Genome Editing of Induced Pluripotent Stem Cells to Decipher Cardiac Channelopathy Variant
Genome Editing of Induced Pluripotent Stem Cells to Decipher Cardiac Channelopathy Variant
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DOI：
10.1016/j.jacc.2018.04.041
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发表时间：
2018-07-03
影响因子：
24
通讯作者：
Wu, Joseph C.
中科院分区：
文献类型：
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作者：
Garg, Priyanka;Oikonomopoulos, Angelos;Wu, Joseph C.
背景技术长QT综合征(LQTS)是一种QT间期延长的致心律失常疾病，使患者容易出现危及生命的室性心律失常，例如尖端扭转型室性心动过速和心源性猝死。临床基因检测已成为识别疑似 LQTS 患者基因变异的护理标准。然而，这些结果常常因意义不确定的变异 (VUS) 的发现而令人困惑，其致病性证据不足。 目的 本研究的目的是证明患者特异性诱导多能干细胞 (iPSC) 的基因组编辑可以成为描述 VUS 在心脏通道病中致病性的有价值的方法。 方法 从具有新型错义变异 (T983I) 的携带者中分离外周血单核细胞KCNH2 (LQT2) 基因和无关的健康对照受试者。使用免整合仙台病毒生成 iPSC，并分化为 iPSC 衍生的心肌细胞 (CM)。 结果 全细胞膜片钳记录显示，与健康对照 iPSC-CM 相比，VUS iPSC-CM 的动作电位持续时间 (APD) 显着延长，快速激活延迟整流 K+ 电流 (I-Kr) 密度降低。 ICA-105574 是一种有效的 I-Kr 激活剂，可增强 VUS iPSC-CM 中的 I-Kr 强度并恢复正常的动作电位持续时间。值得注意的是，与健康对照细胞相比，VUS iPSC-CM 对高风险扭转致伤药物（多非利特、伊布利特和阿齐利特）表现出更大的致心律失常倾向，表明复极储备受损。最后，使用 CRISPR/Cas9 基因编辑（同基因控制）选择性校正 iPSC-CM 中的致病变异，使异常细胞表型正常化，而在健康对照细胞中引入纯合变异则概括了 LQTS 疾病的标志特征。 结论 结果表明，KCNH2(T983I) VUS 可能被归类为潜在致病性。 (C) 2018 年，美国心脏病学会基金会。
BACKGROUND The long QT syndrome (LQTS) is an arrhythmogenic disorder of QT interval prolongation that predisposes patients to life-threatening ventricular arrhythmias such as Torsades de pointes and sudden cardiac death. Clinical genetic testing has emerged as the standard of care to identify genetic variants in patients suspected of having LQTS. However, these results are often confounded by the discovery of variants of uncertain significance (VUS), for which there is insufficient evidence of pathogenicity.OBJECTIVES The purpose of this study was to demonstrate that genome editing of patient-specific induced pluripotent stem cells (iPSCs) can be a valuable approach to delineate the pathogenicity of VUS in cardiac channelopathy.METHODS Peripheral blood mononuclear cells were isolated from a carrier with a novel missense variant (T983I) in the KCNH2 (LQT2) gene and an unrelated healthy control subject. iPSCs were generated using an integration-free Sendai virus and differentiated to iPSC-derived cardiomyocytes (CMs).RESULTS Whole-cell patch clamp recordings revealed significant prolongation of the action potential duration (APD) and reduced rapidly activating delayed rectifier K+ current (I-Kr) density in VUS iPSC-CMs compared with healthy control iPSC-CMs. ICA-105574, a potent I-Kr activator, enhanced I-Kr magnitude and restored normal action potential duration in VUS iPSC-CMs. Notably, VUS iPSC-CMs exhibited greater propensity to proarrhythmia than healthy control cells in response to high-risk torsadogenic drugs (dofetilide, ibutilide, and azimilide), suggesting a compromised repolarization reserve. Finally, the selective correction of the causal variant in iPSC-CMs using CRISPR/Cas9 gene editing (isogenic control) normalized the aberrant cellular phenotype, whereas the introduction of the homozygous variant in healthy control cells recapitulated hallmark features of the LQTS disorder.CONCLUSIONS The results suggest that the KCNH2(T983I) VUS may be classified as potentially pathogenic. (C) 2018 by the American College of Cardiology Foundation.