High-throughput Discovery of Pathogenic Cardiac Sodium Channel Variants

高通量发现致病性心脏钠通道变异体

基本信息

批准号：
9762228
负责人：
Andrew M. Glazer
金额：
$ 1.46万
依托单位：
VANDERBILT UNIVERSITY MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-01 至 2019-10-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9762228
关键词：
Action Potentials Affect American Amino Acids Arrhythmia Benign Biological Assay Brugada syndrome Cardiac Cardiac Myocytes Cell surface Cells Cessation of life Classification Code Coupled Data Diagnosis Disease Disputes Electrophysiology (science)Enhancers Family member Flow Cytometry Gene Transfer Techniques Genes Genetic Genetic screening method Genomic Segment Genomic medicine Genomics Haplotypes Heart Diseases High-Throughput Nucleotide Sequencing Implantable Defibrillators In Vitro Inherited Knowledge Lead Luciferases Medical Genetics Methods Modeling Mutagenesis Operative Surgical Procedures Pathogenicity Patients Population Property Reporting Sampling Sodium Channel Surveys Syndrome Testing United States Untranslated RNA Variant Work accurate diagnosis base cell killing experimental study genome wide association study high throughput screening implantation improved indexing innovation loss of function medical schools mutation screening precision medicine prevent sodium channel proteins sudden cardiac death trafficking voltage

项目摘要

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.

项目概要在美国，心律失常引起的心源性猝死每年夺去超过 25 万人的生命国家。这些死亡的一部分是由高度渗透性遗传性心律失常综合征引起的，例如布鲁格达综合症（BrS）。电压门控心脏钠通道功能变异的丧失， SCN5A 是已知的 BrS 的主要遗传原因。此外，监管变化也会影响 SCN5A 表达与 BrS 相关。如果诊断出 BrS，通常会导致心源性猝死可以通过植入式心脏复律除颤器来避免。因此美国医学院遗传学建议报告 SCN5A 的偶然致病变异，以便患者和家属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 变异的致病性通常是未知的或有争议的，并且通常不能准确地预测心律失常。改进了编码和非编码 SCN5A 变异的致病性分类或良性将使 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 功能丧失变异体。我将追求两个具体目标来检验这个假设：1）识别减少通道活动和贩运的 SCN5A 编码变体，以及 2) 识别增强子以及影响 SCN5A 表达的功能性非编码 SNP。在第一个目标下，我将调查 1920 个可能的编码变体在重要的 96 个氨基酸区域中的活性和运输 SCN5A。我的初步数据显示了原理实验的证明，证明了可行性完成此筛选所需的诱变、转基因和功能测定方法。在下面第二个目标，我实施了基于高通量测序的筛选来发现影响 SCN5A 表达的增强子。我建议完成这个增强器屏幕，然后测试是否影响这些增强子活性的 SNP 会导致 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拓宽和提高我们对重要疾病基因变异的理解。随着基因组医学的不断发展为了变得更加普遍，解释患者变异的挑战将继续增加。该项目为提高技术广度和质量的通用方法提供了一个模板。基因组注释有助于实现基因组和精准医学的承诺。