Cis regulatory variants of haploinsufficiency genes as cardiomyopathy modifiers

Abstract: Cardiomyopathies, occurring in ~1:250 in the population, are disorders with primary impairment of heart muscle structure and function. Approximately 40-50% of the major cardiomyopathy subtypes can be explained as autosomal dominant with incomplete penetrance. Variants causing large subsets of cardiomyopathy are present in genes wherein truncating variants (TVs) are the most common variant class. In particular, MYBPC3TV are the most common cause of hypertrophic cardiomyopathy (HCM), and DSPTV are the most common cause of arrhythmogenic left ventricular cardiomyopathy (ALVC). In prior work and preliminary data, we have clearly shown loss of function to be the primary mechanism for both MYBPC3TV and DSPTV – thus, MYBPC3TV and DSPTV can be considered prototypical causes of haploinsufficiency-associated cardiomyopathy. However, despite knowledge of the genetic mechanisms for these conditions, precision medicine approaches are hindered by the fact that marked variability in clinical severity occurs among patients of all genetic subtypes, likely in large part due to genetic modifiers. Given the strong evidence of haploinsufficiency, noncoding variants that influence the effective “dose” of the causal genes are highly likely to contribute to expressivity of cardiomyopathy for genes such as MYBPC3 and DSP. We show that single nucleotide variants (SNVs) in cis regulatory elements (CREs) of these genes are common in the population, and are associated with allelic imbalance. In addition to these potential cis modifiers, recent genome wide association studies (GWASs) have discovered putative modifiers in protein quality control pathways that could affect gene dose downstream of transcription, but the causative SNVs and their potential additive effects have not been determined. This proposal will test the central hypothesis that variation in CREs strongly impacts the severity of haploinsufficiency-associated cardiomyopathy by altering the effective gene dose. In Aim 1, we will quantify gene-expression effects of all possible SNVs in the major CREs of MYBPC3 and DSP. Using human induced pluripotent stem cell cardiomyocytes (iPSC-CMs), we will perform massively parallel reporter assays (MPRAs) with synthetic saturation mutagenesis of these CREs. We will also determine causal SNVs in linkage disequilibrium with variants found on prior GWAS’s through MPRAs. We will test for additive effects with whole genome sequencing (WGS) and phenotypic data for patients with MYBPC3TV. In Aim 2, we will test whether noncoding variants that disrupt highly conserved transcription factor binding sites within the major CREs of each of MYBPC3 and DSP further reduce mRNA expression and exacerbate disease when superimposed on MYBPC3TV and DSPTV iPSC-CM and mouse models. Taken together, this proposal will generate noncoding regulatory maps covering >30,000 SNVs in the sequence space most likely to influence gene expression of two major prototypes of haploinsufficiency-associated cardiomyopathy, while also establishing concepts and analytical pipelines for broadly advancing precision medicine of cardiomyopathy.