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: 8434923
• 负责人: John R. Giudicessi
• 金额: $ 4.72万
• 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/8434923
• 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; 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.

描述（由申请人提供）：仅在美国，每年心脏性猝死（SCD）索赔估计为300,000例。据估计，这些死亡中有5-10%发生在看似健康的人身上，在尸检调查后，他们的心脏在其他方面结构正常。心脏通道病变，如长qt综合征（LQTS）和Brugada综合征（BrS），由心脏离子通道功能的遗传性缺陷引起，是尸检阴性猝死最常见的可识别原因，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。尽管人们已经付出了相当大的努力来了解通道性猝死的发病机制，但近25%的LQTS、70%的BrS和很大一部分尸检阴性的不明原因猝死在基因上仍然是难以捉摸的。虽然电压门控瞬时外向（Ito）电流大分子复合物内的扰动长期以来一直被假设有助于LQTS和BrS的发病机制，但相对缺乏直接涉及疾病主要Ito分子决定因素遗传变异的分子和功能证据。我们假设kcnd3编码的Kv4.3 1-亚基、kcnip2编码的KChIP2 -亚基或Ito通道表达的调控因子如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）队列中发现了KCND3和KCNIP2中5个可能与疾病相关的非同义突变，以及microRNA-1-2茎中可能与疾病相关的核苷酸替代。我们将首先评估健康和遗传上难以捉摸的SCD中KCND3和KCNIP2遗传变异的全谱和流行程度，通过扩大我们基于PCR/ dhplc的突变分析，包括283例SIDS病例，101例SUDS病例和780例表面健康对照。接下来，为了证明lqts相关的功能丧失突变导致Ito电流减少，而brs相关的功能获得突变导致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发现的突变进行功能表征。最后，我们将利用定量RT-PCR和western blotting技术评估microRNA-1-2中疾病相关核苷酸替换对心脏Ito通道表达的功能影响。