Multisite Validation of a Functional Assay to Adjudicate Brugada Syndrome-Associated Variants.

Background: Brugada syndrome is an inheritable arrhythmia condition that is associated with rare, loss-of-function variants in . Interpreting the pathogenicity of missense variants is challenging, and ≈79% of missense variants in ClinVar are currently classified as variants of uncertain significance. Automated patch clamp technology enables high-throughput functional studies of ion channel variants and can provide evidence for variant reclassification.

Methods: An in vitro -Brugada syndrome automated patch clamp assay was independently performed at Vanderbilt University Medical Center and Victor Chang Cardiac Research Institute. 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. Odds of pathogenicity values were derived from the experimental results according to ClinGen Sequence Variant Interpretation recommendations. The calibrated assay was then used to study variants of uncertain significance observed in 4 families with Brugada syndrome and other arrhythmia phenotypes associated with loss-of-function.

Results: Variant channel parameters generated independently at the 2 research sites showed strong correlations, including peak density (=0.86). The assay accurately distinguished benign controls (24/25 concordant variants) from pathogenic controls (23/24 concordant variants). Odds of pathogenicity values were 0.042 for normal function and 24.0 for abnormal function, corresponding to strong evidence for both American College of Medical Genetics and Genomics/Association for Molecular Pathology benign and pathogenic functional criteria (BS3 and PS3, respectively). Application of the assay to 4 clinical variants of uncertain significance revealed loss-of-function for 3/4 variants, enabling reclassification to likely pathogenic.

Conclusions: This validated high-throughput assay provides clinical-grade functional evidence to aid the classification of current and future -Brugada syndrome variants of uncertain significance.