Identification and characterization of causal genes for LDL cholesterol levels and downstream effects on atherosclerosis

Coronary artery disease is the leading cause of death worldwide and results from progression of atherosclerosis, which is triggered in part by elevated plasma concentrations of LDL cholesterol. Genome-wide association studies have identified many loci that are associated with circulating lipid levels and bioinformatics tools have been implemented to prioritize positional candidate genes. This project aims to better understand the genetics underlying the regulation of plasma LDL levels and their effect of atherosclerosis using a zebrafish (Danio rerio) model system. A multiplex line with the genes abcg5, abcg8, myrf, col4a3bpa, col4a3bpb, st3gal3, ywhaqa and ywhaqb targeted by CRISPR/Cas9 technique was established using zebrafish with fluorescently labeled macrophages (Tg[mpeg1:mCherry]) and neutrophils (Tg[mpo:EGFP]). Monodansylpentane cadaverase was used to visualize lipids droplets, together with macrophages and neutrophils, in 384 overfed larvae, allowing the visualization and quantification of vascular atherogenic traits at 10 days post-fertilization. Euthanized larvae were homogenized for the quantification of triglycerides, total cholesterol, LDL, HDL, glucose and protein levels. DNA was extracted and larvae were paired-end sequenced for the CRISPR-targeted sites. Linear regression analysis to compare the wild-type larvae against homozygous mutants and additive models for orthologous genes were performed. The lower accumulation of lipids and the lower co-localization of macrophages and neutrophils in the vasculature suggested that the larvae with mutations in the gene abcg5, abcg8, col4a3bpb, and ywhaqb resulted in larvae more protected against atherosclerotic phenotype. The study suggested that loss of function of the targeted genes was associated with atherogenic traits, helping to understand the pathophysiology of atherosclerosis