Cellular and clinical phenotypes of novel SCN5a mutations

描述（由申请人提供）：通过SCN5A编码的心脏Na通道a亚基的Na电流（INa）功能障碍参与心律失常、心力衰竭和缺血的致病机制。特定基因型的临床表型机制来自突变引起的潜在分子表型（例如，INa振幅和动力学）和细胞表型（例如，动作电位）。在之前的研究中，我们重点研究了与SCN5A突变相关的异源表达系统的分子表型。我们发现了一种普遍存在的人类剪接变体（Q1077del），它影响其他突变的功能，8种常见多态性（如H558R）的临床相关功能影响，以及贩运缺陷突变的药物拯救。我们还发现了一个Brugada综合征SCN5A等位基因对另一个基因的显性负作用。本提案的两个目的（1和3）对SCN5A遗传变异的致病机制进行了后续研究。另外两个目标（2和4）将项目带入了一个新的方向。越来越清楚的是，INa是SCN5A大分子复合物的一个功能，其中不止一个亚基与¿亚基和其他通道相互作用蛋白（ChIPs）结合，如小窝蛋白和syntrophins。许多疑似遗传性心律失常综合征（LQTS， SIDS， SUDS）患者的致病基因型尚未被发现。从这些队列中的患者中，我们最近发现了假定的芯片（CAV3, SNTA1, GPD1L, SCN4B）的突变，并表明它们以一种可能具有致病性的方式影响INa。在下一阶段，我们将通过在心肌细胞中表达突变来继续最近实施的更综合的研究方法。这种更完整的心脏环境允许在相关条件下（如在完整大分子复合物存在的肾上腺素能状态下）对细胞表型和分子表型进行表征。在目标1中，我们将通过表征分子和细胞表型来发现涉及SCN5A变异的致病机制。在Aim 2中，我们将从疑似遗传性心律失常且未知致病基因型的患者中发现编码疑似SCN5A芯片的新基因突变，并定义分子和细胞表型以建立INa介导的机制。在Aim 3中，我们将研究SCN5A -a亚基相互作用（如显性负作用和拯救效应）。在Aim 4中，我们将探讨两种新型心律失常基因（SNTA1和GPD1L）改变的INa分子表型的分子机制。本提案的综合方法考虑了更多天然系统中的SCN5A大分子复合物，以确定分子和细胞表型，以及分子表型背后的分子机制。除了更好地了解SCN5A大分子复合物外，预期的结果应该有助于深入了解遗传性和获得性心律失常和其他心脏病的基因型-表型联系的发病机制。公共卫生相关性：编码膜离子通道蛋白SCN5A基因的遗传突变可导致婴儿猝死综合征（SIDS）、意外猝死综合征（SUDS）和长QT综合征（LQTS）等综合征中的心律失常损伤和死亡。该项目将发现在这些综合征患者中发现的新基因（不是SCN5A但相关）的突变，以及它们如何与SCN5A相互作用以改变功能并导致心律失常。研究结果有望改善对这些遗传性综合征的诊断，并提出治疗和预防这些综合征的方法。

DESCRIPTION (provided by applicant): Dysfunction of the Na current (INa) flowing through the a subunit of the cardiac Na channel encoded by SCN5A participates in pathogenic mechanisms for arrhythmia, heart failure, and ischemia. The mechanism for the clinical phenotype of a particular genotype comes from the underlying molecular phenotype (e.g., INa amplitude and kinetics) and cellular phenotype (e.g., action potentials) caused by the mutation. In the previous period we focused on the molecular phenotype in heterologous expression systems associated with mutations of SCN5A. We discovered a ubiquitous human splice variant (Q1077del) that affects function of other mutations, clinically relevant functional effects of 8 common polymorphisms (e.g. H558R), and drug rescue of trafficking defective mutations. We also showed a novel dominant negative effect of one Brugada syndrome SCN5A allele on another. Two aims (1 and 3) of this proposal follow up on these pathogenic mechanisms of genetic variation in SCN5A. Two additional aims (2 and 4) take the project in a new direction. It is increasingly clear that INa is a function of the SCN5A macromolecular complex where more than one a subunit combines with ¿ subunits and other channel interacting proteins (ChIPs) such as caveolin and syntrophins. Pathogenic genotypes have yet to be discovered in many patients from cohorts with suspected inherited arrhythmia syndromes (LQTS, SIDS, SUDS). From patients in these cohorts we have recently discovered mutations in putative ChIPs (CAV3, SNTA1, GPD1L, SCN4B) and shown that they affect INa in a way that may be pathogenic. In the next period we will continue a recently implemented more integrative approach to the study by expressing mutations in myocardial cells. This more complete cardiac environment allows for characterization of both the cellular phenotype and the molecular phenotype under relevant conditions such as adrenergic state in the presence of the intact macromolecular complex. In Aim 1 we will discover pathogenic mechanisms involving an SCN5A variant by characterizing the molecular and cellular phenotypes. In Aim 2 we will discover mutations in novel genes encoding putative SCN5A ChIPs from patients with suspected inherited arrhythmia and no known pathogenic genotype, and define molecular and cellular phenotypes to establish an INa mediated mechanism. In Aim 3 we will investigate SCN5A a-a subunit interactions (such as dominant negative and rescue effects). In Aim 4 we will address molecular mechanisms accounting for the molecular phenotype of altered INa for two novel arrhythmia genes (SNTA1 and GPD1L). The integrated approach of this proposal takes into account the SCN5A macromolecular complex in more native systems to determine both molecular and cellular phenotypes, and molecular mechanisms underlying the molecular phenotype. In addition to better understanding of the SCN5A macromolecular complex, the expected results should yield insight into pathogenetic mechanisms of the genotype-phenotype link for inherited and acquired arrhythmia and other cardiac disease. PUBLIC HEALTH RELEVANCE: Inherited mutations in the gene that encodes a membrane ion channel protein called SCN5A cause injury and death from arrhythmia in syndromes such as Sudden Infant Death Syndrome (SIDS), Sudden Unexpected Death Syndrome (SUDS), and long QT syndrome (LQTS). This project will discover mutations in novel genes (not SCN5A but related) discovered in patients with these syndromes and how they interact with SCN5A to alter function and cause arrhythmia. The results are expected to improve diagnosis of these inherited syndromes and suggest ways to treat and prevent them.