Aberrant Splicing in the Cardiac Arrhythmias

心律失常中的异常剪接

基本信息

批准号：
10784588
负责人：
Matthew O'Neill
金额：
$ 3.29万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-07-01 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10784588
关键词：
Academic Medical Centers Adopted Adult Affect Alternative Splicing American Antisense Oligonucleotides Area Arrhythmia Atrial Fibrillation Bar Codes Benign Biological Assay Brugada syndrome CRISPR/Cas technology Cardiac Myocytes Cardiomyopathies Cells Characteristics Classification Clinic Clinical Clustered Regularly Interspaced Short Palindromic Repeats Complement Coupled Data Data Set Development Disease Electrophysiology (science)Environment Etiology Evaluation Exons Genes Genetic Genetic Variation Genomic medicine Goals Heart Abnormalities Human Human Genetics In Vitro Individual Inherited Investigation Knowledge Long QT Syndrome Maps Medical Genetics Mendelian disorder Methods Modality Modeling Molecular Biology Molecular Genetics Mus Mutagens Organic Chemistry Outcome Palpitations Pathogenicity Pathology Patients Pharmaceutical Chemistry Phenotype Physicians Pluripotent Stem Cells Pre-Clinical Model Productivity Protein Isoforms RNA Splicing Research Risk Running Scientist Series Site Syndrome Techniques Technology Testing Therapeutic Trans-Omics for Precision Medicine Transfection Variant Work base editor clinical training clinically actionable cohort design diagnostic value disease phenotype disorder risk drug discovery early onset fetal genetic testing genome editing genome sequencing improved in silico insight interest laboratory experiment loss of function neural network novel therapeutics precision medicine proband rare variant skill acquisition small molecule sudden cardiac death therapeutic target transcriptome sequencing translational genetics variant of unknown significance vector whole genome

项目摘要

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.

项目概要/摘要全基因组测序 (WGS) 的使用日益广泛，发现了大量罕见的非同义基因目前，健康个体和患病个体中的已知疾病基因均存在变异。被归类为意义不确定的变体 (VUS) 并了解这些变体的功能影响变异是实施基因组医学所需的下一步，同时也揭示了变异。导致异常剪接，这是一种日益被广泛认可的疾病机制。孟德尔疾病中 10% 的致病变异源于异常剪接。心律失常疾病不仅阐明了正常和异常的心脏电生理学机制，但推动了对被认为有风险的患者进行越来越常规的临床基因检测心源性猝死（每年导致超过 250,000 名美国人死亡）和早发性心房颤动等后果颤动 (EoAF) 诱发 SCD 的主要遗传性心律失常疾病包括长 QT 综合征。（LQTS）和布鲁格达综合症（BrS），而范德比尔特大学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集中精力来识别变异。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。为了解决我们对心律失常中剪接扰动变异的不完全了解，我匆忙地提出了遗传问题。影响剪接的变异会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来评估异常剪接的研究。我将使用小基因和 CRISPR-Cas9 检测来评估假定的剪接改变 VUS 对 BrS 和 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。我将采用高通量小基因检测来确定遗传变异对 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。针对受发育选择性剪接外显子中罕见变异影响的患者的方法第三，剪接。将在一大群 EoAF 患者中研究心律失常和心肌病基因的变异变化接受过全基因组测序（WGS）的变异体将成为反义的目标。寡核苷酸和小分子可逆转疾病相关模型中变异的表型效应。总的来说，这些研究将显着增进我们对剪接作为贡献机制的理解该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为开发技术和精通转化遗传学，以支持我成为领先的医学科学家的目标。