Genetic and biochemical studies implicate hepatic flavin monooxygenases (FMOs) in TMAO production and atherosclerosis susceptability
Hepatic FMO3 is a known enzymatic source for TMAO in humans, based on the recent recognition of the etiology of an uncommon genetic disorder called trimethylaminuria (also known as fish malodor syndrome)15,17. Subjects with this metabolic condition have impaired capacity to convert TMA, which smells like rotting fish, into TMAO, an odorless stable oxidation product17. We therefore sought to identify possible sources of genetic regulation and role of FMO3 in atherosclerosis using integrative genetics in mice18. Expression levels of FMO3 were determined by microarray analysis in the livers of mice from an F2 intercross between atherosclerosis-prone C57BL/6J.Apoe−/− mice and atherosclerosis-resistant C3H/HeJ.Apoe−/− mice and compared with quantitative measures of atherosclerosis. The expression level of FMO3 showed marked differences between genders (females > 1,000 fold higher than in males). Significant positive correlations were readily found between hepatic FMO3 expression and atherosclerotic lesions (Fig. 4a; Supplementary Fig. 12 top row; Supplementary Fig. 13). Interestingly, a highly significant negative correlation with plasma high density lipoprotein (HDL) cholesterol levels was noted in both male and female mice (Fig. 4b; Supplementary Fig. 12, middle row). Further, plasma levels of the PC metabolite, TMAO, showed a significant positive correlation with hepatic FMO3 expression level in mice (Fig. 4c; Supplementary Fig. 12 bottom row).
FMO3 is one member of a family of FMO enzymes, the majority of which are physically located as a cluster of genes on chromosome 1 in both humans and mice. The various FMOs share sequence homology and overlapping substrate specificities. Further, while rare mutations in or near the FMO3 gene have been identified in individuals with trimethylaminuria19, the impact of these mutations on other FMO genes remains unknown. Examination of the hepatic expression levels of the various FMOs revealed that many are highly correlated with each other in both mice and humans (Supplementary Table 7). Examination of hepatic expression levels of additional FMOs in mice from the atherosclerosis F2 intercross revealed multiple FMOs are significantly correlated with aortic lesion formation, HDL cholesterol concentrations, and plasma TMAO levels (Supplementary Figs. 14–16), suggesting that multiple members of the FMO family of enzymes may participate in atherosclerosis and the PC → TMAO metabolic pathway. To explore the relationship between hepatic FMOs and plasma TMAO levels in humans, paired samples of liver and plasma from subjects undergoing elective liver biopsy were examined. Amongst all of the human FMO monitored, only a trend toward positive association was noted between hepatic expression of FMO3 and plasma TMAO levels (Fig. 4d; Supplementary Fig. 17).
Next, we focused on the genetic regulation of hepatic FMO3 expression (and other FMOs) using eQTL (expression quantitative trait locus) analyses in the F2 mouse cross. The eQTL plot for FMO3 mRNA levels is shown in Supplementary Fig. 18, and demonstrates a strongly suggestive cis locus (LOD=5.9) on mouse chromosome 1 at 151 Mb. FMO3 (and several other FMOs) is located at 164.8 Mb in a region identified as non-identical by descent between C3H/HeJ and C57BL/6 (http://mouse.perlegen.com). This region is just distal to the 95% confidence interval of a previously reported murine atherosclerosis susceptibility locus20. Examining the effect of the closest SNP to FMO3 (rs3689151) as a function of alleles inherited from either parental strain indicated a strong effect on atherosclerosis in both genders of the F2 mice (Kruskal-Wallis test, p<1.×10−6). Bonferroni corrected pair-wise comparisons indicated a dose-dependent significant increase in atherosclerosis in F2 mice heterozygous or homozygous for the C57BL/6J allele (Fig. 4e). Although the resolution on average for an F2 intercross of this size is in excess of 20 Mb and thus does not provide `gene level' resolution, these data show that the locus encompassing the FMO gene cluster on chromosome 1 is associated with atherosclerotic lesion size. Collectively, these results indicate that: (i) hepatic expression levels of multiple FMOs are linked to plasma TMAO levels in mice; (ii) hepatic expression levels of multiple FMOs are associated with both the extent of aortic atherosclerosis and HDL cholesterol levels in mice; (iii) hepatic expression levels of FMO3 suggest an association with plasma TMAO levels in humans; and (iv) a genetic locus containing the FMO gene cluster on chromosome 1 in mice has a strong effect on atherosclerosis.