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

基本信息

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
项目类别：
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

项目摘要

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（由申请人提供）：随着临床重测序在精准医学实践中变得越来越普遍，人类基因组学中的一个主要新挑战是确定罕见变异的功能，我在此提出一种使用诱导多能干细胞（iPSC）来解决这一问题的方法。新的候选疾病基因在人类心律失常综合征中的作用我们已经确定了一个有多位成员患有布鲁格达综合征（BrS）的家庭，这种疾病具有独特的心电图模式，反映了钠电流和猝死风险增加。 SCN5A（编码心脏钠通道的基因以及与 BrS 最常见的单基因相关性）没有出现功能缺失突变，但通过 Sanger 测序在已知的 SCN5A 表达调节因子 TBX5 和 SCN10A 中检测到意义不明的变异。 SCN5A 表达调节网络中的变异对人类心肌细胞电生理学的影响尚不清楚，我推测这些变异单独或共同降低 SCN5A 表达，与家族遗传一致。描述了 TBX5 的 BrS 表型，它含有对哺乳动物上肢和心脏发育过程中的组织模式和细胞分化至关重要的转录因子，在小鼠模型中，Tbx5 的单倍体不足会导致间隙连接蛋白、心房钠尿肽的心脏表达减少。，和心脏常见的人类变体 SCN10A (rs6801957) 位于 SCN5A 增强子中，TBX5 被认为与该增强子相互作用，并可能导致 SCN5A 表达的错误调节。在这项研究中，我将利用最近开发的方法来生成人类心肌细胞。我从患者来源的 iPSC 中分离出真皮成纤维细胞，以便以物种、组织和遗传背景特异性的方式研究遗传变异。家庭成员并将它们重新编程为 iPSC，其他人正在进行将它们强烈分化为心肌细胞，我的初步数据显示与对照细胞相比，受影响患者的细胞中钠电流密度显着降低，这支持了我的工作假设。初步的电生理学结果，以及评估 SCN5A 和 TBX5 的表达重要的是，我使用 RNA 引导的 Cas9 核酸酶将候选变体逆转为野生型序列，从而给了我机会——这是有史以来第一次。心律失常疾病 - 明确确定突变对特定患者细胞表型的作用，了解这些变异对人类心肌细胞心脏钠电流的个体和组合功能影响可能是室性心律失常风险分层的第一步。此外，这项工作通过提供可扩展的方法而具有高度创新性。建立新候选遗传变异功能的方法，以及研究此类变异在多个遗传背景下的影响。