Aberrant Splicing in the Cardiac Arrhythmias

项目摘要/摘要 全基因组测序(WGS)的日益广泛使用揭示了大量罕见的非同义词 在健康和疾病个体中识别的疾病基因的变异。其中大多数目前都是 分类为不确定意义的变体(VU)，并了解这些变体的功能影响 变异体是基因组医学实施的下一步必要步骤。WGS也发现了变化 导致异常剪接，这是一种越来越为人所熟知的疾病机制。目前的估计表明， 在孟德尔病的所有致病变异中，有10%是由异常剪接引起的。孟德尔学派的研究 心律失常不仅照亮了正常和异常的心脏电生理 机制，但推动了越来越常规的临床基因测试，被认为是有风险的患者 结果，例如每年导致25万美国人死亡的心脏性猝死和早发性心房试验 房颤(EoAF)。易患SCD的主要遗传性心律失常疾病包括QT间期延长综合征 (LQTS)和Brugada综合征(BRS)，而Vanderbilt和其他地方的工作涉及一系列 EoAF中的通道病和心肌病综合征。缺乏有重点的努力来确定 导致这些疾病之间的异常剪接构成了临床可操作性的障碍。这项工作将 解决我们对心律失常中剪接扰动变异体的不完全了解。我假设基因是 影响剪接的变异导致遗传性心律失常。因此，我将部署一系列职能部门 使用人类遗传学和分子生物学的最新进展来评估异常剪接的研究。第一, 我将使用微型基因和CRISPR-Cas9检测来评估假定的剪接改变VU在BRS和 LQTS。变异的重新分类对于提高这些疾病的基因检测的效率是至关重要的。第二, 我将采用高通量微基因分析来确定基因变异对SCN5A替代方案的影响 剪接并设计反义寡核苷酸来调节这种剪接。这代表了一种潜在的治疗方法 对于受发育选择性剪接外显子罕见变异影响的患者的方法。第三，拼接- 将在大量EoAF患者中研究心律失常和心肌病基因的改变变异 那些经历过WGS的人。引入神秘剪接位点的变体将成为反义靶点 寡核苷酸和小分子在疾病相关模型中逆转变异的表型效应。 总的来说，这些研究将极大地促进我们对剪接作为一种贡献机制的理解 在遗传性心律失常中。这个项目，辅以严格的人类遗传学和 遗传性心律失常的临床培训，将提供大量机会来发展技术和 精通翻译遗传学，以支持我成为一名领先的内科科学家的目标。

PROJECT SUMMARY/ABSTRACT The increasing use of whole genome sequencing (WGS) is uncovering large numbers of rare non-synonymous variants in recognized disease genes in both healthy and diseased individuals. Most of these are currently classified as Variants of Uncertain Significance (VUS’s) and understanding the functional effects of these variants is a required next step for the implementation of genomic medicine. WGS is also uncovering variation leading to aberrant splicing, an increasingly well-recognized disease mechanism. Current estimates suggest that 10% of all pathogenic variants in Mendelian diseases arise from abnormal splicing. The study of Mendelian cardiac arrhythmia disorders has not only illuminated normal and abnormal cardiac electrophysiologic mechanisms, but has propelled increasingly routine clinical genetic testing for patients thought to be at risk for outcomes such as sudden cardiac death – which kills >250,000 Americans each year – and Early-onset Atrial Fibrillation (EoAF). Key genetic arrhythmia diseases predisposing to SCD include the long QT syndromes (LQTS) and Brugada Syndrome (BrS), while work at Vanderbilt and elsewhere has implicated a range of channelopathy and cardiomyopathy syndromes in EoAF. The absence of a focused effort to identify variants that contribute to aberrant splicing among these diseases constitutes a barrier to clinical actionability. This work will address our incomplete knowledge of splice-perturbing variants in the arrhythmias. I hypothesize that genetic variation affecting splicing contributes to the genetic arrhythmias. I will therefore deploy a series of functional investigations using recent advances in human genetics and molecular biology to assess aberrant splicing. First, I will use minigene and CRISPR-Cas9 assays to assess the impact of putative splice-altering VUS’s in BrS and LQTS. Variant reclassification is essential for improving the yield of genetic testing in these diseases. Second, I will adopt a high throughput minigene assay to determine the impact of genetic variation on SCN5A alternative splicing and design antisense oligonucleotides to modulate this splicing. This represents a potential therapeutic approach for patients affected by rare variants in a developmentally alternatively spliced exon. Third, splice- altering variation will be investigated in arrhythmia and cardiomyopathy genes in a large cohort of EoAF patients who have undergone WGS. Variants introducing cryptic splice sites will be targeted by antisense oligonucleotides and small molecules to reverse the phenotypic effects of variants in a disease-relevant model. Collectively, these studies will significantly advance our understanding of splicing as a contributory mechanism among the genetic arrhythmias. This project, complemented by rigorous coursework in human genetics and clinical training in inherited arrhythmias, will provide substantial opportunities to develop techniques and proficiency in translational genetics to support my goal of becoming a leading physician scientist.