Atrial fibrillation (AF) is an important and increasing public health problem.  Most cases occur in the elderly and so the incidence of the problem is rising; the number of Americans affected with AF is expected to surge to ~16 million by 2050.  Other clinical features include hypertension and underlying heart disease while many relatively young patients have no apparent risk factors and are designated as ? lone?  or early-onset AF (EOAF).  The AF epidemic is further complicated by the lack of effective therapies.  The limited success of treatments stems in part from an incomplete understanding of the pathophysiology of AF and failure to target therapy to the underlying mechanisms.  Traditionally, AF was considered to be a sporadic, non-genetic disorder but we and others have shown that EOAF has a substantial genetic basis.  Positional cloning and candidate gene approaches have linked mutations in cardiac ion channels, and signaling molecules with EOAF.  While these studies have provided important insights into underlying mechanisms, most of the rare variation in susceptibility to EOAF remains unknown.  Given recent advances in next generation sequencing (NGS), discovery of novel genes in other cardiovascular phenotypes and our preliminary data, the overarching goal of this proposal is to use NGS to identify novel AF genes and decipher the underlying genetic mechanisms of EOAF.  Our clinical-DNA registries have systematically enrolled over 60 families from diverse ethnic background with early-onset familial AF.  We propose to use this large and well-characterized cohort to address two specific aims: Aim 1 will conduct a multi-tiered stepwise approach to identify rare genetic variants linked with EOAF.  First whole exome sequencing (WES) will be performed in our existing 60 EOAF families, who do not harbor candidate gene variants, to identify rare variants predicated on rarity, ethnicity, co-segregation with AF, predicted pathogenicity and bioinformatics filtering.  Second, the most promising variants will replicated in AFGen Consortium TOPMed Cohort in which over 3,500 probands with EOAF and 3,500 controls have undergone whole genome sequencing (WGS).  This aim builds on our published study in which we identified five novel candidate AF genes using a WES approach and the recent successful completion of WGS in EOAF probands in the TOPMed Cohort.  Aim 2 will determine the underlying genetic mechanisms of EOAF by functionally characterizing high priority rare variants using in vitro electrophysiology and in vivo functional assays in zebrafish to enable disease-association.  This aim builds upon our prior work where we have functionally characterized a rare AF-linked variant in the Ca channel gene identified by WES and identified rare developmental genes that modulate cardiac conduction in zebrafish.  These studies will not only identify novel AF genes and provide insights into underlying genetic mechanisms of EOAF but will also uncover novel therapeutic approaches for AF.  They also represent the first step in the development of sub-type specific mechanism-based therapies for this common and morbid condition.