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. PUBLIC HEALTH RELEVANCE: Long QT syndrome is a disease associated with delayed cardiac repolarization and prolonged QT intervals on the electrocardiogram, which can lead to ventricular arrhythmias and sudden death. The goal of present proposal is to elucidate how disease-causing mutations lead to hERG channel dysfunction and develop a novel approach to increase hERG channel function. The knowledge gained from this study will strengthen our understanding of the pathogenesis of hERG mutations in long QT syndrome and provide information directed towards the development of new therapeutic strategies for long QT syndrome.