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

描述（由申请人提供）：仅在美国，每年就有30万人死于心脏性猝死（SCD）。据估计，这些死亡中有5-10%发生在看似健康的个体中，在死后调查后心脏结构正常。由心脏离子通道功能的遗传缺陷引起的心脏通道病，如长QT综合征（LQTS）和Brugada综合征（BrS），是导致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。虽然相当大的努力已经致力于了解通道病性猝死的发病机制，近25%的LQTS，70%的BrS，和尸检阴性的原因不明的猝死的很大一部分仍然是遗传难以捉摸。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为有助于LQTS和BrS的发病机制，但存在相对缺乏的分子和功能证据，直接暗示原发性Ito分子决定因素内的遗传变异疾病。我们假设KCND 3编码的Kv4.3 1-亚基、KCNIP 2编码的KChIP 2 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病理学原因。为了支持这一假设，我们先前在临床上稳健的基因型阴性LQTS（n=94）和BrS（n=91）队列中鉴定了KCND 3和KCNIP 2内的五种潜在疾病相关的非同义突变以及microRNA-1-2茎内的一种可能的疾病相关的核苷酸取代。我们将开始通过扩展我们的基于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，评估健康和遗传难以捉摸的SCD中KCND 3和KCNIP 2内遗传变异的全谱和患病率。接下来，为了证明功能丧失型LQTS相关突变导致Ito电流降低，功能获得型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和蛋白质印迹法评估microRNA-1-2内疾病相关核苷酸取代对心脏中Ito通道表达的功能影响。 公共卫生相关性：马约诊所Windland Smith Rice心脏猝死基因组学实验室的主要研究重点是青年心脏猝死（SCD）的发病机制，特别关注原发性通道病，如儿茶酚胺能性多形性室性心动过速（CPVT）、长QT综合征（LQTS）和Brugada综合征（BrS）。揭示基因型阴性心脏性猝死的基础有望通过扩大诊断和治疗选择来加强临床实践，这些诊断和治疗选择需要识别，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折磨的个人和家庭。此外，它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。

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.