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Genome-wide association studies previously identified an association of rs9388451 at chromosome 6q22.3 (near HEY2) with Brugada syndrome. The causal gene and underlying mechanism remain unresolved.We used an integrative approach entailing transcriptomic studies in human hearts and electrophysiological studies in Hey2+/− (Hey2 heterozygous knockout) mice to dissect the underpinnings of the 6q22.31 association with Brugada syndrome.We queried expression quantitative trait locus data acquired in 190 human left ventricular samples from the genotype-tissue expression consortium for cis-expression quantitative trait locus effects of rs9388451, which revealed an association between Brugada syndrome risk allele dosage and HEY2 expression (β=+0.159; P=0.0036). In the same transcriptomic data, we conducted genome-wide coexpression analysis for HEY2, which uncovered KCNIP2, encoding the β-subunit of the channel underlying the transient outward current (Ito), as the transcript most robustly correlating with HEY2 expression (β=+1.47; P=2×10−34). Transcript abundance of Hey2 and the Ito subunits Kcnip2 and Kcnd2, assessed by quantitative reverse transcription–polymerase chain reaction, was higher in subepicardium versus subendocardium in both left and right ventricles, with lower levels in Hey2+/− mice compared with wild type. Surface ECG measurements showed less prominent J waves in Hey2+/− mice compared with wild-type. In wild-type mice, patch-clamp electrophysiological studies on cardiomyocytes from right ventricle demonstrated a shorter action potential duration and a lower Vmax in subepicardium compared with subendocardium cardiomyocytes, which was paralleled by a higher Ito and a lower sodium current (INa) density in subepicardium versus subendocardium. These transmural differences were diminished in Hey2+/− mice because of changes in subepicardial cardiomyocytes.This study uncovers a role of HEY2 in the normal transmural electrophysiological gradient in the ventricle and provides compelling evidence that genetic variation at 6q22.31 (rs9388451) is associated with Brugada syndrome through a HEY2-dependent alteration of ion channel expression across the cardiac ventricular wall.