Multi-site validation of a functional assay to adjudicate Brugada Syndrome-associated variants.

Brugada Syndrome (BrS) is an inheritable arrhythmia condition that is associated with rare, loss-of-function variants in the cardiac sodium channel gene, . Interpreting the pathogenicity of missense variants is challenging and ~79% of missense variants in ClinVar are currently classified as Variants of Uncertain Significance (VUS). An -BrS automated patch clamp assay was generated for high-throughput functional studies of Na1.5. The assay was independently studied at two separate research sites - Vanderbilt University Medical Center and Victor Chang Cardiac Research Institute - revealing strong correlations, including peak density (=0.86). The assay was calibrated according to ClinGen Sequence Variant Interpretation recommendations using high-confidence variant controls (n=49). Normal and abnormal ranges of function were established based on the distribution of benign variant assay results. The assay accurately distinguished benign controls (24/25) from pathogenic controls (23/24). Odds of Pathogenicity values derived from the experimental results yielded 0.042 for normal function (BS3 criterion) and 24.0 for abnormal function (PS3 criterion), resulting in up to strong evidence for both ACMG criteria. The calibrated assay was then used to study VUS observed in four families with BrS and other arrhythmia phenotypes associated with loss-of-function. The assay revealed loss-of-function for three of four variants, enabling reclassification to likely pathogenic. This validated APC assay provides clinical-grade functional evidence for the reclassification of current VUS and will aid future -BrS variant classification.