A rare non-coding enhancer variant in SCN5A contributes to the high prevalence of Brugada syndrome in Thailand

Brugada syndrome (BrS) is a cardiac arrhythmia disorder that causes sudden death in young adults. Rare genetic variants in the SCN5A gene, encoding the Nav1.5 sodium channel, and common non-coding variants at this locus, are robustly associated with the condition. BrS is particularly prevalent in Southeast Asia but the underlying ancestry-specific factors remain largely unknown. Methods: Genome sequencing of BrS probands and population-matched controls from Thailand was performed to identify rare non-coding variants at the SCN5A-SCN10A locus that were enriched in BrS cases. A likely causal variant was prioritised by computational methods and introduced into human pluripotent stem cell (hiPSC) lines using CRISPR-Cas9. The effect of the variant on SCN5A expression and Nav1.5 sodium channel current was then assessed in hiPSC-derived cardiomyocytes (hiPSC-CMs). Results: A rare non-coding variant in an SCN5A intronic enhancer region was highly enriched in BrS cases (detected in 3.9% of cases with a case-control odds ratio of 45.2). The variant affects a nucleotide conserved across all mammalian species and predicted to disrupt a Mef2 transcription factor binding site. Heterozygous introduction of the enhancer variant in hiPSC-CMs caused significantly reduced SCN5A expression from the variant-containing allele and a 30% reduction in Nav1.5-mediated sodium current density compared to isogenic controls, confirming its pathogenicity. Patients with the variant had severe phenotypes, with 89% experiencing cardiac arrest. Conclusions: This is the first example of a functionally validated rare non-coding variant at the SCN5A locus and highlights how genome sequencing in understudied populations can identify novel disease mechanisms. The variant partly explains the increased prevalence of BrS in this region and enables the identification of at-risk variant carriers to reduce the burden of sudden cardiac death in Thailand. Overall design: The RE5 GRCh38:chr3-38580380-A-C variant was introduced in heterozygous state in the PGP1 hiPSC line by CRISPR/Cas9-mediated editing (Synthego). In order to quantify the potential cis-regulatory effect of the RE5 variant ( intronic) on SCN5A allelic expression we isolated bulk messanger RNA from cardiomyocytes of the control PGP1-SV1 hiPSC line and isogenic variant-carrying A7 hiPSC line, followed by RNAseq analysis for quantification of the allele-specific transcript outputs. To distinguish transcripts originating from the two alleles, since the intronic variant was not identifiable from the messenger RNA, we took advantage of a heterozygous exonic variant (c.3183A>G ) located 596bp from the intronic variant and determined their phase by Sanger sequencing of the genomic fragment.