Cellular & Clinical Phenotypes of Novel SCN5a Mutations

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• 批准号: 6944832
• 负责人: JONATHAN C MAKIELSKI
• 金额: $ 36.38万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-15 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6944832
• 关键词: arrhythmia; gene mutation; human data; human genetic material tag; immunocytochemistry; protein isoforms; protein structure function; sodium channel; voltage /patch clamp; voltage gated channel

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.

描述（由申请人提供）：SCN 5A编码心脏中发现的人电压依赖性Na通道（hNaV1.5）的α亚基。 我们发现，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。 该通道的突变也会导致先天性获得性长QT综合征（LQT 3）和Brugada综合征（BS）的心脏性猝死。 我们最近发现了四种新的SCN 5A突变：1）婴儿猝死综合征（A997 S，R1826 H）中的两种是LQT 3。2）LQT 3（M1766 L）和BS（G1743）突变具有被抗肿瘤药物“拯救”的表达缺陷。 3)M1766 L在不同的NaV1.5背景下是否有正常或缺失的电流，我们建议进一步研究表达缺陷的程度和机制，以及它们的“拯救”和“背景”的重要性。 通过与马约诊所的阿克曼博士合作，我们还发现了20多个新的SCN 5A突变，以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来定义功能，并通过心律失常机制将分子功能与临床表型相关联。 在目标1中，我们将研究野生型变体的表达和功能，以及突变通道的表达和功能如何依赖于背景克隆。 在目标2中，我们将研究新的突变体。 在目标3中，我们将研究具有“功能获得”的突变体，并检验LQT 3中的晚电流衰减与增强的频率依赖性QT间期适应、晚发作和比无晚电流衰减的突变更好的预后相关的假设。 在目标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，并检验这种丧失可以通过药物“拯救”的假设。 在目标5中，我们将与β 1和β 3亚基共表达突变，并评估PKA刺激的影响，以检验这些区域对作用机制至关重要的假设。 这些对新发现的研究将对突变心律失常综合征中的心律失常机制和基因型-表型相关性产生影响，更广泛地说，对“正常”心脏中的变体产生影响，这些变体可能会深入了解获得性心律失常的遗传易感性。 在更基本的水平上，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。