High-throughput Discovery of Pathogenic Cardiac Sodium Channel Variants

项目总结 在美国，每年有超过25万人死于心律失常导致的心脏性猝死 各州。这些死亡中的一部分是由高度渗透性遗传性心律失常综合征引起的，例如 如Brugada综合征(BRS)。电压门控心脏钠通道功能变异的丧失， SCN5A是BRS的主要致病基因。此外，影响 SCN5A的表达与BRS有关。如果诊断为BRS，心源性猝死通常 使用植入型心律转复除颤器可以避免。因此，美国医学院 遗传学建议报告SCN5A的偶然致病变异，以便患者和 家庭成员可以得到准确的诊断和治疗。不幸的是，我们和其他人发现 SCN5A变异体的致病性通常是未知的或有争议的，而且往往不准确 预测心律失常。改进了编码和非编码SCN5A变体作为致病基因的分类 或良性将使BRS的诊断和治疗更加准确。我的假设是，在体外 高通量筛选方法可以准确识别广泛的致病编码和 调节性SCN5A功能变异缺失。我将追求两个具体目标来检验这一假设：1) 确定可减少通道活动和贩运的SCN5A编码变体，以及2)确定增强子 以及影响SCN5A表达的功能性非编码SNP。在第一个目标下，我将调查 1920个可能的编码变异体在一个重要的96个氨基酸区域的活性和贩运 SCN5A。我的初步数据显示了原理实验的证据，这些实验证明了 完成这项筛查所需的诱变、转基因和功能分析方法。在……下面 第二个目标，我已经实现了一个基于高通量测序的筛查来发现 影响SCN5A表达的增强子。我建议完成这个增强剂筛选，然后测试 影响这些增强子活性的SNP有助于BRS。这些研究具有创新性，因为 他们利用最近开发的基于高通量测序的方法来扩大和 提高我们对一种重要疾病基因变异的理解。随着基因组医学的继续 为了变得更加常见，解释患者变体的挑战将继续增长。 该项目提供了一个模板，用于改进 基因组注释，以帮助实现基因组和精确医学的承诺。

PROJECT SUMMARY Arrhythmia-induced sudden cardiac death claims more than 250,000 lives each year in the United States. A subset of these deaths result from highly penetrant inherited arrhythmia syndromes, such as Brugada Syndrome (BrS). Loss of function variants in the voltage-gated cardiac sodium channel, SCN5A, are the major known genetic cause of BrS. Additionally, regulatory variation that affects SCN5A expression has been implicated in BrS. If BrS is diagnosed, sudden cardiac death can often be averted with an implantable cardioverter-defibrillator. Therefore the American College of Medical Genetics recommends that incidental pathogenic variants in SCN5A be reported so that patients and family members can be accurately diagnosed and treated. Unfortunately, we and others have found that the pathogenicity of SCN5A variants is often unknown or disputed and often does not accurately predict arrhythmias. Improved classification of coding and non-coding SCN5A variants as pathogenic or benign would enable more accurate diagnosis and treatment of BrS. My hypothesis is that in vitro high-throughput screening methods can accurately identify a broad set of pathogenic coding and regulatory SCN5A loss of function variants. I will pursue two specific aims to test this hypothesis: 1) Identify SCN5A coding variants that reduce channel activity and trafficking, and 2) Identify enhancers and functional non-coding SNPs affecting SCN5A expression. Under the first aim, I will survey the activity and trafficking of the 1920 possible coding variants in an important 96 amino acid region of SCN5A. My preliminary data shows proof of principle experiments that demonstrate the feasibility of mutagenesis, transgenesis, and functional assay methods necessary to complete this screen. Under the second aim, I have implemented a high-throughput sequencing-based screen to discover enhancers that affect SCN5A expression. I propose to finish this enhancer screen, then test whether SNPs that affect these enhancers' activity contribute to BrS. These studies are innovative because they leverage recently developed high-throughput sequencing-based methods to broaden and improve our understanding of variants in an important disease gene. As genomic medicine continues to become more commonplace, the challenge of interpreting patients' variants will continue to grow. This project provides a template for a general approach for improving the breadth and quality of genomic annotations to help deliver on the promise of genomic and precision medicine.