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.

SCN5A部分或完全功能丧失变异是心律失常疾病 Brugada 综合征 (BrS1) 最常见的遗传原因。然而， SCN5A变异体的致病性常常未知或存在争议；迄今为止，在至少一个个体中观察到的 1,390 个SCN5A错义变异中，80% 是意义不确定的变异 (VUS)。 VUS 的指定是在临床护理中使用序列数据的障碍。我们选择了 83 个变异：10 个先前研究的对照变异、10 个疑似良性变异和 63 个疑似 Brugada 综合征相关变异，这些变异是根据其在一般人群和 Brugada 综合征个体中的频率进行选择的。我们使用高通量自动膜片钳来研究 83 个变体的功能，目的是利用功能数据对变体进行重新分类。先前研究的十个对照的功能特性与已发布的手动膜片钳数据一致。所有 10 种疑似良性变异均具有野生型样功能。 22 个可疑 BrS 变体具有通道功能丧失（<10 id=46>SCN5A .