Establishing Pathogenicity Of Novel Lqts8 Variant Via Genomic Editing Of Human Ipsc

Long QT syndrome (LQTS) is a major cause for sudden cardiac death (SCD) in the young. The use of commercial genetic testing for LQTS has rapidly expanded, but inability to predict whether a rare variant is pathogenic has limited clinical benefit. Novel missense variants are routinely reported as “Variants of Unknown Significance (VUS)”, which cannot be used to screen family members at-risk for SCD. The development of new approaches to determine pathogenicity of rare variants is a major unmet need. Here, we studied a novel heterozygous missense variant (p.N639T) detected in CACNA1C (encodes CaV1.2 Ca2+ channel) by commercial genetic testing and reported as a VUS in a family with LQTS. We first studied electrophysiological characteristics of L-type Ca2+ current (LTCC) using HEK293 cells expressing wild type and mutant (N639T) channel. We found significant decrease in the rate of voltage-dependent inactivation (using Ba2+ as a charge carrier) in N639T cells and right shift in steady-state inactivation, but current density was also significantly reduced in N639T. We then used CRISPR/Cas9 to introduce heterozygous N639T into control hiPSC from a healthy volunteer, which were then differentiated into cardiomyocytes (CM). N639T hiPSC-CM were compared against isogenic (iCTRL) and population control (pCTRL) hiPSC-CM expressing WT CACNA1C, by measuring the extracellular field potential (EFP) of paced hiPSC-CM monolayers, and by standard single-cell patch-clamp. EFP was significantly prolonged in N639T hiPSC-CM. Verifying our data from HEK293 experiments, patch-clamp studies showed that N639T prolongs ventricular action potential (APD 50=131±27 ms (iCTRL) vs. 254±53 ms (N639T), p<0.05) by slowing voltage-dependent LTCC inactivation (τ=74±16 ms (iCTRL) vs. 150±29 ms (N639T), p<0.01), without affecting current density. We conclude that genetic editing of hiPSC-CM can rapidly and efficiently establish pathogenicity of a VUS in LQTS such as the CACNA1C-p.N639T mutation.