Genetic and Pharmacologic Inactivation of ANGPTL3 and Cardiovascular Disease

Abstract

Background: Loss-of-function variants in the angiopoietin-like 3 gene (ANGPTL3) have been associated with decreased plasma levels of triglycerides, low-density lipoprotein (LDL) cholesterol, and high-density lipoprotein (HDL) cholesterol. It is not known whether such variants or therapeutic antagonism of ANGPTL3 are associated with a reduced risk of atherosclerotic cardiovascular disease.

Methods: We sequenced the exons of ANGPTL3 in 58,335 participants in the DiscovEHR human genetics study. We performed tests of association for loss-of-function variants in ANGPTL3 with lipid levels and with coronary artery disease in 13,102 case patients and 40,430 controls from the DiscovEHR study, with follow-up studies involving 23,317 case patients and 107,166 controls from four population studies. We also tested the effects of a human monoclonal antibody, evinacumab, against Angptl3 in dyslipidemic mice and against ANGPTL3 in healthy human volunteers with elevated levels of triglycerides or LDL cholesterol.

Results: In the DiscovEHR study, participants with heterozygous loss-of-function variants in ANGPTL3 had significantly lower serum levels of triglycerides, HDL cholesterol, and LDL cholesterol than participants without these variants. Loss-of-function variants were found in 0.33% of case patients with coronary artery disease and in 0.45% of controls (adjusted odds ratio, 0.59; 95% confidence interval, 0.41 to 0.85; P=0.004). These results were confirmed in the follow-up studies. In dyslipidemic mice, inhibition of Angptl3 with evinacumab resulted in a greater decrease in atherosclerotic lesion area and necrotic content than a control antibody. In humans, evinacumab caused a dose-dependent placebo-adjusted reduction in fasting triglyceride levels of up to 76% and LDL cholesterol levels of up to 23%.

Conclusions: Genetic and therapeutic antagonism of ANGPTL3 in humans and of Angptl3 in mice was associated with decreased levels of all three major lipid fractions and decreased odds of atherosclerotic cardiovascular disease. (Funded by Regeneron Pharmaceuticals and others; ClinicalTrials.gov number, NCT01749878 .).

Figure 1. Loss-of-Function Variants in ANGPTL3 and Lipid Levels in Humans

The top of the diagram shows the organization of the ANGPTL3 protein, which is 460 amino acids long. Functional sections of the protein are marked in varying colors and demarcated with lines labeled to show the numbering of the amino acid sequence for each section. Beneath the protein diagram, the individual protein-sequence variants are listed in order of their position in the protein. Beneath the protein-sequence variants are plots of ANGPTL3 concentration and lipid levels. The red lines indicate the median value for each level among all carriers of loss-of-function variants. Each point represents a trait value for a single carrier of the loss-of-function variant specified above each box. The blue line indicates the median value for each trait for all sequenced persons not carrying a loss-of-function variant in ANGPTL3. To convert the values for cholesterol to millimoles per liter, multiply by 0.02586. To convert the values for triglycerides to millimoles per liter, multiply by 0.01129. HDL denotes high-density lipoprotein, LDL low-density lipoprotein, and SP signal peptide.

Figure 2. Association of ANGPTL3 Loss-of-Function Variants and Coronary Artery Disease

The association between the ANGPTL3 loss-of-function variants and coronary artery disease (CAD) was tested in each study with the use of logistic regression or Firth’s penalized likelihood logistic regression. Combined effects were determined by inverse-variance– weighted fixed-effects meta-analysis. For each study, the square indicates the odds ratio and the line indicates the 95% confidence interval. The square size is proportional to the precision of the estimate. For the combined estimate, the center of the diamond indicates the odds ratio for the meta-analysis, with the left and right corners of the diamond indicating the boundaries of the 95% confidence interval. ANGPTL3 loss-of-function variants were ascertained by means of targeted genotyping in the Copenhagen General Population Studies (CGPS) and by means of exome sequencing in the other studies. Duke denotes the Duke Catheterization Genetics cohort, GHS Geisinger Health System (DiscovEHR study), Penn the University of Pennsylvania Medicine BioBank, and TAICHI the Taiwan Metabochip consortium.

Figure 3. (facing page). Effect of Inhibition of Angptl3 with a Monoclonal Antibody in APOE*3Leiden.CETP Mice

Evinacumab reduced plasma levels of total cholesterol (Panel A) and triglycerides (Panel B) in APOE*3Leiden.CETP mice throughout the 13 weeks of the study. Plasma lipoproteins were separated on fast protein liquid chromatography at the end of the study and assessed for total cholesterol (Panel C) and triglycerides (Panel D). Representative images of atherosclerotic lesions (hematoxylin–phloxine–saffron stain) in a cross section of the aortic root area that was used for area quantification are shown (Panel E); the total atherosclerotic lesion area per cross section was evaluated at the end of the study (Panel F). Macrophage and necrotic content (indicators of lesion inflammation) (Panel G) and smooth muscle cells (SMC) and collagen content (indicators of lesion stability) (Panel H) were determined in the severe (type IV and V) lesions and normalized to lesion size. All values in Panels A, B, and F through H are means ±SE. For Panels A through D, 14 mice were given the control antibody and 22 were given evinacumab. The analysis conducted in Panels E through H involved 14 mice given the control antibody and 15 given evinacumab. VLDL denotes very-low-density lipoprotein.

Figure 4. Effects of Inhibition of ANGPTL3 with a Monoclonal Antibody on Triglyceride Levels in Human Volunteers

The median percent change from baseline in the triglyceride level (measured in milligrams per deciliter) is shown for the placebo group and for the groups receiving evinacumab at various doses. Individual data points are median values, with I bars showing interquartile ranges.