The Role of the Transient Outward Current in Genetically Elusive Sudden Death

瞬时外向电流在遗传性猝死中的作用

• 批准号: 8056419
• 负责人: John R. Giudicessi
• 金额: $ 3.65万
• 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 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8056419
• 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; Property; 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; clinical practice; cohort; density; gain of function; genetic variant; loss of function; member; novel; 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）：每年仅在美国就有 300,000 人因心源性猝死 (SCD) 死亡。根据尸检调查，这些死亡中估计有 5-10% 发生在看似健康但心脏结构正常的个体中。由心脏离子通道功能遗传性缺陷引起的心脏离子通道病，如长 QT 综合征 (LQTS) 和布鲁格达综合征 (BrS)，代表了尸检阴性猝死的最常见可识别原因，包括 35% 的不明原因猝死综合征 (SUDS) 和 10% 的婴儿猝死综合征 (SIDS) 病例。尽管人们在了解通道病性猝死的发病机制方面付出了相当大的努力，但近 25% 的 LQTS、70% 的 BrS 和大部分尸检阴性的不明原因猝死在遗传学上仍然难以捉摸。虽然长期以来人们一直假设电压门控瞬态外向 (Ito) 电流大分子复合物内的扰动有助于 LQTS 和 BrS 的发病机制，但直接暗示疾病的主要 Ito 分子决定因素内的遗传变异的分子和功能证据相对较少。我们假设 KCND3 编码的 Kv4.3 1- 亚基、KCNIP2 编码的 KChIP2 2- 亚基或 Ito 通道表达调节因子（如 microRNA-1-2）的突变可能导致猝死的心律失常原因。为了支持这一假设，我们之前在临床稳健、基因型阴性的 LQTS (n=94) 和 BrS (n=91) 队列中鉴定了 KCND3 和 KCNIP2 内的 5 个潜在与疾病相关的非同义突变，以及 microRNA-1-2 茎内可能与疾病相关的核苷酸取代。我们将首先评估健康和遗传上难以捉摸的 SCD 中 KCND3 和 KCNIP2 遗传变异的全谱和患病率，方法是将我们基于 PCR/DHPLC 的突变分析扩展到包括 283 个 SIDS 病例、101 个 SUDS 病例和 780 个表面上健康的对照。接下来，为了证明功能丧失的 LQTS 相关突变导致 Ito 电流减少，而功能获得性 BrS 相关突变导致 Ito 电流增加，分别导致复极延长或加速，我们将使用全细胞膜片钳技术对所有发现的突变进行功能表征。最后，我们将使用定量 RT-PCR 和蛋白质印迹评估 microRNA-1-2 内与疾病相关的核苷酸取代对心脏中 Ito 通道表达的功能影响。 公共健康相关性：Mayo Clinic Windland Smith Rice 心脏猝死基因组学实验室的主要研究重点是青少年心源性猝死 (SCD) 的发病机制，特别关注原发性离子通道病，例如儿茶酚胺能多形性室性心动过速 (CPVT)、长 QT 综合征 (LQTS) 和布鲁格达综合征 (BrS)。揭开基因型阴性心源性猝死的基础，有望通过扩大识别、风险分层和有效治疗受这些潜在致命但高度可治疗的疾病困扰的个人和家庭所需的诊断和治疗选择来加强临床实践。此外，它还提供了通过探索仍然神秘的病理学来揭示新生物学的可能性。