Genetic analysis in UK Biobank links insulin resistance and transendothelial migration pathways to coronary artery disease

Abstract

UK Biobank is among the world's largest repositories for phenotypic and genotypic information in individuals of European ancestry. We performed a genome-wide association study in UK Biobank testing ∼9 million DNA sequence variants for association with coronary artery disease (4,831 cases and 115,455 controls) and carried out meta-analysis with previously published results. We identified 15 new loci, bringing the total number of loci associated with coronary artery disease to 95 at the time of analysis. Phenome-wide association scanning showed that CCDC92 likely affects coronary artery disease through insulin resistance pathways, whereas experimental analysis suggests that ARHGEF26 influences the transendothelial migration of leukocytes.

Figure 1. Study Design

Stage 1 consisted of a genome-wide association study for the coronary artery disease phenotype performed in UK Biobank; variants below a threshold P value < 0.05 moving forward to meta-analysis with CARDIoGRAM Exome (Stage 2) or CARDIoGRAMplusC4D summary statistics (Stage 3). Abbreviations: 1000G, 1000 Genomes; CARDIoGRAMplusC4D, Coronary ARtery DIsease Genome-wide Replication and Meta-analysis

Figure 2. Phenome-wide association results for 15 novel loci

For the 15 novel CAD risk variants identified in our study, Z-scores (aligned to the CAD risk allele) were obtained from the Genomics plc Platform and UK Biobank. A positive Z-score (red) indicates a positive association between the CAD risk allele and the disease/trait, while a negative Z-score (blue) indicates an inverse association. Boxes are outlined in green if the variant is significantly (P < 0.00013) associated with the given trait. Abbreviations: Adj, Adjusted; BMI, Body Mass Index; BP, Blood Pressure; crea, Creatinine; cys, cystatin-c; COPD, chronic obstructive pulmonary disease; eGFR, estimated Glomerular Filtration Rate; HDL, High Density Lipoprotein; LDL, Low Density Lipoprotein;

Figure 3. Biological pathways underlying genetic loci associated with coronary artery disease

CAD GWAS loci identified to date are depicted along with the plausible relationship to the underling biological pathway. The 15 new loci described in this paper are shown in bold. Loci names are based on the nearest genes; however, the causal gene(s) remains unclear for most associated loci and as such, the resultant annotation may prove incorrect in some cases. Adapted from Ref. .

Figure 4. Functional assessment of ARHGEF26 p.Val29Leu in vitro

a) ARHGEF26-29Leu increases leukocyte transendothelial migration. HAEC were transfected with non-targeting siRNA and empty vector (control), siRNA against ARHGEF26 3′-UTR and empty vector, siRNA and ARHGEF26-WT, or siRNA and ARHGEF26-29Leu. Transfected HAEC were plated on transwell inserts and treated with 10 ng/mL TNF-α. Differentiated HL60 cells were loaded on the upper chambers of transwells and allowed to transmigrate across HAEC towards vehicle (blue) or 50 ng/mL SDF-1 (red). The migrated cells were quantified as percentage of input cells per well (n=5 or 6; mean±s.d.; F=11.89, DF=3 by two-way ANOVA within vehicle and SDF-1 subgroups with Fisher’s LSD test; variance among vehicle subgroups non-significant; NS, not significant; representative of 3 independent experiments). b) ARHGEF26-29Leu increases leukocyte adhesion on endothelial cells. HAEC were transfected as 2a) and cultured on 96-well plates until confluent and treated with 10 ng/mL TNF-α. Calcein-AM-labeled THP-1 cells were incubated with HAEC and washed to remove non-adherent cells. The adherent cells were lysed, quantified by Calcein-AM fluorescence and compared to siRNA+WT (n=25, 17, 20, and 17; mean±s.d.; F=14.53, DF=3 by one-way ANOVA; NS, not significant; * P<0.0001 compared to siRNA+WT; representative of 3 independent experiments). c) ARHGEF26-29Leu increases vascular smooth muscle cell proliferation. HCASMC were transfected as 2a) and made quiescent by serum starvation for 48 h, followed by 72-h proliferation in normal serum medium. Cell proliferation was quantified by a luminescent assay and compared to siRNA+WT (n=20; mean±s.d.; F=197.5, DF=3 by one-way ANOVA; NS, not significant; * P<0.0001 compared to siRNA+WT; representative of 3 independent experiments).