Abstract 405: Functional Characterization of Coronary Artery Disease (CAD) Associated SNPs at the ADAMTS7 Locus
SNPs in the genomic region containing the gene ADAMTS7, a gene that codes for a metalloproteinase involved in vascular smooth muscle cell migration, are significantly associated with CAD in humans. Recently, our lab demonstrated that knock out of Adamts7 in mice is protective against atherosclerosis, suggesting that ADAMTS7 is involved in the pathogenesis of cardiovascular disease. Several of the top CAD-associated SNPs (p<5e-8) identified by CARDIoGRAMplusC4D consortium lie in putative enhancer regions of ADAMTS7.. We investigated the functionality of CAD associated SNPs that overlap 5 active regulatory elements as predicted by vascular smooth muscle cell (VSMC) ChIP-seq signals, ENCODE histone methylation peaks, and aortic smooth muscle cell DNase hypersensitivity peaks. Several regions containing association SNPs showed enhancer activity in minimal promoter luciferase reporter assays. Region 2 showed an 8.2 (p<0.01) and a 32.4 (p<0.005) fold increase in luciferase activity in HEK293T and A7r5 rat aortic smooth muscle cell lines, respectively, when normalized to empty vector control, while Region 5 demonstrated a 4.9 (p<0.01) and 1.8 (p<0.01) normalized fold increase in 293T and A7r5 cells, respectively. Both of these regions contain binding motifs for TCF21, a transcription factor involved in the development of the human coronary vasculature that is also a GWAS locus for CAD, that is disrupted by genotype of the CAD-associated SNP. We performed electrophoretic mobility shift assays (EMSAs) using purified TCF21 protein and showed altered binding in Region 5 based on disease associated SNP variation. Additionally, EMSAs using human coronary artery smooth muscle cell (hCASMC) nuclear extract detected a band shift demonstrating protein binding at both predicted enhancer regions 2 and 5. However, we were not able to detect a significant difference in protein binding between the major and minor allele, suggesting transcription factors other than TCF21 may be regulating these regions. More work is being done using Capture-C technology to interrogate physical interactions between these regions and the promoter of ADAMTS7, but our current data suggest that top CAD-associated SNPs alter regulatory activity at the ADAMTS7 locus.
Author Disclosures: K. Ou: None. S. Nuernberg: None. R.C. Bauer: None. D.J. Rader: None. M.P. Reilly: None.
- © 2015 by American Heart Association, Inc.