Genetic inhibition of angiopoietin-like protein-3, lipoprotein-lipid levels and cardiometabolic diseases

Background: RNA-based, antibody-based and gene editing-based therapies are currently under investigation to determine if the inhibition of angiopoietin-like protein-3 (ANGPTL3) could reduce lipoprotein-lipid levels and atherosclerotic cardiovascular diseases (ASCVD) risk. We used Mendelian randomization (MR) to determine whether genetic variations influencing ANGPTL3 liver gene expression, blood levels and protein structure could causally influence triglyceride and apolipoprotein B (apoB) levels as well as coronary artery disease (CAD), ischemic stroke (IS) and other cardiometabolic diseases. Methods: We performed RNA-sequencing of 246 explanted liver samples to identify single-nucleotide polymorphisms (SNPs) associated with liver expression of ANGPTL3 . We used genome-wide summary statistics of plasmatic protein levels of ANGPTL3 from the deCODE study (n=35,359). We also identified 647 carriers of ANGPTL3 protein-truncating variants (PTVs) associated with lower plasma triglyceride levels in the UK Biobank. We performed two-sample MR using SNPs that influence ANGPTL3 liver expression or ANGPTPL3 blood protein levels as exposure and cardiometabolic diseases as outcomes (CAD, IS, heart failure, non-alcoholic fatty liver disease, acute pancreatitis, and type 2 diabetes). The impact of rare variants influencing plasma triglyceride levels on apoB levels and CAD was also investigated. Results: In two-sample MR studies, common genetic variants influencing ANGPTL3 hepatic or blood expression levels of ANGPTL3 had a very strong effect on plasma triglyceride levels, a more modest effect on LDL cholesterol, a weaker effect on apoB levels and no effect on CAD or other cardiometabolic diseases. In the UK Biobank, carriers of rare ANGPTL3 PTVs providing lifelong reductions in median plasma triglyceride levels (-0.37 [interquartile range=0.41] mmol/L) had slightly lower apoB levels (-0.06 [interquartile range=0.32] g/L) and similar CAD event rate compared to noncarriers (10.2% versus 10.9% in carriers versus noncarriers, p=0.60). Conclusions: PTVs influencing ANGPTL3 DNA sequence as well as common genetic variants influencing ANGPTL3 hepatic expression and/or blood protein levels exhibit a strong effect on circulating plasma triglyceride levels, a weak effect on circulating apoB levels and no effect on ASCVD. Near-complete inhibition of ANGPTL3 function will likely be required to decrease apoB levels and reduce ASCVD risk. ### Competing Interest Statement BJA is a consultant for Novartis, Editas Medicine, and Silence Therapeutics and has received research contracts from Pfizer, Ionis Pharmaceuticals, and Silence Therapeutics. AT receives research funding from Johnson & Johnson, Medtronic, and G.I. windows for studies related to bariatric surgery as well as consulting fees from Bausch Health and Novo Nordisk. The remaining authors disclose no conflicts. ### Funding Statement This study was funded by Pfizer with additional support from the Canadian Institutes of Health Research (CIHR) and the Foundation of the IUCPQ. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish results. The Genotype-Tissue Expression (GTEx) Project was supported by the Common Fund of the Office of the Director of the National Institutes of Health, and by National Cancer Institute, National Human Genome Research Institute, National Heart, Lung and Blood Institute, National Institute on Drug Abuse, National Institute of Mental Health, and National Institute of Neurological Disorders and Stroke. ÉG holds a Doctoral Research Award from the CIHR. JB holds a Masters′ Research Award from the Fonds de recherche du Québec: Santé (FRQS). EG holds a Doctoral Research Award from the FRQS. BJA holds a Senior Scholar Award from the FRQS. YB holds a Canada Research Chair in Genomics of Heart and Lung Diseases. PM holds a FRQS Research Chair on the Pathobiology of Calcific Aortic Valve Disease. MCV is Canada Research Chair in Genomics applied to Nutrition and Metabolic Health. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Ethics committee/IRB of Institut Universitaire de Cardiologie et de Pneumologie de Québec - Université Laval gave ethical approval for this work (Number of approval: 2021-3656, 22070). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes R version 4.0.4 and RStudio Server 1.3.1093 was used to analyze data and create plots50. TwoSampleMR 0.5.6 (https:// github.com/MRCIEU/TwoSampleMR)51, the MendelianRandomization 0.5.1 (https://cran.r-project.org/web/packages/ MendelianRandomization), the ieugwasr (https://mrcieu.github.io/ieugwasr/), the data.table 1.14.0 (https://github. com/Rdatatable/data.table), the coloc (https://cran.r-project.org/ web/packages/coloc/) and the HyPrColoc (https://github.com/ jrs95/hyprcoloc) packages in R. The alignment pipeline is available at https://github.com/broadinstitute/gtex-pipeline/tree/master/rnaseq. Code for analysis is available at the associated website of each software package. All genome-wide association study summary statistics used are publicly available. Summary statistics for ANGPTL3 levels from deCODE genetics were downloaded from https://www.decode.com/summarydata/. The full summary statistics for the genome-proteome-wide association study for ANGPTL3 from the Fenland cohort were downloaded from: https://omicscience. org/apps/pgwas/.