Objective: Genome-wide association studies (GWAS) identified 56 loci that are robustly associated with the risk of coronary artery disease (CAD). Only a handful of genes in these loci are currently known to influence CAD. We previously showed that: 1) CAD-associated loci exert their effect at least in part by influencing atherosclerotic plaque formation; 2) zebrafish larvae represent a robust model system for high-throughput, image-based screens of early-stage atherosclerosis. We now aim to identify causal genes in GWAS-identified loci for CAD using zebrafish model systems.
Methods: Heterozygous carriers of nonsense (sa1516 in cxcl12b; fh336 in col4a1) or essential splice site (sa1504 in flt1) mutations were in-crossed, and offspring were fed on a normal or high amount of a normal or high cholesterol diet from 5 to 10 days post-fertilisation. Vascular lipid deposition (monodansylpentane cadaverase) and co-localisation of lipids with macrophages (Tg:mpeg1-mCherry) and/or neutrophils (Tg:mpo-EGFP) were subsequently visualised using an automated positioning system, fluorescence microscope and CCD camera, and objectively quantified using a custom-written image analysis pipeline in ilastik, CellProfiler and ImageJ. After imaging, whole-body total cholesterol and triglyceride levels were assessed using enzymatic assays, and larvae were genotyped using KASP technology. A multilevel (hierarchical) mixed model analysis was performed to examine the effect of the mutations (additive model) on atherogenic traits, while adjusting for diet and time-of-imaging, as well as for batch (random factor).
Results: In data from up to 253 larvae, sa1504 in flt1 was associated with less vascular lipid deposition (P=2E-5), less co-localisation of lipids and macrophages (P=9E-3), and lower whole body total cholesterol levels (P=2E-2). No significant effects were identified for the sa1516 and fh336 mutations (N up to 223 and 344, respectively).
Discussion: Our results represent the first mutant model demonstrating that flt1 plays a role in early-stage atherosclerosis, implying that FLT1 is the culprit for CAD in the 13q12 locus. Based on the results of a priori performed bioinformatics analyses, we believe that CXCL12 and COL4A1 remain the most promising candidates in their respective loci. The lack of effect for these genes in our screen may reflect genetic redundancy, adaptation and/or a late onset effect.