Multi-site validation of a functional assay to adjudicate SCN5A Brugada Syndrome-associated variants

Abstract Background: Brugada Syndrome (BrS) is an inherited arrhythmia condition that increases the risk of sudden cardiac death. BrS is associated with rare, loss-of-function variants in the cardiac sodium channel gene, SCN5A. Interpreting the pathogenicity of SCN5A missense variants is challenging and ~79% of SCN5A missense variants in ClinVar are currently classified as Variants of Uncertain Significance (VUS). Automated patch clamp (APC) technology enables high-throughput functional studies of ion channel variants and can provide evidence for variant reclassification. Methods: An APC assay was developed to provide calibrated functional evidence of the impact of SCN5A missense variants on channel function. The assay was validated according to the American College of Medical Genetics and Genomics/Association for Molecular Pathology variant interpretation guidelines. Stably transfected SCN5A-expresing Human Embryonic Kidney 293 cell lines were independently generated and studied at two separate research sites: Vanderbilt University Medical Center and Victor Chang Cardiac Research Institute. For each variant, multiple channel parameters were measured using the SyncroPatch 384PE APC platform. The assay was calibrated using high-confidence variant controls (n=25 benign and n=24 likely pathogenic or pathogenic). 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 SCN5A VUS observed in four families with BrS and other arrhythmia phenotypes associated with SCN5A loss-of-function. Results: Variant channel parameters generated independently at the two research sites showed strong correlations, including peak INa density (R2=0.86). The assay accurately distinguished benign controls (24/25 concordant variants) from pathogenic controls (23/24 concordant variants). The assay yielded Odds of Pathogenicity of 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. Application of the assay to four clinical SCN5A VUS revealed loss-of-function for three of four variants, enabling reclassification to likely pathogenic. Conclusion: This APC assay provides clinical-grade functional evidence for the classification of current VUS and will aid future SCN5A-BrS variant classification. ### Competing Interest Statement Dr. Glazer is a consultant for BioMarin, Inc. Victoria Parikh is a scientific advisory board member (Lexeo Therapeutics), clinical advisor (Constantiam Biosciences) and consultant (BioMarin, Inc., Viz.ai). Victoria Parikh also receives research support from BioMarin, Inc. ### Funding Statement This study was funded by the National Institutes of Health (NIH): R01 HL164675 (DMR), R01 HL149826 (DMR), R00 HG010904 (AMG), R01 HG013025 (ABS), and R35 GM150465 (AMG), a New South Wales Cardiovascular Disease Senior Scientist grant (JIV), and a MRFF Genomics Health Futures Mission grant MRF2016760 (JIV and CAN). MJO received support from NIH grants F30HL163923 and T32GM007347. We also acknowledge support from the Victor Chang Cardiac Research Institute Innovation Centre, funded by the NSW Government. VUMC flow cytometry experiments were performed in the Vanderbilt Flow Cytometry Shared Resource. The Vanderbilt Flow Cytometry Shared Resource is supported by the Vanderbilt Ingram Cancer Center (NIH P30 CA68485) and the Vanderbilt Digestive Disease Research Center (NIH DK058404). The VUMC Nanion SyncroPatch 384PE is housed and managed within the Vanderbilt High-Throughput Screening Core Facility, an institutionally supported core, and was funded by NIH Shared Instrumentation Grant 1S10OD025281. The HTS Core receives support from the Vanderbilt Institute of Chemical Biology and the Vanderbilt Ingram Cancer Center (NIH P30 CA68485). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The IRB of University of Washington Human Subjects Division gave ethical approval for this work. The IRB of Stanford University School of Medicine gave ethical approval for this work. The IRB of Vanderbilt University Medical Center gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors.