诱导多能干细胞的基因组编辑以解析心脏通道病变体。
Genome Editing of Induced Pluripotent Stem Cells to Decipher Cardiac Channelopathy Variant.
机构信息
Stanford Cardiovascular Institute and Department of Medicine, Division of Cardiology, Stanford University, Stanford, California.
Stanford Cardiovascular Institute and Department of Medicine, Division of Cardiology, Stanford University, Stanford, California; Center for Inherited Cardiovascular Disease, Division of Cardiovascular Medicine, Stanford University, Stanford, California.
出版信息
J Am Coll Cardiol. 2018 Jul 3;72(1):62-75. doi: 10.1016/j.jacc.2018.04.041.
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) density in VUS iPSC-CMs compared with healthy control iPSC-CMs. ICA-105574, a potent I activator, enhanced I 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 VUS may be classified as potentially pathogenic.
背景
长 QT 综合征（LQTS）是一种 QT 间期延长的心律失常疾病，使患者易患尖端扭转型室性心动过速和心脏性猝死等致命性室性心律失常。临床基因检测已成为识别疑似 LQTS 患者遗传变异的标准护理方法。然而，这些结果常常因发现意义不明的变异（VUS）而变得复杂，对于这些变异，其致病性证据不足。
目的
本研究旨在证明，对患者特异性诱导多能干细胞（iPSC）进行基因组编辑可能是一种有价值的方法，可以阐明心脏通道病中 VUS 的致病性。
方法
从携带 KCNH2（LQT2）基因新型错义变异（T983I）的携带者和无关的健康对照个体中分离外周血单核细胞。使用无整合的 Sendai 病毒生成 iPSC，并将其分化为 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）显著延长，快速激活延迟整流钾电流（I）密度降低。强效 I 激活剂 ICA-105574 增强了 I 的幅度，并在 VUS iPSC-CM 中恢复了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 在响应高风险致扭转型药物（多非利特、伊布利特和阿齐利特）时表现出更大的致心律失常倾向，表明复极化储备受损。最后，使用 CRISPR/Cas9 基因编辑（同基因对照）对 iPSC-CM 中的因果变异进行选择性校正，使异常的细胞表型正常化，而在健康对照细胞中引入纯合变异则再现了 LQTS 疾病的标志性特征。
结论
结果表明，KCNH2 VUS 可能被归类为潜在致病性。
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