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 ge...