PO-691-03 FUNCTIONAL ASSAYS IDENTIFY SPLICE-ALTERING VARIANTS IN MENDELIAN CHANNELOPATHIES

Abstract

Rare protein-altering variants in SCN5A, KCNQ1, and KCNH2 are major causes of Brugada Syndrome (BrS) and the Long QT Syndrome (LQTS). While splice-altering variants lying outside the 2 bp canonical splice sites can cause these diseases, their role remains poorly described in these arrhythmia syndromes. We implemented two functional assays to assess pathogenicity of 11 putative splice-altering variants of uncertain significance (VUS) in LQTS and BrS patients; 8/11 were intronic. We identified putative variants for study from a recent curation of rare variants detected in BrS and LQTS patients. We deployed the well established in vitro minigene assays to assess the splicing consequences of 9 VUS in the 3 genes. Two SCN5A variants were incompatible with the minigene approach, so we introduced each into a healthy control induced pluripotent stem cell line (iPSC) by CRISPR-Cas9 gene editing. We differentiated cells into iPSC cardiomyocytes (iPSC-CMs) and studied splicing by RT-PCR. We used the American College of Medical Genetics and Genomics (ACMG) functional assay criteria to reclassify VUS. We compared our assays to splicing predictions from the in silico tool SpliceAI. We identified aberrant splicing, with presumed disruption of protein sequence, in 7/9 VUS studied using the minigene assay and 1/2 in iPSC-CMs. We were able to reclassify 11 VUS, 10 to likely pathogenic, and 1 to likely benign. The computational tool SpliceAI had moderate sensitivity for the in vitro splicing results above a predictive threshold of 0.5 (7/11). VUS that are not located in canonical splice sites can nevertheless disrupt splicing and confer pathogenicity in BrS and LQTS.