9p21 DNA variants associated with coronary artery disease impair interferon-γ signalling response

Abstract

Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) in the 9p21 gene desert associated with coronary artery disease (CAD) and type 2 diabetes. Despite evidence for a role of the associated interval in neighbouring gene regulation, the biological underpinnings of these genetic associations with CAD or type 2 diabetes have not yet been explained. Here we identify 33 enhancers in 9p21; the interval is the second densest gene desert for predicted enhancers and six times denser than the whole genome (P < 6.55 × 10(-33)). The CAD risk alleles of SNPs rs10811656 and rs10757278 are located in one of these enhancers and disrupt a binding site for STAT1. Lymphoblastoid cell lines homozygous for the CAD risk haplotype show no binding of STAT1, and in lymphoblastoid cell lines homozygous for the CAD non-risk haplotype, binding of STAT1 inhibits CDKN2BAS (also known as CDKN2B-AS1) expression, which is reversed by short interfering RNA knockdown of STAT1. Using a new, open-ended approach to detect long-distance interactions, we find that in human vascular endothelial cells the enhancer interval containing the CAD locus physically interacts with the CDKN2A/B locus, the MTAP gene and an interval downstream of IFNA21. In human vascular endothelial cells, interferon-γ activation strongly affects the structure of the chromatin and the transcriptional regulation in the 9p21 locus, including STAT1-binding, long-range enhancer interactions and altered expression of neighbouring genes. Our findings establish a link between CAD genetic susceptibility and the response to inflammatory signalling in a vascular cell type and thus demonstrate the utility of genome-wide association study findings in directing studies to novel genomic loci and biological processes important for disease aetiology.

Figure 1. Functional annotation of the 9p21 interval

The locations of the core CAD and T2D associated intervals (track A) and the predicted insulators (brown), enhancers (orange) and promoters (green) in HeLa cells are indicated (track B). The enhancers are distributed (track C) between the CAD interval (red), the T2D interval (blue) or located outside (orange). The location of the binding sites for FoxA1 in MCF7 cells (track D2) and STAT1 in IFNγ treated and non-treated HeLa (track D3) as well as the distribution of 9p21 chromatin marks in the ENCODE data (tracks E1 and E2 – Supplemental Methods) are indicated.

Figure 2. LD analysis of the 9p21 interval

(a) The sequenced interval shows three LD blocks. The 53-kb CAD interval is located at the 3′ end of the first block (A), the second block (B) spans 11-kb corresponding to the T2D interval and the third block (C) spans 63-kb. LD map (D′) based on variants identified in the 50 sequenced samples. (b) Number of variants in LD at various r² thresholds (y axis) with any CAD (red) or T2D associated variants (blue). The distance spanned by the SNPs in LD is indicated (x axis).

Figure 3. In vivo effects of the ECAD9 variants

(a) Enrichment of the ECAD9 STAT1 binding site by anti-STAT1 ChIP in HUVEC cells untreated or treated with IFNγ. (b) Changes in level of expression of CDKN2B and CDKN2BAS genes upon treatment with IFNγ in HeLa and HUVEC. (c) Enrichment of the ECAD9 STAT1 binding site by anti-STAT1 ChIP in LCL homozygous for the CAD non-risk or CAD risk haplotypes. (d) Expression level changes of CDKN2BAS in LCL homozygous for non-risk or risk CAD haplotype after STAT1 knock-down by siRNA. (*) and (**) indicate two-tailed Student T-test p <0.05 and p<0.01, respectively.

Figure 4. Long-Range interaction with the enhancer locus

(a) Circular representation of the 3D-DSL results. The scale shows chromosome 9 position (hg18) in 100kb increments. RefSeq genes (dark blue) and HeLa predicted enhancers (orange) are displayed with the CAD (red) and T2D (blue) associated enhancers. The histogram represents the normalized average number of reads mapping to each BamH1 donor site. The inner circle links connect BamH1 acceptor sites for the 9 most strongly interacting donor sites. (b) PCR validation of the long-range interaction between ECAD9 and CDKN2A/B (upper panel) or MTAP (lower panel). Arrow indicates the specific product.