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.