2. Academic Validation
  3. Multi-site validation of a functional assay to adjudicate SCN5A Brugada Syndrome-associated variants

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

  • medRxiv. 2023 Dec 20:2023.12.19.23299592. doi: 10.1101/2023.12.19.23299592.
Joanne G Ma 1 2,  Matthew J O'Neill 3,  Ebony Richardson 4,  Kate L Thomson 5,  Jodie Ingles 4,  Ayesha Muhammad 3,  Joseph F Solus 6,  Giovanni Davogustto 7,  Katherine C Anderson 7,  M Benjamin Shoemaker 7,  Andrew B Stergachis 8,  Brendan J Floyd 9,  Kyla Dunn 9,  Victoria N Parikh 9,  Henry Chubb 9,  Mark J Perrin 10,  Dan M Roden 11,  Jamie I Vandenberg 1 2,  Chai-Ann Ng 1 2,  Andrew M Glazer 6
Affiliations

Affiliations

  • 1 Mark Cowley Lidwill Research Program in Cardiac Electrophysiology, Victor Chang Cardiac Research Institute, Darlinghurst, NSW, Australia.
  • 2 School of Clinical Medicine, UNSW Sydney, Darlinghurst, NSW, Australia.
  • 3 Vanderbilt University School of Medicine, Nashville, TN, USA.
  • 4 Clinical Genomics Laboratory, Centre for Population Genomics, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia and Murdoch Children Research Institute, Melbourne, Australia.
  • 5 Oxford Genetics Laboratories, Churchill Hospital, Oxford, UK.
  • 6 Vanderbilt Center for Arrhythmia Research and Therapeutics (VanCART), Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA.
  • 7 Division of Cardiovascular Medicine, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA.
  • 8 University of Washington School of Medicine, Department of Medicine, Seattle, WA, USA.
  • 9 Stanford Center for Inherited Cardiovascular Disease, Stanford University School of Medicine, Stanford, CA, USA.
  • 10 Department of Genomic Medicine, Royal Melbourne Hospital, Victoria, Australia.
  • 11 Vanderbilt Center for Arrhythmia Research and Therapeutics (VanCART), Departments of Medicine, Pharmacology, and Biomedical Informatics, Vanderbilt University Medical Center, Nashville, TN, USA.
PMID: 38196587 DOI: 10.1101/2023.12.19.23299592
Abstract

Brugada Syndrome (BrS) is an inheritable arrhythmia condition that 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). An in vitro SCN5A-BrS automated patch clamp assay was generated for high-throughput functional studies of NaV1.5. 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 INa density (R2=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 SCN5A VUS observed in four families with BrS and Other arrhythmia phenotypes associated with SCN5A 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 SCN5A-BrS variant classification.

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