Investigating the Role of a Putative Heart Enhancer Upstream of NFATC1 in Congenital Heart Disease

ABSTRACT

Congenital heart disease (CHD) is a group of common birth defects that despite advances in care, still carry significant morbidity and mortality. Many genes have been implicated in CHD, but for most individuals a genetic cause remains unknown. However, the studies of these genes have largely focused on coding regions, leaving the contribution of non-coding variants to the disease poorly understood. Non-coding regions contain distal cis-regulatory elements (CRE) that control the temporal and spatial expression of genes during embryonic development. Recently, thousands of CRE’s suspected to alter expression in many genes important for heart development have been catalogued, referred to as putative heart enhancers (HE). Our laboratory performs genome sequencing on CHD patients and has identified a single-nucleotide variant (SNV) in a putative HE. This HE is located upstream of NFATC1, a gene identified to be critical for proper valve formation in animal models. To investigate the effect of this SNV and its encompassing region on NFATC1 expression, we designed reporter plasmids containing the SNV and sequential deletions of the region upstream of the NFATC1 promoter and transcription start site. The plasmids were transfected into immortalized cell lines (EA.hy926, HL-1, H9c2) and assayed using an in vitro dual-luciferase reporter system. This revealed that the SNV, along with several encompassing regions, play an important role in controlling NFATC1 expression. Furthermore, using in silico tools, we show that the SNV introduces several transcription factor (TF) motifs, potentially explaining the observed changes in gene expression. This work reveals that the identified SNV and several non-coding regions upstream of NFATC1 may contribute to CHD. Identifying the causes of CHD remains important in the care of affected individuals. This project demonstrates that non-coding variants in genes involved in heart development may underlie the causes of some CHDs.