Aberrant Splicing in the Cardiac Arrhythmias

项目概要/摘要 全基因组测序（WGS）的使用越来越多，发现了大量罕见的非同义突变。 健康和患病个体中已识别的疾病基因的变异。其中大部分目前 被归类为不确定意义的变体（VUS），并了解这些变体的功能效果 变异体是实施基因组医学所需的下一步。WGS还发现了 导致异常剪接，这是一种越来越被广泛认识的疾病机制。目前的估计表明， 孟德尔疾病中所有致病性变体的10%来自异常剪接。孟德尔的研究 心律失常不仅阐明了正常和异常的心脏电生理， 机制，但推动了越来越多的常规临床基因检测的患者被认为是在风险， 结果，如心脏性猝死-每年杀死> 250，000美国人-和早发性心房颤动 房颤（EoAF）。易患SCD的关键遗传性心律失常疾病包括长QT综合征 （LQTS）和Brugada综合征（BrS），而在范德比尔特和其他地方的工作涉及一系列的 EoAF中的通道病和心肌病综合征。没有集中精力确定 导致这些疾病之间异常剪接的基因突变构成了临床可操作性的障碍。这项工作将 解决了我们对心律失常中剪接干扰变体的不完整知识。我假设基因 影响剪接的变异导致遗传性心律失常。因此，我将部署一系列功能 利用人类遗传学和分子生物学的最新进展进行研究，以评估异常剪接。第一、 我将使用minigene和CRISPR-Cas9测定来评估推定的剪接改变VUS在BrS中的影响， LQTS。变异重新分类对于提高这些疾病的基因检测的产量至关重要。第二、 我将采用高通量微基因检测来确定遗传变异对SCN 5A替代物的影响， 剪接和设计反义寡核苷酸以调节这种剪接。这代表了一种潜在的治疗方法 一种用于受发育性选择性剪接外显子中罕见变异影响的患者的方法。第三，拼接- 将在一个大的EoAF患者队列中研究心律失常和心肌病基因的改变变异 谁经历了WGS。引入隐蔽剪接位点的变体将被反义寡核苷酸靶向。 寡核苷酸和小分子来逆转疾病相关模型中变体的表型效应。 总的来说，这些研究将大大推进我们对剪接作为一种贡献机制的理解 遗传性心律失常这个项目，辅以严格的人类遗传学课程， 遗传性心律失常的临床培训，将提供大量的机会， 熟练的转化遗传学，以支持我成为一名领先的医学科学家的目标。

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.