Cellular & Clinical Phenotypes of Novel SCN5a Mutations

描述（由申请人提供）：SCN5A编码心脏中发现的人类电压依赖性钠通道（hNaV1.5）的α亚基。我们进行了新的观察，发现人类心脏中存在多达四种非常常见的NaV1.5变体，其中至少有一些具有功能意义。该通道的突变也可导致先天性获得性长QT综合征（LQT3）和Brugada综合征（BS）的心源性猝死。我们最近对四种新的SCN5A突变进行了表征，发现：1)在婴儿猝死综合征（A997S, R1826H）中，LQT3.2、LQT3 （M1766L）和BS （G1743）突变的表达缺陷被抗心律失常药物“拯救”。3) M1766L的电流是否正常取决于测试所用的NaV1.5背景的不同。我们建议进一步探讨表达缺陷的程度和机制及其“拯救”和“背景”的重要性。通过与梅奥诊所的Ackerman博士的合作，我们还获得了大约20个新的SCN5A突变来研究新的功能缺陷和心律失常机制。我们将在细胞培养中制造和表达这些通道，通过电压钳和免疫细胞化学确定功能，并通过心律失常机制将分子功能与临床表型联系起来。在目标1中，我们将研究野生型变异的表达和功能，以及突变体通道的表达和功能如何取决于背景克隆。在目标2中，我们将研究新的突变体。在Aim 3中，我们将研究具有“功能增益”的突变，并验证LQT3的晚期电流衰减与增强的速率依赖性QT间期适应、晚发病和比没有晚期电流衰减的突变更好的预后相关的假设。在Aim 4中，我们将研究具有“功能丧失”的突变，功能丧失的机制包括新的运输缺陷，并验证这种丧失可以通过药物“拯救”的假设。在Aim 5中，我们将与beta1和beta3亚基共表达突变，并评估PKA刺激的效果，以验证这些区域对作用机制至关重要的假设。这些新发现的研究将对心律失常机制和突变心律失常综合征的基因型-表型相关性以及更普遍的“正常”心脏变异产生影响，这可能会对获得性心律失常的遗传易感性产生影响。在更基本的层面上，这些“自然”实验将有助于理解这一重要通道的结构-功能关系。

DESCRIPTION (provided by applicant): SCN5A encodes the alpha subunit of the human voltage-dependent Na channel (hNaV1.5) found in heart. We have made the novel observation that up to four very common variants of NaV1.5 exist in human heart and at least some have functional implications. Mutations in this channel also cause sudden cardiac death in the congenitally acquired long QT syndrome (LQT3) and the Brugada Syndrome (BS). We have recently characterized four novel SCN5A mutations and found: 1) Two in Sudden Infant Death Syndrome (A997S, R1826H) are LQT3.2) LQT3 (M1766L) and BS (G1743) mutations have expression defects "rescued" by antiarrhythmic drugs. 3) M1766L has normal or absent current depending on the variant NaV1.5 background used to test it. We propose to investigate further the extent and mechanisms of expression defects and their "rescue" and the importance of "background". Through collaboration with Dr. Ackerman at Mayo Clinic we also have >20 additional novel SCN5A mutations to investigate for novel functional defects and arrhythrnia mechanism. We will make and express these channels in cell culture, define function by voltage clamp and immunocytochemistry, and correlate molecular function with clinical phenotype through arrhythmia mechanism. In Aim 1 we will investigate the expression and function of wild type variants, and also how mutant channel expression and function depends upon the background clone. In Aim 2 we will study novel mutants. In Aim 3 we will investigate mutants with "gain of function" and test the hypothesis that late current decay in LQT3 correlates with enhanced rate dependent QT interval adaptation, later onset, and better prognosis than mutations without late current decay. In Aim 4 we will investigate mutations with "loss of function", the mechanism for loss of function including novel trafficking defects, and test the hypothesis that this loss can be "rescued" by drugs. In Aim 5 we will co-express mutations with the beta1 and beta3 subunit, and assess effects of PKA stimulation, to test the hypothesis that these areas are critical to the mechanism of action. These studies on the novel findings will have implications for arrhythmia mechanism and genotype-phenotype correlation in both mutation arrhythmia syndromes and more generally for the variants in "normal' hearts that may generate insight into genetic predisposition to acquired arrhythmia. At a more basic level these "natural" experiments will contribute to understanding the structure-function relationship of this important channel.