Abstract 9803: Most SCN5A Missense Loss-of-Function Variants Also Have a Dominant Negative Effect

Abstract

Introduction: Up to 30% of patients with Brugada Syndrome (BrS) carry loss of function (LoF) variants in the cardiac sodium channel gene SCN5A . Recent studies have suggested that the protein product Na V 1.5 can form dimers, suggesting the possibility that LoF variants may have dominant negative effects. Methods: We identified 36 SCN5A missense variants previously found to have peak current <10% compared to wild-type (WT). HEK293T cells were engineered to stably express one or two copies of SCN5A using AttB-AttP landing pad and Sleeping Beauty transposon systems. Cell lines were created expressing the missense LoF SCN5A variants alone or in heterozygous co-expression with WT SCN5A . Channel expression was validated by flow cytometry and peak sodium currents were measured using automated patch clamp (>20 replicate cells/variant). To assess clinical risk, we compared case versus control ratios among different variant mechanisms using a published BrS case consortium and the gnomAD population database. Results: 32/36 LoF variants studied alone had peak current <10% as expected. In heterozygous experiments, WT alone was normalized to 100% and WT+WT showed ~200% peak current. In heterozygous expression with WT, 29/32 LoF variants showed a reduction to less than 75% normalized peak current, demonstrating a dominant negative effect. We next tested the hypothesis that dominant negative variants carry a higher disease risk than other variant classes. We observed a 5.0-fold enrichment for BrS cases among carriers of dominant negative missense variants compared to putative haploinsufficient variants (frameshift/splice site) (p=0.003). Conclusions: Most SCN5A missense LoF variants also have a dominant negative effect. Case-control comparisons reveal that this variant class carries an outsized BrS risk compared to other classes of pathogenic variants. Figure . A) SCN5A homozygous expression. B) SCN5A heterozygous expression. C) Case-control analysis.