Vanderbilt University School of Medicine, Nashville, Tennessee; Vanderbilt Center for Arrhythmia Research and Therapeutics, Department of Medicine, Nashville, Tennessee.
Vanderbilt Center for Arrhythmia Research and Therapeutics, Department of Medicine, Nashville, Tennessee.
Heart Rhythm. 2019 Nov;16(11):1686-1695. doi: 10.1016/j.hrthm.2019.04.031. Epub 2019 Apr 18.
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 the pathogenicity of VUS are needed.
The purpose of this study was to rapidly determine the pathogenicity of a CACNA1C variant reported by commercial genetic testing as a VUS using a patient-independent human induced pluripotent stem cell (hiPSC) model.
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.
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 Ca1.2 currents. Heterologous expression studies confirmed the effect of N639T on Ca1.2 inactivation.
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 the functional significance of LQTS VUS in high-throughput hiPSC-CM assays.
商业性长 QT 综合征（LQTS）基因检测迅速发展，但由于无法准确预测罕见变异是否具有致病性，其临床获益有限。新型错义变异通常被报告为意义不明的变异（VUS），不能用于筛查有发生心源性猝死风险的家庭成员。因此，需要更好的方法来确定 VUS 的致病性。
本研究旨在使用患者独立的人诱导多能干细胞（hiPSC）模型快速确定商业性基因检测报告为 VUS 的 CACNA1C 变异的致病性。
使用 CRISPR/Cas9 基因组编辑，将 CACNA1C-p.N639T 引入来自无关健康志愿者的先前建立的 hiPSC 中，从而产生患者独立的 hiPSC 模型。生成了 3 个独立的杂合性 N639T hiPSC 系，并将其分化为心肌细胞（CM）。通过测量 96 孔 hiPSC-CM 单层的细胞外场电位（EFP）和膜片钳技术，比较 N639T hiPSC-CM 的电生理特性与同基因和人群对照 hiPSC-CM。
仅在光刺激但非自发搏动的 N639T hiPSC-CM 中观察到 EFP 显著延长。膜片钳研究表明，N639T 通过减缓 Ca1.2 电流的电压依赖性失活而延长心室动作电位。异源表达研究证实了 N639T 对 Ca1.2 失活的影响。
患者独立的 hiPSC 模型能够快速生成功能数据，支持将 CACNA1C VUS 重新分类为可能的致病性，从而确立了一种新的 LQTS 8 型突变。此外，我们的结果表明，在高通量 hiPSC-CM 测定中，控制搏动率对于评估 LQTS VUS 的功能意义非常重要。
