Pathogenesis of hERG Mutations in Human Long QT Syndrome

描述（由申请人提供）：长QT综合征（LQTS）是一种以心脏复极延迟和心律失常和猝死风险增加为特征的疾病。长QT综合征2型（LQT 2）是由人类ether-a-go-go相关基因（hERG）突变引起的。hERG编码心脏中快速激活延迟整流钾通道的成孔亚基。LQT 2是LQTS的第二大流行形式，占LQTS基因分型病例的35%至40%。LQT 2突变可通过多种机制引起hERG通道功能障碍。在之前的资助期间，我们已经证明无义介导的mRNA衰变和剪接缺陷是LQT 2中hERG通道功能障碍的重要机制。我们还表明，hERG C-末端亚型的产生是由hERG内含子9的选择性剪接和多聚腺苷酸化之间的竞争决定的，并且hERG C-末端亚型的相对表达在调节hERG通道功能中起着重要作用。在本申请中，我们将使用全长hERG基因构建体来研究hERG C-末端亚型表达调节的基础机制，表征LQT 2中hERG通道功能障碍的两种新机制，并开发一种调节hERG C-末端亚型相对表达的新方法。此外，我们将使用患者特异性诱导多能干（iPS）细胞衍生的心肌细胞作为模型来研究LQT 2的病理生理学。本申请的具体目的是：目的1）研究新鉴定的LQT 2剪接位点突变，其破坏内含子9的3'剪接位点并改变hERG C-末端同种型的相对表达。目的2）建立一种通过诱导hERG C端亚型表达由非功能性亚型向功能性亚型转变来增加hERG电流的反义方法。目的3）研究LQT 2的一种新机制，即LQT 2的翻译提前终止，随后翻译重新启动，导致产生N端截短的hERG通道，并改变其门控特性。目的4）建立LQT 2患者特异性iPS细胞系并表征iPS细胞衍生的心肌细胞中的LQT 2突变。这项研究将增加我们对LQT 2突变如何在转录后和翻译水平导致hERG通道功能障碍的认识。我们相信，这项工作将对我们理解和治疗长QT综合征产生持续和重大的影响。

DESCRIPTION (provided by applicant): Long QT syndrome (LQTS) is a disorder characterized by delayed cardiac repolarization and an increased risk of arrhythmias and sudden death. Long QT syndrome type 2 (LQT2) is caused by mutations in the human ether-a-go-go-related gene (hERG). hERG encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel in the heart. LQT2 is the second most prevalent form of LQTS, accounting for 35% to 40% of genotyped cases of LQTS. LQT2 mutations can cause hERG channel dysfunction by a variety of mechanisms. In the previous funding period, we have shown that nonsense-mediated mRNA decay and splicing defects are important mechanisms of hERG channel dysfunction in LQT2. We have also shown that generation of hERG C-terminal isoforms is determined by competition between alternative splicing and polyadenylation of hERG intron 9 and that the relative expression of hERG C-terminal isoforms plays an important role in regulation of hERG channel function. In the present application, we will use full-length hERG gene constructs to study mechanisms that underlie the regulation of hERG C-terminal isoform expression, characterize two new mechanisms of hERG channel dysfunction in LQT2, and develop a novel approach to modulate the relative expression of hERG C-terminal isoforms. In addition, we will use patient-specific induced pluripotent stem (iPS) cell-derived cardiomyocytes as a model to study pathophysiology of LQT2. The specific aims of this application are: Aim 1) To study a newly identified LQT2 splice site mutation that disrupts the 3' splice site of intron 9 and alters the relative expression of hERG C-terminal isoforms. Aim 2) To develop an antisense approach to increase hERG current by inducing a shift in hERG C-terminal isoform expression from the nonfunctional isoform to the functional isoform. Aim 3) To characterize a new mechanism of LQT2 in which premature termination followed by the reinitiation of translation results in the generation of N-terminally truncated hERG channels with altered gating properties. Aim 4) To create LQT2 patient-specific iPS cell lines and characterize LQT2 mutations in iPS cell-derived cardiomyocytes. This study will increase our knowledge of how LQT2 mutations lead to hERG channel dysfunction at the posttranscriptional and translational level. We believe that this work will have a sustained and significant impact on our understanding and treatment of long QT syndrome.