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.

随着临床基因检测的广泛部署，可疑的孟德尔疾病患者与广泛人口一样，一个重大的新兴挑战是DNA致病性的准确性预测变体。多个功能，包括种群中的等位基因频率，家族史和功能研究目前已用于将已知或新变体分配给三个广泛类别：良性，致病性或（最常见的）不确定意义（VU）的变体。我们在这里建议测试部署新型表型方法将改善变异分类的假设，我们将重点在这里在SCN5A上，编码心脏钠通道。尽管该渠道在正常的心脏功能，SCN5A变体非常普遍，并且与严重和有时，威胁生命的表型，包括1型Brugada综合征（BRS1），类型3长QT 综合征（LQT3），传导系统疾病，心力衰竭和心房颤动。在AIM 1中，我们将确定电子健康记录（EHR）中表型的实用性与功能研究结合以评估 SCN5A VUS致病性。我们的电子病历和基因组学（出现）网络参与者是否正在完成包括SCN5A在内的109个Mendelian疾病基因的测序，其中25,000个具有EHR的受试者。我们将通过分析与SCN5A相关的EHR表型评估VUS致病性具有和不具有SCN5A稀有变体的受试者并为新发现的变体建立体外功能使用多路复用的半自动电生理方法。在AIM 2中，我们将基于使用初步数据深突变扫描（DMS） - 一种使目标中每个核苷酸化的高通量方法基因组序列并建立其功能后果 - 以确定SCN5A编码变体 BRS1或LQT3功能。在试点实验中，我们开发了一种药物挑战，使细胞生存偏见不会在其表面表达钠电流（BRS表型），并且针对显示的细胞增强的持续钠电流（LQT3）。然后，我们生成了所有252个可能的非同义词或横跨SCN5A的12AA重物的废话单氨基酸（AA）变体暴露了转染的细胞池在药物挑战中使用一个突变体/细胞，使用了下一代测序前和药后挑战识别具有BRS1和LQT3特征的变体，并用常规斑块夹进行验证的预测方法。现在，我们将扩展到扫描较大区域，从253 AA编码跨膜开始域IV，已知具有数十种致病性变体。此外，我们将DMS结果映射到一个模型探测功能变体损失和增益的结构基础的通道。我们将合并DMS数据进入可用的新统计模型，以建立改进的SCN5A变体疾病风险模型。结果这项工作将是建立SCN5A中罕见变体功能后果的新方法（和最终其他基因），从而实现基因组医学。