The patient-independent human iPSC model – a new tool for rapid determination of genetic variant pathogenicity in long QT syndrome

Abstract Background Commercial genetic testing for Long QT Syndrome (LQTS) has rapidly expanded, but the inability to accurately predict whether a rare variant is pathogenic has limited its clinical benefit. Novel missense variants are routinely reported as "Variant of Unknown Significance (VUS)" and cannot be used to screen family members at-risk for sudden cardiac death. Better approaches to determine pathogenicity of VUS are needed. Objective To rapidly determine the pathogenicity of a CACNA1C variant reported by commercial genetic testing as a VUS using a patient-independent induced pluripotent stem cell (hiPSC) model. Methods Using CRISPR/Cas9 genome editing, CACNA1C-p.N639T was introduced into a previously-established hiPSC from an unrelated healthy volunteer, thereby generating a patient-independent hiPSC model. Three independent heterozygous N639T hiPSC lines were generated and differentiated into cardiomyocytes (CM). Electrophysiological properties of N639T hiPSC-CM were compared to those of isogenic and population control hiPSC-CM by measuring the extracellular field potential (EFP) of 96-well hiPSC-CM monolayers, and by patch-clamp. Results Significant EFP prolongation was observed only in optically-stimulated but not in spontaneously-beating N639T hiPSC-CM. Patch clamp studies revealed that N639T prolonged the ventricular action potential by slowing voltage-dependent inactivation of Ca V 1.2 currents. Heterologous expression studies confirmed the effect of N639T on Ca V 1.2 inactivation. Conclusion The patient-independent hiPSC model enabled rapid generation of functional data to support reclassification of a CACNA1C VUS to "likely pathogenic", thereby establishing a novel LQTS type 8 mutation. Furthermore, our results indicate the importance of controlling beating rates to evaluate functional significance of LQTS VUS in high-throughput hiPSC-CM assays.