Functional and Transcriptional Mechanisms of Familial Brugada Syndrome

家族性布鲁格达综合征的功能和转录机制

• 批准号: 9272264
• 负责人: Kevin R. Bersell
• 金额: $ 4.61万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-02-03 至 2020-02-02
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9272264
• 关键词: Acute myocardial infarction; Address; Affect; Animals; Arrhythmia; Atrial Natriuretic Factor; Binding; Biochemical; Brugada syndrome; CRISPR/Cas technology; Candidate Disease Gene; Cardiac; Cardiac Electrophysiologic Techniques; Cardiac Myocytes; Cells; Clinical; Complex; Connexins; DNA; DNA Binding; DNA Resequencing; Data; Dermal; Development; Dideoxy Chain Termination DNA Sequencing; Disease; Electrocardiogram; Electrophysiology (science); Enhancers; Family; Family Study; Family member; Fibroblasts; Gene Targeting; Genes; Genetic; Genetic Transcription; Genetic Variation; Genetic study; Guide RNA; Heart; Human; Impairment; Individual; Investigation; Mediating; Methods; Modeling; Muscle Cells; Mutation; Myocardium; Patients; Pattern; Pharmaceutical Preparations; Phenotype; Physiology; Population; Predisposition; Public Health; Regulation; Regulator Genes; Reporter; Research; Risk; Role; Sodium; Sodium Channel; Stratification; Syndrome; Testing; Time; Tissues; Transcriptional Regulation; Translations; United States; Upper Extremity; Variant; Ventricular Arrhythmia; Ventricular Tachycardia; Work; clinical decision-making; clinically relevant; combinatorial; density; genetic association; genetic variant; genome editing; human genomics; indium arsenide; induced pluripotent stem cell; innovation; loss of function mutation; member; method development; mouse model; mutant; novel; nuclease; precision medicine; prevent; public health relevance; rare variant; sudden cardiac death; transcription factor; variant of unknown significance

 DESCRIPTION (provided by applicant): As clinical resequencing becomes increasingly common in the practice of precision medicine, a major emerging challenge in human genomics is establishing function of rare variants. I present here an approach using induced pluripotent stem cells (iPSC) to address the role of new candidate disease genes for a human arrhythmia syndrome. We have identified a family with multiple members affected by Brugada syndrome (BrS), a condition with a distinctive ECG pattern reflecting decreased sodium current and increased risk of sudden cardiac death. There were no loss of function mutations in SCN5A, the gene encoding the cardiac sodium channel and the most common monogenic association with BrS. However, variants of unknown significance were detected by Sanger sequencing in TBX5 and SCN10A, known regulators of SCN5A expression. The influence of variants in the regulation network of SCN5A expression on human cardiomyocyte electrophysiology is undefined. I hypothesize that these variants, individually or together, reduce SCN5A expression, consistent with the familial BrS phenotype described. TBX5 is a T-box containing transcription factor critical to mammalian tissue patterning and cellular differentiation during development of the upper extremities and the heart. Haploinsufficiency of Tbx5 in murine models results in diminished cardiac expression of gap junction proteins, atrial natriuretic peptide, and the cardiac sodium channel. The common human variant SCN10A (rs6801957) is located in an SCN5A enhancer with which TBX5 is thought to interact and likely contributes to misregulation of SCN5A expression. In this research I will take advantage of the recent development of methods to generate human cardiomyocytes from patient-derived iPSCs to allow for the study of genetic variants in a species, tissue, and genetic background-specific manner. I have isolated dermal fibroblasts from an affected family member and reprogrammed them to iPSCs, and others are underway. I have differentiated these into cardiomyocytes, and my preliminary data show strikingly reduced sodium current density in cells from affected patients compared to control cells, strongly supporting my working hypothesis. I will extend my preliminary electrophysiologic findings, as well as assess SCN5A and TBX5 expression. Importantly, I have used RNA-guided Cas9 nuclease to reverse the candidate variants to wild-type sequence thus giving me the opportunity - for the first time in any arrhythmia disease - to definitively establish the role of he mutations to the cellular phenotype in a specific patient. Understanding the individual and combinatorial functional effects of these variants on cardiac sodium current in human cardiomyocytes can be an initial step in the stratification of ventricular arrhythmia risk to influence clinical decision making. Further, this work is highly innovative by providing a scalable approach to establish function of new candidate genetic variants, as well as to study the effects of such variants across multiple genetic backgrounds.

 描述（由适用提供）：随着临床重新方程在精确医学的实践中变得越来越普遍，人类基因组学的重大挑战是建立稀有变体的功能。我在这里提出了一种使用诱导多能干细胞（IPSC）来解决人类心律不齐综合征的作用的方法。我们已经确定了一个患有多个受Brugada综合征（BRS）的家庭的家庭，这种疾病具有独特的ECG模式，反映了钠电流降低并增加心脏死亡的风险。 SCN5A，编码心脏钠通道的基因以及与BRS最常见的单基因关联，没有功能突变的损失。然而，通过在SCN5A表达的已知调节剂TBX5和SCN10A中，Sanger测序检测到了未知显着性的变异。 SCN5A表达调节网络中变体对人心肌细胞电生理学的影响不确定。我假设这些变体单独或一起降低了SCN5A表达，与所描述的家族BRS表型一致。 TBX5是一种T-box，其中包含在上肢和心脏发育过程中对哺乳动物组织模式和细胞分化至关重要的转录因子。鼠模型中TBX5的单倍不足会导致间隙连接蛋白，心房纳地尿肽和心脏的心脏表达降低 钠通道。普通的人类变体SCN10A（RS6801957）位于SCN5A增强子中，TBX5被认为可以相互作用，并可能导致SCN5A表达的不正常。在这项研究中，我将利用最近开发的方法来从患者衍生的IPSC中产生人类心肌细胞，以研究一种物种，组织和遗传背景特异性方式中的遗传变异。我已经将它们分化为心肌细胞，而我的初步数据显示，与对照细胞相比，受影响患者的细胞中的钠电流密度大大降低，从而强烈支持了我的工作假设。我将扩展我的初步电生理发现，并评估SCN5A和TBX5表达。重要的是，我已经使用RNA引导的CAS9核酸酶将候选变体扭转为野生型序列，从而使我在任何心律不齐疾病中首次获得了机会，以确定在特定患者的细胞表型中确定HE突变对细胞表型的作用。了解这些变体对人心肌细胞中心脏钠电流的个体和组合功能效应可能是室性心律失常风险分层以影响临床决策的第一步。此外，通过提供可扩展性，这项工作具有很高的创新性 建立新候选遗传变异的功能的方法，以及研究这些变异在多种遗传背景之间的影响。