TMAO and its precursors in relation to host genetics, gut microbial composition, diet, and clinical outcomes: Meta-analysis of 5 prospective population-based cohorts

Trimethylamine N-oxide (TMAO) is a circulating microbiome-derived metabolite implicated in the development of atherosclerosis and cardiovascular disease (CVD). We investigated whether plasma levels of TMAO, its precursors (betaine, carnitine, deoxycarnitine, choline) and TMAO-to-precursor ratios associate with clinical outcomes, including CVD and mortality. This was followed by an in-depth analysis of their genetic, gut microbial and dietary determinants. The analyses were conducted in five Dutch prospective cohort studies including 7,834 individuals. To further investigate association results, Mendelian Randomization (MR) was also explored. We found only plasma choline levels (hazard ratio (HR) 1.17, (95% CI 1.07; 1.28)) and not TMAO to be associated with CVD risk. Our association analyses uncovered 10 genome-wide significant loci, including novel genomic regions for betaine (6p21.1, 6q25.3), choline (2q34, 5q31.1) and deoxycarnitine (10q21.2, 11p14.2) comprising several metabolic gene associations, e.g., CPS1 or PEMT . Furthermore, our analyses uncovered 68 gut microbiota associations, mainly related to TMAO-to-precursors ratios and the Oscillospiraceae family and 16 associations of food groups and metabolites including fish-TMAO, meat-carnitine and plant-based food-betaine associations. No significant association was identified by MR approach. Our analyses provide novel insights into the TMAO pathway, its determinants and pathophysiological impact in the general population. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This project is supported by the Netherlands Heart Foundation (IN-CONTROL CVON grant 2012-03 to T.H., M.G.N., L.A.B.J., F.K., A.Z., P.E.S., C.M.D. and J.F. and IN-CONTROL CVON grant 2018-27 to M.G.N., N.P.R., L.A.B.J., F.K., A.Z., and J.F.). The research is supported by Medical Delta, scientific program METABODELTA: Metabolomics for clinical advances in the Medical Delta. This research was part of the Netherlands X-omics Initiative and partially funded by NWO, project 184.034.019. ### 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 Leiden Longevity study protocol was approved by the ethical committee of the Leiden University Medical Center before the start of the study (P01.113). The LifeLines-DEEP study is approved by the Ethical Committee of the University Medical Center Groningen. The Rotterdam Study has been approved by the Medical Ethics Committee of the Erasmus MC (registration number MEC 02.1015) and by the Dutch Ministry of Health, Welfare and Sport (Population Screening Act WBO, license number 1071272-159521-PG). The Ethical Committee of Radboud University Nijmegen approved the 300-Obesity study. 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 and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes The data is available from the author on request.