Regulatory variants in TCF7L2 are associated with thoracic aortic aneurysm

Abstract

Thoracic aortic aneurysm (TAA) is characterized by dilation of the aortic root or ascending/descending aorta. TAA is a heritable disease that can be potentially life threatening. While 10%-20% of TAA cases are caused by rare, pathogenic variants in single genes, the origin of the majority of TAA cases remains unknown. A previous study implicated common variants in FBN1 with TAA disease risk. Here, we report a genome-wide scan of 1,351 TAA-affected individuals and 18,295 control individuals from the Cardiovascular Health Improvement Project and Michigan Genomics Initiative at the University of Michigan. We identified a genome-wide significant association with TAA for variants within the third intron of TCF7L2 following replication with meta-analysis of four additional independent cohorts. Common variants in this locus are the strongest known genetic risk factor for type 2 diabetes. Although evidence indicates the presence of different causal variants for TAA and type 2 diabetes at this locus, we observed an opposite direction of effect. The genetic association for TAA colocalizes with an aortic eQTL of TCF7L2, suggesting a functional relationship. These analyses predict an association of higher expression of TCF7L2 with TAA disease risk. In vitro, we show that upregulation of TCF7L2 is associated with BCL2 repression promoting vascular smooth muscle cell apoptosis, a key driver of TAA disease.

Keywords: GWAS; TCF7L2; VSMC; apoptosis; regulatory variant; thoracic aortic aneurysm.

Figure 1

TCF7L2 locus in TAA and T2D (A) LocusZoom plot of TCF7L2 locus in TAA GWAS from CHIP+MGI. (B) LocusZoom plot of TCF7L2 locus in T2D GWAS from DIAGRAM. LD colors are with respect to the TAA index variant rs4073288. (C) Conditional effect sizes (beta, in SD units) of TAA index variant rs4073288 in TAA GWAS. Eight independent T2D variants were used for conditioning. Error bars represent 95% confidence intervals. (D) Comparison of effect sizes (beta) in TAA and T2D GWAS. Variants in this locus have opposite direction in effect sizes in TAA versus T2D GWAS.

Figure 2

Functional characterization of TCF7L2 locus (A) LocusZoom plot of aortic eQTL of TCF7L2. LD colors are with respect to the TAA index variant rs4073288. (B) Comparison of p values in TAA GWAS and aortic eQTL from GTEx. These two associations colocalize with posterior probability 0.96 by COLOC. The solid shapes represent 23 variants that were in both GWAS and eQTL 95% credible sets. Two variants were prioritized by RegulomeDB (rs4074718 and rs4077527). (C) Regulatory landscape of TCF7L2 gene body in thoracic aorta/ascending aorta from ENCODE. The fine-mapped variant rs4077527 intersects with DNase I hypersensitive site (DHS) and H3K27ac in this tissue. Using promoter capture Hi-C in aorta, we highlighted only genomic bins that significantly interact (p < 0.01) with TCF7L2 promoter. Promoter capture Hi-C p values for 23 variants are given in Figure S4B.

Figure 3

TCF7L2 expression is directly associated with vascular smooth muscle cell apoptosis in vitro HASMCs were infected with adenovirus (30 MOI)-encoding LacZ (control) or myc-tagged TCF7L2, and after 48 h, (A) the abundance of the indicated proteins in the cell extracts was determined by immunoblot (representative of three independent experiments) and (B) the mRNA expression of the indicated genes, relative to ACTB, by quantitative real-time PCR. A LacZ control was set at 1 as reference (n = 3; representative of three independent experiments). Data are mean ± SEM. ^∗∗p ≤ 0.0004. HASMCs were transfected with siRNA control or siRNA against TCF7L2 via Lipofectamine RNAiMax, and after 72 h, (C) the abundance of the indicated proteins and (D) the mRNA expression of the indicated genes were determined as described above with an siRNA control set at 1 as reference. ^∗∗p ≤ 0.0002. HASMCs were infected with adenovirus as in (A). After 48 h in serum-free medium, they were treated with Fas ligand (FasL, 100 ng/mL) in serum-free medium and apoptosis was assessed by (E) annexin V via FACS analysis after treatment for 4 h (n = 3; representative of three independent experiments) or (F) the abundance of the indicated proteins in the cell extracts after 6 h as determined by immunoblot (representative of three independent experiments). HASMCs were transfected with the indicated siRNA as in (C). After 72 h in serum-free medium, they were treated with FasL (100 ng/mL) for (G) annexin V assay after 4 h as in (E) or (H) immunoblot after 6 h as in (F) and subjected to immunoblot as in (E). Densitometry for (A), (B), (E), (F), and mRNA expression of BAX are provided in Figure S5.