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。2型长QT综合征（LQT2）是由人类以太-a-go-go相关基因（hERG）突变引起的。hERG编码心脏中快速激活的延迟整流钾通道的成孔亚基。LQT2是第二常见的LQTS形式，占LQTS基因型病例的35%至40%。LQT2突变可通过多种机制引起hERG通道功能障碍。在之前的资助期内，我们已经证明无义介导的mRNA衰变和剪接缺陷是LQT2中hERG通道功能障碍的重要机制。我们还发现，hERG c末端异构体的产生是由hERG内含子9的选择性剪接和聚腺苷化之间的竞争决定的，并且hERG c末端异构体的相对表达在hERG通道功能的调节中起着重要作用。在目前的应用中，我们将使用全长hERG基因构建体来研究hERG c -末端异构体表达调控机制，表征LQT2中hERG通道功能障碍的两种新机制，并开发一种调节hERG c -末端异构体相对表达的新方法。此外，我们将使用患者特异性诱导多能干细胞（iPS）衍生的心肌细胞作为模型来研究LQT2的病理生理。本申请的具体目的是：目的1)研究一个新发现的LQT2剪接位点突变，该突变破坏了内含子9的3'剪接位点，并改变了hERG c端亚型的相对表达。目的2)开发一种反义方法，通过诱导hERG c端异构体从非功能异构体向功能异构体的表达转变来增加hERG电流。目的3)表征LQT2的新机制，其中过早终止随后重新启动翻译导致n端截断的hERG通道的产生，并改变门控特性。目的4)建立LQT2患者特异性iPS细胞系，并在iPS细胞衍生的心肌细胞中表征LQT2突变。这项研究将增加我们对LQT2突变如何在转录后和翻译水平上导致hERG通道功能障碍的认识。我们相信这项工作将对我们对长QT综合征的理解和治疗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.