Po-01-240 utility of functional characterization in the promotion or demotion of clinically encountered variants of uncertain significance in the three most common long qt syndrome-causative genes: , kcnh, and

Genetic testing for cardiac channelopathies such as long QT syndrome (LQTS) and Brugada syndrome (BrS) is standard of care. Although the American College of Medical Genetics (ACMG) guidelines for variant interpretation have provided a framework for distinguishing pathogenic variants from benign, many rare genetic variants remain classified as a variant of uncertain significance (VUS) due to lack of epidemiologic or functional data to either promote or demote the variant to pathogenic or benign status, respectively. To functionally characterize clinically encountered VUSs in the three most common LQTS-causative genes: KCNQ1 (LQT1), KCNH2 (LQT2), and SCN5A (LQT3 and BrS1) in vitro by patch clamp technique to provide additional evidence for variant promotion/demotion. A total of 89 rare nonsynonymous VUS (15 KCNQ1, 19 KCNH2, and 55 SCN5A) were engineered by site directed mutagenesis and expressed in either HEK293 cells or TSA201 cells. Whole cell patch-clamp technique was used to functionally characterize these 89 variants to determine gain-of-function (GOF), loss-of-function (LOF), or normal channel function. Overall, the majority of VUS (50/89; 56.2%) studied displayed a wild-type-like electrophysiological phenotype and enabled demotion to likely benign status. The remaining VUS (39/89; 43.8%) were promoted to likely pathogenic status based on either a GOF or LOF electrophysiological signature following patch-clamp analysis. Among 15 KCNQ1 variants, 11 (73.3%) had normal channel function, 3 (20.0%) LOF, and 1 (6.7%) GOF. Of the 19 KCNH2 variants, 11 (57.9%) displayed normal channel function, 5 (26.3%) LOF, and 3 (15.8%) GOF. Finally, among the 55 SCN5A variants, 28 (50.9%) exhibited normal channel function, 19 (34.5%) LOF, and 8 (14.5%) GOF. Here, we provide functional characterization of 89 nonsynonymous VUSs identified during the course of clinically indicated genetic testing. Consistent with the clinical index of suspicion in the VUS-positive subject, 44% of the VUS were promoted to a likely pathogenic variant while 56% were demoted to a likely benign variant. Whether genetic test companies will incorporate this functionally characterized compendium of VUS and issue amended genetic test reports awaits their review and incorporation into their internal framework for variant annotation.