Abstract 16901: Utilizing In Silico Pathogenicity Prediction Tools and Heterologous Expression Patch Clamp Studies to Identify Bona Fide Pathogenic KCNH2 Variants Among Publically Available Exomes Derived From Subjects Without Evidence for Either Type 2 Long QT Syndrome or Type 1 Short QT Syndrome

Abstract

Introduction: Loss-of-function (LOF) and gain-of-function (GOF) pathogenic variants in the KCNH2 -encoded Kv11.1 potassium channel cause type 2 long QT syndrome(LQT2) and type 1 short QT syndrome (SQT1), respectively. Given the prevalence of KCNH2- mediated heart disease (1 in 8000 for LQT2,1 in 100,000 for SQT1) and the reduced penetrance/variable expressivity seen in LQTS and SQTS, large databases such as the Genome Aggregation Database (gnomAD, n=141,352 individuals) might harbor subjects with unidentified LQT2/SQT1-causative mutations. Objective: To determine which “ultra-rare” KCNH2 missense variants (MVs) in gnomAD are most likely to be LQT2- or SQT1-causative. Methods: A list of gnomAD-derived MVs in KCNH2 was compiled. The frequency of each MV in gnomAD was assessed. Each MV was analyzed by 8 in silico pathogenicity prediction tools. MVs that were seen once in gnomAD and predicted damaging by all 8 tools were considered possible LQT2/SQT1-causative MVs worthy of functional validation via whole-cell patch clamp. Results: Overall, 233/474 (49%) of gnomAD KCNH2 MVs were seen in just 1 of >140,000 subjects. Of these, 2 (0.4%) MVs (S320W, S379Y) were predicted damaging by 8 in silico tools. Both S320W and S379Y localize to the N-terminus of the Kv11.1 potassium channel. There was no significant change in peak or tail current density between heterozygously expressed KCNH2-S320W (n=13, p=NS) and KCNH2-WT (n=13). However, heterozygous KCNH2-S379Y (n=12) produced a marked increase in peak current density across the range -30 mV to +20 mV and shifted the V 1/2 of activation by -17 mV (p < 0.001). Also, tail current density of S379Y channels significantly increased across the range -20 mV to +20 mV and the V 1/2 of deactivation shifted by -26 mV (p < 0.001). Conclusion: With this stringent requirement of ultra-rarity, universal prediction of damaging impact, and functionally abnormal electrophysiological properties, one SQT1-causative pathogenic variant in gnomAD was identified consistent with the predicted frequency of SQTS. The search continues among the remaining 32 ultra-rare MVs in KCNH2 where at least 75% of the in silico tools predicted deleterious impact of the MV to find the 15-20 subjects in gnomAD’s population who ought to have an LQT2-causative MV.