Cellular and clinical phenotypes of novel SCN5a mutations

描述(由申请人提供)：由SCN5A编码的流经心脏钠通道a亚单位的钠电流(Ina)功能障碍参与了心律失常、心力衰竭和缺血的致病机制。一种特定基因的临床表型的机制来自于由该突变引起的潜在的分子表型(例如，INA幅度和动力学)和细胞表型(例如，动作电位)。在前一阶段，我们重点研究了与SCN5A突变相关的异源表达系统中的分子表型。我们发现了一个普遍存在的人类剪接变异体(Q1077del)，它影响其他突变的功能，8个常见多态(如H558R)的临床相关功能效应，以及贩卖缺陷突变的药物拯救。我们还显示了一个Brugada综合征SCN5A等位基因对另一个等位基因的新的显性负效应。这项建议的两个目标(1和3)跟进了SCN5A基因变异的这些致病机制。另外两个目标(2和4)将该项目带向一个新的方向。越来越清楚的是，INA是SCN5A大分子复合体的功能，其中一个以上的亚基与亚基和其他通道相互作用蛋白(CHIP)如小窝蛋白和合营养素结合。在许多疑似遗传性心律失常综合征(LQTS、SID、SODS)的队列患者中，尚未发现致病基因类型。从这些队列中的患者中，我们最近发现了可能的芯片(CAV3、SNTA1、GPD1L、SCN4B)的突变，并表明它们以一种可能致病的方式影响INA。在下一阶段，我们将继续最近实施的更综合的方法，通过在心肌细胞中表达突变来进行研究。这种更完整的心脏环境允许在相关条件下表征细胞表型和分子表型，例如在完整的大分子复合体存在的情况下的肾上腺素能状态。在目标1中，我们将通过分子和细胞表型的特征来发现涉及SCN5A变异的致病机制。在目标2中，我们将从疑似遗传性心律失常和未知致病基因的患者中发现新的编码SCN5A芯片的基因突变，并定义分子和细胞表型以建立INA介导的机制。在目标3中，我们将研究SCN5Aα-α亚基之间的相互作用(如显性负效应和挽救效应)。在目标4中，我们将阐述两个新的心律失常基因(SNTA1和GPD1L)的INA改变的分子表型的分子机制。该建议的综合方法考虑了更多自然系统中的SCN5A大分子复合体，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大分子复合体外，预期的结果还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-表型联系的致病机制。与公共卫生相关：编码一种名为SCN5A的膜离子通道蛋白的基因的遗传突变会导致婴儿猝死综合征(SID)、猝死综合征(SUDS)和长QT综合征(LQTS)等综合征中心律失常造成的损伤和死亡。该项目将发现在这些综合征患者中发现的新基因(不是SCN5A，而是相关基因)的突变，以及它们如何与SCN5A相互作用改变功能并导致心律失常。这些结果有望改善对这些遗传综合征的诊断，并提出治疗和预防它们的方法。

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.