ASHG 2017 Annual Meeting - The American Society of Human Genetics - (-) - [2017-10-17; online 2017-10-17]
Background: Genome-wide association studies have identified 77 loci that are robustly associated with coronary artery disease (CAD). In all but a few of these loci the causal genes and mechanisms remain unknown. Results from small-scale studies suggest that zebrafish larvae represent a promising model system for genetic screens in dyslipidemia, early-stage atherosclerosis and CAD. We aim to confirm or refute these results in a large-scale study, expand the phenotypic pipeline, and increase the throughput. Methods: At the core of our setup is an automated positioning and imaging system that allows visualization and quantification of atherogenic traits in ~100 zebrafish larvae per day at 10 days post-fertilization, by making use of fluorescent transgenes and dyes. We used a three-tiered approach to validate the zebrafish model system: 1) a dietary intervention to examine the effect of overfeeding and cholesterol supplementation (N=2193); 2) a treatment regime with atorvastatin and ezetimibe (N=956); and 3) a genetic screen for zebrafish orthologues of LDLR, PCSK9, APOB and APOE using a multiplex CRISPR-Cas9 approach (N=2x384). After imaging, whole-body lipid and glucose levels were assessed using enzymatic assays, and CRISPR-Cas9 target sites were sequenced on a MiSeq. Results: Overfeeding and cholesterol supplementation have independent pro-atherogenic effects, including elevated total cholesterol and triglyceride levels, more vascular deposition of lipids and oxidized LDLc, and more co-localization of lipids with macrophages and neutrophils. Treatment with atorvastatin and ezetimibe results in lower whole-body total cholesterol, LDLc and triglyceride levels, as well as in less vascular lipid deposition and less co-localization of lipids and macrophages. Finally, mutations in APOE orthologues result in higher whole-body LDLc levels and more co-localization of lipids with macrophages or neutrophils compared with wildtypes. Mutations in APOB orthologues tend to result in higher LDLc levels, more vascular lipid deposition, and more co-localizing lipids and neutrophils. Treatment with lipid lowering drugs and mutations in pcsk9 both result in higher whole-body glucose levels. Data from all larvae combined show that atherosclerosis in 10-day-old zebrafish larvae is mainly driven by higher triglyceride but not LDLc levels. Conclusion: Zebrafish larvae can be used to systematically identify and characterize causal genes for CAD.