The Role of The Transient Outward Current In Genetically Elusive Sudden Death

瞬态外向电流在遗传性猝死中的作用

• 批准号: 8620703
• 负责人: John R. Giudicessi
• 金额: $ 1.32万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2014-05-16
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8620703
• 关键词: 3' Untranslated Regions; Action Potentials; Attention; Autopsy; Binding; Biogenesis; Biology; Cardiac; Cell surface; Cells; Cessation of life; Clinic; Collection; Complex; Defect; Development; Diagnostic; Disease; Event; Family; Functional RNA; Genes; Genetic Variation; Genomics; Genotype; Health; Heart; Heart Atrium; Height; Hepatic Stellate Cell; Individual; Investigation; Ion Channel; Kv channel-interacting protein 2; Laboratories; Lead; Long QT Syndrome; Macromolecular Complexes; MicroRNAs; Missense Mutation; Molecular; Mutation; Myocardium; Nucleotides; Patch-Clamp Techniques; Pathogenesis; Pathologic; Pathology; Phase; Potassium Channel; Prevalence; Regulation; Relative (related person); Research; Reverse Transcriptase Polymerase Chain Reaction; Rice; Risk; Role; Romano-Ward Syndrome; Shapes; Site; Sudden Death; Sudden infant death syndrome; Syndrome; Therapeutic; Time; Transcription Repressor/Corepressor; United States; Variant; Ventricular; Ventricular Arrhythmia; Ventricular Tachycardia; Western Blotting; base; biophysical properties; clinical practice; cohort; density; gain of function; genetic variant; loss of function; member; novel; public health relevance; stem; sudden cardiac death; voltage

DESCRIPTION (provided by applicant): Each year sudden cardiac death (SCD) claims an estimated 300,000 in the United States alone. An estimated 5-10% of these deaths occur in seemingly healthy individuals with otherwise structurally normal hearts following post-mortem investigation. Cardiac channelopathies such as Long-QT Syndrome (LQTS) and Brugada Syndrome (BrS) which arise from heritable defects in cardiac ion channel function represent the most common identifiable causes underlying autopsy negative sudden death, including 35% of Sudden Unexplained Death Syndrome (SUDS) and 10% of Sudden Infant Death Syndrome (SIDS) cases. While considerable effort has been devoted to understanding the pathogenesis of channelopathic sudden death, nearly 25% of LQTS, 70% of BrS, and a large proportion of autopsy negative sudden unexplained deaths still remain genetically elusive. While perturbations within the voltage-gated transient outward (Ito) current macromolecular complex have long been hypothesized to contribute to the pathogenesis of LQTS and BrS, there exists a relative paucity of molecular and functional evidence directly implicating genetic variation within primary Ito molecular determinants to disease. We hypothesize that mutations in the KCND3-encoded Kv4.3 1- subunit, KCNIP2-encoded KChIP2 2-subunit, or regulators of Ito channel expression such as microRNA-1-2 might lead to arrhythmic causes of sudden death. In support of this hypothesis we previously identified five potentially disease-associated non-synonymous mutations within KCND3 and KCNIP2 as well as a possible disease-associated nucleotide substitution within the stem of microRNA-1-2 in clinically robust, genotype negative LQTS (n=94) and BrS (n=91) cohorts. We will begin by assessing the full spectrum and prevalence of genetic variation within KCND3 and KCNIP2 in health and genetically elusive SCD by expanding our PCR/DHPLC-based mutational analysis to include 283 SIDS cases, 101 SUDS cases, and 780 ostensibly healthy controls. Next, to demonstrate that loss-of-function LQTS-associated mutations result in reduced Ito current and gain-of-function BrS-associated mutations result in increased Ito current, leading to prolonged or accelerated repolarization respectively we will functionally characterize all discovered mutations using the whole cell patch clamp technique. Finally, we will assess the functional impact of a disease-associated nucleotide substitution within microRNA-1-2 on Ito channel expression in the heart using quantitative RT-PCR and western blotting. PUBLIC HEALTH RELEVANCE: The primary research focus of the Mayo Clinic Windland Smith Rice Sudden Cardiac Genomics Laboratory is the pathogenetics of youthful sudden cardiac death (SCD), with particular attention to primary channelopathies such as catecholaminergic polymorphic ventricular tachycardia (CPVT), Long-QT Syndrome (LQTS), and Brugada Syndrome (BrS). Unraveling the basis of genotype-negative sudden cardiac death promises to enhance clinical practice by expanding the diagnostic and therapeutic options needed to identify, risk stratify, and effectively treat individuals and families afflicted by these potentially lethal, yet highly treatable conditions. Further, it offers the possibility of unveiling novel biology through the exploration of still enigmatic pathology.

描述(由申请人提供)：仅在美国，每年就有大约30万人因心脏性猝死(SCD)而死亡。据估计，这些死亡中有5%-10%发生在经过尸检后心脏结构正常的看似健康的人身上。长QT间期综合征(LQTS)和Brugada综合征(BRS)等心脏通道病是最常见的尸检阴性猝死原因，包括35%的不明原因猝死综合征(SODS)和10%的婴儿猝死综合征(SIDS)。虽然人们已经致力于了解通道病理性猝死的发病机制，但近25%的LQT、70%的BRS和很大一部分尸检阴性的原因不明的猝死仍然难以从基因上得到解释。虽然电压门控瞬时外向电流(ITO)电流大分子复合体内的扰动长期以来被认为与LQTS和BRS的发病有关，但直接涉及主要ITO分子决定因素中的遗传变异与疾病有关的分子和功能证据相对较少。我们推测，KCND3编码的Kv4.3 1亚基、KCNIP2编码的KChIP2 2亚基的突变，或Ito通道表达的调节因子，如microRNA-1-2，可能导致心律失常导致猝死。为了支持这一假设，我们先前在临床健壮的、基因阴性的LQTS(n=94)和BRS(n=91)队列中发现了KCND3和KCNIP2中五个潜在的与疾病相关的非同义突变，以及可能与microRNA-1-2的茎内的疾病相关的核苷酸替换。我们将首先评估KCND3和KCNIP2基因变异在健康和遗传难以捉摸的SCD中的全谱和流行率，将我们的基于PCR/DHPLC的突变分析扩展到283例SID病例、101例SODS病例和780名表面上健康的对照。接下来，为了证明功能缺失的LQTS相关突变导致Ito电流降低，以及功能获得的BRS相关突变导致Ito电流增加，分别导致延长或加速复极，我们将使用全细胞膜片钳技术对所有已发现的突变进行功能表征。最后，我们将使用定量RT-PCR和Western blotting评估microRNA-1-2中与疾病相关的核苷酸替换对心脏Ito通道表达的功能影响。 公共卫生相关性：梅奥诊所温德兰·史密斯·赖斯心脏突然基因组实验室的主要研究重点是青年心脏性猝死(SCD)的病因学，特别关注原发通道病变，如儿茶酚胺能多形性室性心动过速(CPVT)、长QT综合征(LQTS)和Brugada综合征(BRS)。揭开基因阴性心脏性猝死的基础有望通过扩大所需的诊断和治疗选择来加强临床实践，以识别、风险分层和有效地治疗患有这些潜在致命但高度可治疗的疾病的个人和家庭。此外，它还提供了通过探索仍然谜一般的病理学来揭示新生物学的可能性。