Functional Assays Reclassify Suspected Splice-Altering Variants of Uncertain Significance in Mendelian Channelopathies

Abstract

Background: Rare protein-altering variants in SCN5A, KCNQ1, and KCNH2 are major causes of Brugada Syndrome (BrS) and the congenital Long QT Syndrome (LQTS). While splice-altering variants lying outside 2-bp canonical splice sites can cause these diseases, their role remains poorly described. Objective: We implemented two functional assays to assess 12 recently reported putative splice-altering variants of uncertain significance (VUS) and 1 likely pathogenic (LP) variant without functional data observed in BrS and LQTS probands. Methods: We deployed minigene assays to assess the splicing consequences of 10 variants. Three variants incompatible with the minigene approach were introduced into control induced pluripotent stem cells (iPSCs) by CRISPR genome editing. We differentiated cells into iPSC-derived cardiomyocytes (iPSC-CMs) and studied splicing outcomes by reverse transcription-polymerase chain reaction (RT-PCR). We used the American College of Medical Genetics and Genomics functional assay criteria (PS3/BS3) to reclassify variants. Results: We identified aberrant splicing, with presumed disruption of protein sequence, in 8/10 variants studied using the minigene assay and 1/3 studied in iPSC-CMs. We reclassified 9 VUS to LP, 1 VUS to Likely Benign, and 1 LP variant to pathogenic. Conclusions: Functional assays reclassified splice-altering variants outside canonical splice sites in BrS- and LQTS-associated genes.

How Functional Genomics Can Keep Pace With VUS Identification

“…While the tools described herein primarily cover variants in coding-regions, non-coding VUS are also prevalent in cardiac disease and several in silico tools have been developed to assess those, notably the neural network-based splice variant predictor SpliceAI ( 117 ). These tools along with reporter assays should help toward pathogenic assessment of splice variants via ACMG guidelines ( 118 ).…”