Unraveling interindividual variation of trimethylamine N-oxide and its precursors at the population level

Abstract

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 are associated 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 7834 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, for example, CPS1 or PEMT. Furthermore, our analyses uncovered 68 gut microbiota associations, mainly related to TMAO-to-precursors ratios and the Ruminococcaceae 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 the MR approach. Our analyses provide novel insights into the TMAO pathway, its determinants, and pathophysiological impact on the general population.

Keywords: TMAO; diet; genetics; gut microbiome; meta‐analysis; population cohort.

Figure 1

Overview of study design and performed analyses. The study population included participants from Rotterdam Study I‐4 (RSI‐4), Rotterdam Study III‐2 (RSIII‐2), Leiden Longevity Study (LLS), LifeLines‐DEEP (LLD), and 300‐Obese cohort (300‐OB).

Figure 2

Variance explained in gut microbiome‐related metabolite levels and ratios by different data layers. The X‐axis shows the coefficient of determinations R ² gained with each additional data layer (gray ‐ anthropometrics; red ‐ genetics; blue ‐ diet; purple ‐ microbiome). Two models were trained, using LLD (A and C) or Rotterdam Study III‐2 (named RS) (B and D) as the train set, and the left‐out cohort as the test set respectively.

Figure 3

Results of genome‐wide association analyses for (A) individual metabolites and (B) TMAO‐to‐precursor ratios. Each dot represents a genetic variant. Genetic variants surpassing the Bonferroni corrected significance threshold (p value < 8.33 × 10⁻⁹) are highlighted in red. Genetic variants showing suggestive evidence of association (p value < 1.7 × 10⁻⁷) are highlighted in blue.

Figure 4

Microbial associations with plasma metabolic concentrations (A) Heatmap showing results of the association analysis between metabolites and gut microbial taxa. Displayed results are after adjustment for age, sex, BMI, and study‐specific covariates. Metabolites are displayed on the x‐axis and gut microbial taxa are shown on the y‐axis. Red color denotes positive associations and blue color stands for negative associations. Hash symbol (#) represents the Bonferroni significant associations (p value < 6.2 × 10^–5), while star denotes suggestive associations (p value < 1.2 × 10^–3). (B) Boxplots displaying the distribution of normalized TMAO concentration per clr‐transformed abundance quartiles from cutC, measured from metagenomic shotgun sequencing in LLD.