p.Arg814Gln, which was reclassified from likely pathogenic for Brugada syndrome to likely pathogenic for long QT syndrome. (B) Patch clamp results varied depending on the number of observed individuals with Brugada syndrome in the literature^, and the gnomAD allele frequency. LoF, loss of function.

Figure 4

Structural Basis of SCN5A Loss-of-Function Variants (A) Two-dimensional schematic of Na_V1.5 structure. All previously unstudied variants are shown and color-coded based on peak current density (white 75%–125%, gray 50%–75%, orange 10%–50%, red < 10%). (B and C) Three-dimensional homology model of Na_V1.5. Variants are colored as in (A). (D) Top-down view of WT (top) and p.Glu901Lys (bottom), as modeled using Rosetta. The lysine residue projects into the pore, likely disrupting sodium passage. (E) View of WT and p.Cys335Arg, as modeled using Rosetta. The WT protein has a disulfide bond between Cys335 (left) and Cys280 (right), which was inferred from the spatial proximity of these two residues and the fact that the corresponding residues in the template structures also form a disulfide bond; this bond is disrupted by p.Cys335Arg. The disulfide bond is indicated with an asterisk (^∗). (F) Four leucine -> proline variants in this study. p.Leu136Pro is a partial loss-of-function variant and p.Leu839P, p.Leu928Pro, and p.Leu1340Pro are loss of function. The structures of these four variants were not modeled because modeling drastic structural changes involving prolines that are part of a helix usually cannot be reliably modeled in Rosetta. However, these variants likely cause loss of protein function by causing a kink in the alpha helix and protein misfolding.