Large-scale validation of zebrafish larvae as a model system for genetic screens in dyslipidaemia, atherosclerosis and coronary artery disease

Bandaru M, Emmanouilidou A, Ranefall P, von der Heyde B, Klingström T, Ledin J, Larsson A, Wählby C, Ingelsson E, den Hoed M

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.

BioImage Informatics [Collaborative]

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