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Functional Genomics of Cardiac Sodium Channel Variants

心脏钠通道变异的功能基因组学

基本信息

批准号：
10538620
负责人：
DAN M RODEN
金额：
$ 73.54万
依托单位：
VANDERBILT UNIVERSITY MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-01 至 2024-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10538620
关键词：
Action Potentials American Amino Acids Atrial Fibrillation BRCA1 gene Benign Brugada syndrome CALM1 gene Cardiac Categories Cells Classification ClinVar Code Communities Computerized Medical Record Coupled DNA Data Data Set Databases Disease Electronic Health Record Electronic Medical Records and Genomics Network Electrophysiology (science)Family Gene Frequency General Population Genes Genetic Diseases Genomic medicine Genomics Health Heart Heart failure In Vitro Individual Ion Channel Lead Learning Life Long QT Syndrome Mainstreaming Maps Medical Genetics Medicine Mendelian disorder Methods Modeling Mutagens Mutation Nucleotides PTEN gene Participant Pathogenicity Patient Care Patients Persons Pharmaceutical Preparations Phenotype Play Population Proteins Provider Recommendation Recording of previous events Reporting Risk Role Scanning Sodium Sodium Channel Statistical Models Structural Models Structure Surface Syncope System TPMT gene Tachyarrhythmias Testing Transfection Transmembrane Domain United States National Institutes of Health Validation Variant Ventricular Work body system candidate identification clinical decision-making disorder risk experimental study falls functional genomics gain of function genetic testing genetic variant genome sequencing heart function heart rhythm human disease improved indium arsenide loss of function medical schools mutant mutation screening next generation sequencing novel novel strategies patch clamp protein function protein structure public health relevance rare variant response scale up sudden cardiac death variant detection variant of unknown significance

项目摘要

As clinical genetic testing is becoming widely deployed for patients with suspected Mendelian diseases as well as in the broad population, a major emerging challenge is the accurate prediction of pathogenicity of DNA variants. Multiple features, including allele frequencies across populations, family history, and functional studies, are currently being used to assign known or new variants to three broad categories: benign, pathogenic, or (most commonly) variant of uncertain significance (VUS). We propose here to test the hypothesis that deploying novel phenotyping methods will improve variant classification, and we will focus here on SCN5A, encoding the cardiac sodium channel. Despite the fact that this channel plays a critical role in normal heart function, SCN5A variants are surprisingly common and have been associated with serious and occasionally life-threatening phenotypes including type 1 Brugada Syndrome (BrS1), type 3 Long QT Syndrome (LQT3), conduction system disease, heart failure, and atrial fibrillation. In aim 1, we will determine the utility of phenotyping in the electronic health record (EHR) coupled to functional studies to assess SCN5A VUS pathogenicity. The Electronic Medical Records and Genomics (eMERGE) network in which we are participants is completing sequencing of 109 Mendelian disease genes, including SCN5A, in 25,000 subjects with EHRs. We will assess VUS pathogenicity by analyzing SCN5A-related EHR phenotypes in subjects with and without SCN5A rare variants and establishing in vitro function for newly-detected variants using multiplexed semi-automated electrophysiologic methods. In aim 2, we will build on preliminary data using deep mutational scanning (DMS) – a high-throughput method to mutagenize each nucleotide in a target genomic sequence and establish its functional consequences – to identify SCN5A coding variants with BrS1 or LQT3 features. In a pilot experiment, we developed a drug challenge that biases survival toward cells that do not express sodium current at their surface (the BrS phenotype) and against cells that display enhanced sustained sodium current (LQT3). We then generated all 252 possible non-synonymous or nonsense single amino acid (aa) variants across a 12aa regon of SCN5A, exposed a pool of cells transfected with one mutant/cell to the drug challenge, used next-generation sequencing pre- and post-drug challenge to identify variants with BrS1 and LQT3 features, and validated predictions with conventional patch clamp methods. We will now scale up to scan larger regions, starting with the 253 aa encoding transmembrane domain IV, known to harbor dozens of pathogenic variants. Further, we will map DMS results onto a model of the channel to probe the structural basis of loss and gain of function variants. We will incorporate DMS data into available and new statistical models to build an improved model of SCN5A variant disease risk. The result of this work will be new approaches to establish functional consequences of rare variants in SCN5A (and ultimately other genes), and thus enable genomic medicine.

随着临床基因检测也广泛应用于疑似孟德尔疾病的患者与广大人群一样，新出现的一个主要挑战是准确预测 DNA 的致病性变种。多种特征，包括人群中的等位基因频率、家族史和功能研究目前正用于将已知或新的变异分为三大类：良性、致病性或（最常见）意义不确定的变异（VUS）。我们建议在这里测试假设部署新的表型分析方法将改善变异分类，我们将在这里重点关注位于 SCN5A 上，编码心脏钠通道。尽管这个渠道在正常心脏功能中，SCN5A 变异非常常见，并且与严重和严重的心脏功能相关。偶尔危及生命的表型，包括 1 型布鲁格达综合征 (BrS1)、3 型长 QT 综合征 (LQT3)、传导系统疾病、心力衰竭和心房颤动。在目标 1 中，我们将确定电子健康记录 (EHR) 中表型分析的效用与功能研究相结合以评估 SCN5A VUS 致病性。我们在其中的电子病历和基因组学 (eMERGE) 网络参与者正在 25,000 人中完成 109 个孟德尔疾病基因的测序，其中包括 SCN5A 具有 EHR 的受试者。我们将通过分析 SCN5A 相关 EHR 表型来评估 VUS 致病性具有和不具有 SCN5A 罕见变异的受试者，并建立新检测到的变异的体外功能使用多重半自动电生理学方法。在目标 2 中，我们将利用初步数据深度突变扫描 (DMS) – 一种诱变靶标中每个核苷酸的高通量方法基因组序列并确定其功能结果 – 识别 SCN5A 编码变体 BrS1 或 LQT3 功能。在一项试点实验中，我们开发了一种药物挑战，使细胞的存活率偏向其表面不表达钠电流（BrS 表型），并且针对显示钠电流的细胞增强的持续钠电流（LQT3）。然后我们生成了所有 252 个可能的非同义或 SCN5A 12aa 区域的无义单氨基酸 (aa) 变体，暴露了一组转染的细胞使用一种突变体/细胞进行药物挑战，在药物挑战前和后使用下一代测序来识别具有 BrS1 和 LQT3 特征的变异，并使用传统膜片钳验证预测方法。我们现在将扩大扫描更大的区域，从 253 个氨基酸编码跨膜开始 IV 域，已知含有数十种致病变异。此外，我们将把 DMS 结果映射到一个模型上探索功能变体的损失和增益的结构基础的通道。我们将合并 DMS 数据入可用的新统计模型，以建立 SCN5A 变异疾病风险的改进模型。结果这项工作的重点将是建立 SCN5A 中罕见变异的功能后果的新方法（以及最终是其他基因），从而使基因组医学成为可能。