Since 2005, genome wide association studies (GWAS) have identified thousands of associations between genetic loci and complex traits, but functional mechanistic studies have lagged. I propose to resolve the mechanism of the most robust GWAS signal for the QT interval at chromosome 1q23. Discovered in 2006, chr1q23 is associated with mortality, drug response, and arrhythmia risk in otherwise healthy individuals and in those with congenital long QT syndrome (cLQTS). The chr1q23 haplotype explains variation in mRNA levels of the nearest mapped gene NOS1AP, suggesting that it affects QT via altered NOS1AP expression. In animal cardiomyocytes and neurons, NOS1AP binds NOS1, a nitric oxide synthase that catalyzes the S-nitrosylation (SNO) reaction between NO and thiol groups on cysteine residues. Some rare genetic variants cause cLQTS by altering proteins that interact with the sodium channel, NaV1.5, increasing SNO of Nav1.5 to enhance late sodium current. Thus, I hypothesize that the GWAS signal at chr1q23 increases the QT through greater S-nitrosylation of the cardiac sodium channel and enhanced late sodium current. My preliminary data support this hypothesis: SNO block blunts late sodium current to wild-type levels in some but not all gain-of-function variants in NaV1.5, keeping action potential durations (APD) short. I will test my hypothesis with 3 aims. First, I will delete a 150 base pair DNA segment surrounding the lead polymorphism in chr1q23 using CRISPR-Cas9. I will do this in induced pluripotent stem cells differentiated to cardiomyocytes (iPSC-CMs) having SNO-sensitive NaV1.5 variants. I will then measure APD and NOS1AP levels to establish chr1q23's role in NOS1AP expression, expecting longer APD and reduced expression. In Aim 2, I will study how NOS1AP modulates late current. In iPSC-CMs treated with siRNA targeting NOS1AP, I will measure SNO of NaV1.5 and APD, expecting increased SNO and longer APD. Finally, I will test the generalizability of this mechanism using a high-throughput electrophysiological platform. I will measure late sodium current, at baseline and upon treatment with a SNO blocker, in ~40 NaV1.5 variants strongly or weakly associated with cLQTS. If successful, this work will explain the mechanism of the most robust GWAS signal for the QT interval and will identify a novel therapeutic target, SNO inhibition, for congenital and drug-induced long QT. It will also help stratify arrhythmia risk to inform clinical decisions.
|Program type||Predoctoral Fellowship|
|Effective start/end date||01/01/2020 → 12/31/2021|