Trimethylamine-N-oxide, a New Risk Factor for Non-alcoholic Fatty Liver Disease Changes the Expression of miRNA-34a, and miRNA-122 in the Fatty Liver Cell Model.

Non-alcoholic fatty liver disease is a multifactorial disorder with complicated pathophysiology ranging from simple steatosis to steatohepatitis and liver fibrosis. Trimethylamine-N-oxide (TMAO) production is believed to be correlated with choline deficiency. This study investigated the expression of miRNA-34a, miRNA-122, and miRNA-192 in the fatty liver cell model treated with different concentrations of TMAO. A fatty liver cell model was developed by exposing HepG2 cells to a mixture of palmitate and oleate in a ratio of 1:2 at a final concentration of 1200 µM for 24 h. The confirmed fatty liver cells were treated with 37.5, 75, 150, and 300 µM of TMAO for 24 h. RT-qPCR was used to quantify the expression of microRNAs in a cellular model. The cellular expression of all microRNAs was significantly higher in treated fatty liver cells compared to normal HepG2 cells (P < 0.05). Only 75 and 150 µM of TMAO significantly increased the expression of miRNA-34a and miRNA-122 compared to both fatty and normal control cells (P < 0.05). Our results provided an experimental documentation for the potential effect of TMAO to change the expression of miR-34a and miR-22 as a mechanism for contributing to the pathogenesis of non-alcoholic fatty liver disease.

Novel Association between Flavin-Containing Monooxygenase 3 Gene Polymorphism and Antithyroid Drug-Induced Agranulocytosis in the Han Population.

Some effective antithyroid drugs (ATDs) have been widely used for patients with Graves' disease (GD) but are associated with ATD-induced agranulocytosis. We selected 29 ATD-induced agranulocytosis patients, 44 ATD-induced neutropenia patients, and 140 GD controls among the Chinese Han population who were recruited at the First Affiliated Hospital of Xi'an Jiao Tong University. We assessed their response to ATDs treatment by performing genotyping for a candidate gene association study of samples from patients receiving treatment. Human flavin-containing monooxygenase 3 (FMO3), which is the major hepatic enzyme involved in the production of N-oxide of trimethylamine, catalyzes the oxygenation of a variety of drug compounds. Six single SNP, genotype, haplotype (HAP), and association analyses of the FMO3 gene with ATD-induced agranulocytosis/neutropenia under different models (i.e., additive, dominant, and recessive models) were performed. Rs1736557, which caused an amino acid variation V257M, showed a strong association between ATD-induced agranulocytosis and GD controls after Bonferroni correction (p = 0.011, OR 2.301, 95% CI 1.201-4.409). The presence of HAP 3 (HAP3) in the FMO3 gene HAP was statistically associated with ATD-induced agranulocytosis (p = 0.038, permutation p value). Our findings indicate that genetic variations in the FMO3 gene are associated with the response to ATDs maintenance treatment in ATD-induced agranulocytosis patients of -Chinese Han population.

Metabolic phenotyping and systems biology approaches to understanding metabolic syndrome and fatty liver disease.

Metabolic syndrome, a cluster of risk factors for type 2 diabetes mellitus and cardiovascular disease, is becoming an increasing global health concern. Insulin resistance is often associated with metabolic syndrome and also typical hepatic manifestations such as nonalcoholic fatty liver disease. Profiling of metabolic products (metabolic phenotyping or metabotyping) has provided new insights into metabolic syndrome and nonalcoholic fatty liver disease. Data from nuclear magnetic resonance spectroscopy and mass spectrometry combined with statistical modeling and top-down systems biology have allowed us to analyze and interpret metabolic signatures in terms of metabolic pathways and protein interaction networks and to identify the genomic and metagenomic determinants of metabolism. For example, metabolic phenotyping has shown that relationships between host cells and the microbiome affect development of the metabolic syndrome and fatty liver disease. We review recent developments in metabolic phenotyping and systems biology technologies and how these methodologies have provided insights into the mechanisms of metabolic syndrome and nonalcoholic fatty liver disease. We discuss emerging areas of research in this field and outline our vision for how metabolic phenotyping could be used to study metabolic syndrome and fatty liver disease.

Association of Flavin Monooxygenase Gene E158K Polymorphism with Chronic Heart Disease Risk.

We studied the relationship between the risk of chronic heart disease and FMO3 gene polymorphism E158K analyzed by PCR and restriction fragment length polymorphism (RFLP) analysis. The homozygous 158KK genotype of FMO3 gene is associated with high risk of chronic heart disease in women, but not in men. FMO3 gene polymorphism E158K is a significant predictor of predisposition to chronic heart disease in women.

Analysis of changes in hepatic gene expression in a murine model of tolerance to acetaminophen hepatotoxicity (autoprotection).

Pretreatment of mice with a low hepatotoxic dose of acetaminophen (APAP) results in resistance to a subsequent, higher dose of APAP. This mouse model, termed APAP autoprotection was used here to identify differentially expressed genes and cellular pathways that could contribute to this development of resistance to hepatotoxicity. Male C57BL/6J mice were pretreated with APAP (400mg/kg) and then challenged 48h later with 600mg APAP/kg. Livers were obtained 4 or 24h later and total hepatic RNA was isolated and hybridized to Affymetrix Mouse Genome MU430_2 GeneChip. Statistically significant genes were determined and gene expression changes were also interrogated using the Causal Reasoning Engine (CRE). Extensive literature review narrowed our focus to methionine adenosyl transferase-1 alpha (MAT1A), nuclear factor (erythroid-derived 2)-like 2 (Nrf2), flavin-containing monooxygenase 3 (Fmo3) and galectin-3 (Lgals3). Down-regulation of MAT1A could lead to decreases in S-adenosylmethionine (SAMe), which is known to protect against APAP toxicity. Nrf2 activation is expected to play a role in protective adaptation. Up-regulation of Lgals3, one of the genes supporting the Nrf2 hypothesis, can lead to suppression of apoptosis and reduced mitochondrial dysfunction. Fmo3 induction suggests the involvement of an enzyme not known to metabolize APAP in the development of tolerance to APAP toxicity. Subsequent quantitative RT-PCR and immunochemical analysis confirmed the differential expression of some of these genes in the APAP autoprotection model. In conclusion, our genomics strategy identified cellular pathways that might further explain the molecular basis for APAP autoprotection.

Deciphering the diurnal rhythm regulating mechanism of flavin-containing monooxygenase 3 in mouse liver.

Circadian genes play an important role in the field of drug metabolism. Flavin-containing monooxygenase 3 is a well-known phase I enzyme which participates in metabolism of many exogenous and endogenous substances, especially production of trimethylamine N-oxide. Here, we aimed to decipher diurnal rhythms of flavin-containing monooxygenase 3 expression and activity, and explore the regulation mechanism by clock genes. Our results showed that its mRNA and protein exhibited robust diurnal rhythms in mouse liver and cell lines. Consistently, significant alterations were observed for in vitro microsomal N-oxidation rates of procainamide, which kept in line with its protein expression at different time in wild-type and reverse erythroblastosis virus α knockout mice. Further, flavin-containing monooxygenase 3 was negatively regulated by E4 promoter-binding protein 4 in AML12 and Hepa1-6 cells, while it was positively influenced by reverse erythroblastosis virus α and brain and muscle ARNT-like protein-1. Moreover, luciferase reporter assays and electrophoretic mobility shift assays showed E4 promoter-binding protein 4 inhibited the transcription of flavin-containing monooxygenase 3 by binding to a D-box1 element (-1606/-1594 bp), while brain and muscle ARNT-like protein-1 positively activated the transcription via direct binding to three E-boxes (-863/-858 bp, -507/-498 bp, and -115/-104 bp) in this enzyme promoter. Taken together, this study would be helpful to reveal the mechanism of clock-controlled drug metabolism and facilitate the practice of chrono-therapeutics.

Targeting Trimethylamine N-Oxide: A New Therapeutic Strategy for Alleviating Atherosclerosis.

Atherosclerosis (AS) is one of the most common cardiovascular diseases (CVDs), and there is currently no effective drug to reverse its pathogenesis. Trimethylamine N-oxide (TMAO) is a metabolite of the gut flora with the potential to act as a new risk factor for CVD. Many studies have shown that TMAO is involved in the occurrence and development of atherosclerotic diseases through various mechanisms; however, the targeted therapy for TMAO remains controversial. This article summarizes the vital progress made in relation to evaluations on TMAO and AS in recent years and highlights novel probable approaches for the prevention and treatment of AS.

Associations of the T329S Polymorphism in Flavin-Containing Monooxygenase 3 With Atherosclerosis and Fatty Liver Syndrome in 90-Week-Old Hens.

This study aimed to evaluate the effects of the spontaneous genetic mutation T329S in flavin-containing monooxygenase 3 (FMO3) on atherosclerosis (AS), fatty liver syndrome (FLS), and adiposity in 90-week-old layers. At 90 weeks of age, 27 FMO3 genotyped Rhode Island White chickens (consisting of nine AA hens, nine AT hens, and nine TT hens) with normal laying performance were selected. The AS lesions, incidence of FLS, fat deposition, metabolic characteristics, and production performance of these egg-layers with different FMO3 genotypes were assessed. The T329S mutation in TT hens reduced the AS lesions (P < 0.01) and altered the plasma metabolic indices more than it did in the AA and AT hens. Furthermore, it reduced the incidence of FLS, hepatic triglyceride deposition (P < 0.05), liver indices (P < 0.05), and fat deposition (P < 0.05) in the subcutis and abdomen of TT hens compared to those of AA and AT hens. Moreover, as an effect of T329S, TT hens laid a higher than average number of eggs and maintained a higher egg-laying rate from 68 to 90 weeks than AA and AT hens. Our study confirmed that the T329S mutation in FMO3 could reduce the development of AS lesions, the incidence of FLS, and fat deposition, which are associated with changes in plasma and hepatic metabolic indices and improvements in the laying performance of older layers. Our results may provide a new strategy for using the T329S mutation to improve the health status and production performance of layers during the late laying period.

FMO3 deficiency of duck leads to decreased lipid deposition and increased antibacterial activity.

BACKGROUND: Most duck eggs possess a fishy odor, indicating that ducks generally exhibit impaired trimethylamine (TMA) metabolism. TMA accumulation is responsible for this unpleasant odor, and TMA metabolism plays an essential role in trimethylaminuria (TMAU), also known as fish odor syndrome. In this study, we focused on the unusual TMA metabolism mechanism in ducks, and further explored the unclear reasons leading to the debilitating TMA metabolism. METHODS: To achieve this, transcriptome, proteome, and metagenome analyses were first integrated based on the constructed duck populations with high and low TMA metabolism abilities. Additionally, further experiments were conducted to validate the hypothesis regarding the limited flavin-containing monooxygenase 3 (FMO3) metabolism ability of ducks. RESULTS: The study demonstrated that liver FMO3 and cecal microbes, including Akkermansia and Mucispirillum, participated in TMA metabolism in ducks. The limited oxidation ability of FMO3 explains the weakening of TMA metabolism in ducks. Nevertheless, it decreases lipid deposition and increases antibacterial activity, contributing to its survival and reproduction during the evolutionary adaptation process. CONCLUSIONS: This study demonstrated the function of FMO3 and intestinal microbes in regulating TMA metabolism and illustrated the biological significance of FMO3 impairment in ducks.

Rapid detection of FMO3 single nucleotide polymorphisms using a pyrosequencing method.

The present study aimed to develop a reliable pyrosequencing method to detect four single nucleotide polymorphisms (SNPs) of the flavin­containing monooxygenase 3 (FMO3) gene and to compare the ethnic differences in their allelic frequencies. The pyrosequencing method was used to detect four FMO3 SNPs, namely, c.855C>T (N285N, rs909530), c.441C>T (S147S, rs1800822), c.923A>G (E308G, rs2266780) and c.472G>A (E158K, rs2266782). The allelic frequencies of these SNPs in 122 unrelated Korean subjects were as follows: i) 44.7% for c.855C>T; ii) 23.4% for c.441C>T; iii) 23.0% for c.923A>G; and iv) 27.1% for c.472G>A. Linkage disequilibrium (LD) analysis revealed that the SNPs c.923A>G and c.472G>A exhibited a strong LD (D'=0.8289, r2=0.5332). In conclusion, the pyrosequencing method developed in this study was successfully applied to detect the c.855C>T, c.441C>T, c.923A>G and c.472G>A SNPs of FMO3.

Bile acid coordinates microbiota homeostasis and systemic immunometabolism in cardiometabolic diseases.

Cardiometabolic disease (CMD), characterized with metabolic disorder triggered cardiovascular events, is a leading cause of death and disability. Metabolic disorders trigger chronic low-grade inflammation, and actually, a new concept of metaflammation has been proposed to define the state of metabolism connected with immunological adaptations. Amongst the continuously increased list of systemic metabolites in regulation of immune system, bile acids (BAs) represent a distinct class of metabolites implicated in the whole process of CMD development because of its multifaceted roles in shaping systemic immunometabolism. BAs can directly modulate the immune system by either boosting or inhibiting inflammatory responses via diverse mechanisms. Moreover, BAs are key determinants in maintaining the dynamic communication between the host and microbiota. Importantly, BAs via targeting Farnesoid X receptor (FXR) and diverse other nuclear receptors play key roles in regulating metabolic homeostasis of lipids, glucose, and amino acids. Moreover, BAs axis per se is susceptible to inflammatory and metabolic intervention, and thereby BAs axis may constitute a reciprocal regulatory loop in metaflammation. We thus propose that BAs axis represents a core coordinator in integrating systemic immunometabolism implicated in the process of CMD. We provide an updated summary and an intensive discussion about how BAs shape both the innate and adaptive immune system, and how BAs axis function as a core coordinator in integrating metabolic disorder to chronic inflammation in conditions of CMD.

T329S Mutation in the FMO3 Gene Alleviates Lipid Metabolic Diseases in Chickens in the Late Laying Period.

The T329S mutation in flavin-containing monooxygenase 3 (FMO3) impairs the trimethylamine (TMA) metabolism in laying hens. The TMA metabolic pathway is closely linked to lipid metabolic diseases, such as atherosclerosis and fatty liver disease. We aimed to evaluate the effects of the T329S mutation in FMO3 on lipid metabolism in chickens during the late laying period. We selected 18 FMO3 genotyped individuals (consisting of six AA, six AT, and six TT hens) with similar body weight and production performance. The lipid metabolism and deposition characteristics of the laying hens with different genotypes were compared. The T329S mutation moderated the serum-lipid parameters in TT hens compared to those in AA and AT hens from 49 to 62 weeks. Furthermore, it reduced the serum trimethylamine N-oxide concentrations and increased the serum total bile acid (p < 0.05) and related lipid transporter levels in TT hens. Moreover, it significantly (p < 0.01) decreased atherosclerotic lesions and hepatic steatosis in TT hens compared to those in the AA and AT hens. Our findings may help improve the health status in laying hens during the late laying period.

Experimental Periodontitis Deteriorated Atherosclerosis Associated With Trimethylamine N-Oxide Metabolism in Mice.

BACKGROUND: Periodontitis is considered a risk factor for atherosclerosis, but the mechanism is not clear. It was reported that oral administration of Porphyromonas gingivalis altered the gut microbiota in mice. Gut dysbiosis and the intestinal metabolite trimethylamine N-oxide (TMAO) were verified to be associated with atherosclerosis. Therefore, the possible TMAO-related mechanism between periodontitis and atherosclerosis needs to be explored. METHODS: Experimental periodontitis was established by oral administration of P. gingivalis for 2 months in ApoE-/- mice. Mouse hemi-mandibles were scanned using Micro-CT. Quantification of TMAO was performed using liquid chromatography-tandem mass spectrometry. Mouse feces were collected and the bacterial DNA was extracted, then the gut microbiota was analyzed using 16S rRNA genes. Atherosclerotic lesion areas were quantified. Livers, small intestines, and large intestines were analyzed for gene expression. RESULTS: Aggravated atherosclerosis plaques were found in experimental periodontitis mice. Plasma TMAO, a pathogenic factor of atherosclerosis, was initially found to be increased in periodontitis mice. Changes in the composition and abundance of the intestinal microflora of periodontitis mice were found. Flavin monooxygenase 3 (FMO3), the catalyzing enzyme of TMAO in the liver, was significantly increased, accompanied by an increase of IL-6 in liver, the abnormal intestinal integrity and enhanced plasma LPS. The IL-6 and LPS were verified to be able to increase FMO3 in HepG2 cells. CONCLUSION: Our research discovered that experimental periodontitis in ApoE-/- mice induced gut dysbiosis and an increase in TMAO. These results suggest a possible mechanism by which periodontitis may accelerate atherosclerosis by influencing the intestinal microbes and the metabolism, which were triggered by inflammation of the liver and intestine.

Upregulation of flavin-containing monooxygenase 3 mimics calorie restriction to retard liver aging by inducing autophagy.

Flavin-containing monooxygenase 3 (FMO3) gene expression is often upregulated in long-lived murine models. However, the specific relationship between FMO3 and aging remains unknown. Here, we show that 40% calorie restriction (CR), which is considered to be one of the most robust interventions to delay aging progression, markedly upregulates FMO3. Most importantly, upregulation of hepatocyte FMO3 in murine models prevented or reversed hepatic aging. Accordingly, the upregulation of FMO3 mimicked the effects of CR: reduced serum levels of pro-inflammatory cytokine interleukin-6 and fasting insulin; relief of oxidative stress, with lower hepatic malondialdehyde levels and higher superoxide dismutase activity; reduced serum and hepatic levels of total cholesterol and triglyceride, as well as reduced lipid deposition in the liver; and diminished levels of aging-related markers ß-gal and p16. There were also synergistic effects between FMO3 upregulation and CR. Inhibition of autophagy blocked the anti-aging effects of upregulation of hepatocyte FMO3, including reversing the amelioration of the serum and hepatic parameters related to inflammation, oxidative stress, lipid metabolism, liver function, and hepatocyte senescence. Our results suggest that the upregulation of FMO3 mimics CR to prevent or reverse hepatic aging by promoting autophagy.

Goose FMO3 gene cloning, tissue expression profiling, polymorphism detection and association analysis with trimethylamine level in the egg yolk.

Flavin-containing monooxygenase 3 (FMO3) plays a critical role in catalyzing the conversion of trimethylamine (TMA) to trimethylamine-N-oxide (TMAO) in vivo. Despite the well-documented association between FMO3 mutations and a 'fishy' off-flavor eggs in chicken and quail, little information is available regarding the molecular characteristic of goose (Anser cygnoides) FMO3 and its relationship with the yolk TMA content. To fill these gaps, we cloned the full-length cDNA sequence of goose FMO3, which comprised 1851bp encoding 531 amino acids. FMO3 mRNA was dramatically expressed in liver than in other tissues in the geese. Eight single nucleotide polymorphisms (SNPs) were detected in the entire coding region. The CC genotype at the T669C site, GG at the A723G site, and AA at the G734A site of FMO3 were highly significantly associated with elevated TMA content in goose egg yolk (P<0.001). Carriers of the A allele of G734A or C allele of T885C had yolk TMA content that had a high probability of being elevated after feeding with additional choline chloride (P=0.0429, OR=4.1300, 95%CI=1.0390-16.4270, and P=0.0251, OR=4.6060, 95%CI=1.1620-18.2620, respectively). This work lays a foundation for studying the function of FMO3 and yolk TMA content in goose. However, studies using larger sample sizes and more goose breeds are required to determine whether the fishy off-flavor trait exists in goose.

The TMAO-Producing Enzyme Flavin-Containing Monooxygenase 3 Regulates Obesity and the Beiging of White Adipose Tissue.

Emerging evidence suggests that microbes resident in the human intestine represent a key environmental factor contributing to obesity-associated disorders. Here, we demonstrate that the gut microbiota-initiated trimethylamine N-oxide (TMAO)-generating pathway is linked to obesity and energy metabolism. In multiple clinical cohorts, systemic levels of TMAO were observed to strongly associate with type 2 diabetes. In addition, circulating TMAO levels were associated with obesity traits in the different inbred strains represented in the Hybrid Mouse Diversity Panel. Further, antisense oligonucleotide-mediated knockdown or genetic deletion of the TMAO-producing enzyme flavin-containing monooxygenase 3 (FMO3) conferred protection against obesity in mice. Complimentary mouse and human studies indicate a negative regulatory role for FMO3 in the beiging of white adipose tissue. Collectively, our studies reveal a link between the TMAO-producing enzyme FMO3 and obesity and the beiging of white adipose tissue.

Trimethylamine-N-Oxide: Friend, Foe, or Simply Caught in the Cross-Fire?

Trimethylamine-N-oxide (TMAO), a gut-derived metabolite, has recently emerged as a candidate risk factor for cardiovascular disease and other adverse health outcomes. However, the relation between TMAO and chronic disease can be confounded by several factors, including kidney function, the gut microbiome, and flavin-containing monooxygenase 3 (FMO3) genotype. Thus, whether TMAO is a causative agent in human disease development and progression, or simply a marker of an underlying pathology, remains inconclusive. Importantly, dietary sources of TMAO have beneficial health effects and provide nutrients that have critical roles in many biological functions. Pre-emptive dietary strategies to restrict TMAO-generating nutrients as a means to improve human health warrant careful consideration and may not be justified at this time.

Effects of dietary rapeseed meal supplementation on cecal microbiota in laying hens with different flavin-containing monooxygenase 3 genotypes.

To evaluate the effect of dietary rapeseed meal (RM) supplementation on cecal trimethylamine and bacteria in laying hens with different flavin-containing monooxygenase 3 (FMO3) genotypes, a 3 × 2 2-factorial arrangement was employed using FMO3 genotypes (AA, AT, and TT) and dietary RM (0 and 14% of diet) as the main effects. At 50 wk of age, 36 hens of AT genotype and 36 hens of TT genotype were randomly allotted to one of the 2 dietary treatments, and each dietary treatment consisted of 3 replicates with 6 birds each. A total of 12 hens with AA genotype were allotted to one of the 2 dietary treatments that consisted of 3 replicates with 2 hens. Hens were fed 0% RM in a corn-soybean (SM) diet for one wk before the 6-week feeding trial period. Dietary RM supplementation increased trimethylamine (TMA) concentrations in both egg yolks (P < 0.0001) and cecal chyme (P < 0.0001). Dietary RM supplementation increased bacterial abundance and diversity (P < 0.0001). Weighted UniFrac, Nonmetric Multidimensional Scaling, and analysis of similarity (R-ANOSIM = 0.1516; P-value = 0.014) indicated distinct clustering was dependent on diets rather than FMO3 genotypes. Twenty-four phyla (most dominant, Bacteroides, Firmicutes, and Proteobacteria) and 229 genera were identified in the cecal samples. Compared with the SM diets, RM diets increased the proportion of Firmicutes (P = 0.004), Proteobacteria (P = 0.006), and Firmicutes:Bacteroides (P = 0.001), and some low-abundance phyla (P < 0.01), whereas the abundance of Bacteroides was lower (P = 0.0002). The abundance of 42 genera varied with dietary types. Six phyla and 35 genera were positively correlated with TMA concentration in the cecal chyme. In conclusion, the major TMA-producing bacteria in cecal were from Firmicutes and Proteobacteria phyla. The major TMA-producing bacterial genera could be from the genera that positively correlated with TMA concentration.

Genetic Variant in Flavin-Containing Monooxygenase 3 Alters Lipid Metabolism in Laying Hens in a Diet-Specific Manner.

Genetic variant T329S in flavin-containing monooxygenase 3 (FMO3) impairs trimethylamine (TMA) metabolism in birds. The TMA metabolism that under complex genetic and dietary regulation, closely linked to cardiovascular disease risk. We determined whether the genetic defects in TMA metabolism may change other metabolic traits in birds, determined whether the genetic effects depend on diets, and to identify genes or gene pathways that underlie the metabolic alteration induced by genetic and diet factors. We used hens genotyped as FMO3 c.984 A>T as well as those with the homozygous normal genotype. For each genotype, hens were provided with either a corn-soybean meal basal diets (SM), which contains lower levels of TMA precursor, or the basal diets supplemented with 21% of rapeseed meal (RM), which contains higher levels of TMA precursor. An integrative analysis of metabolomic and transcriptomic was used to explore the metabolic patterns of FMO3 genetic variant in hens that were fed the two defined diets. In birds that consumed SM diets, the T329S mutation increased levels of plasma TMA and lipids, FMO3 mRNA levels, and the expression of genes involved in long chain polyunsaturated fatty acid biosynthesis. In birds that consumed RM diets, the T329S mutation induced fishy odor syndrome, a repression in LXR pathway and a reciprocal change in lipid metabolism. Variations in TMA and lipid metabolism were linked to the genetic variant in FMO3 in a diet-specific manner, which suggest FMO3 functions in TMA metabolism and lipid homeostasis. LXR pathway and polyunsaturated fatty acid metabolism are two possible mechanisms of FMO3 action in response to dietary TMA precursor.

Analysis of six novel flavin-containing monooxygenase 3 (FMO3) gene variants found in a Japanese population suffering from trimethylaminuria.

Polymorphic human flavin-containing monooxygenase 3 (FMO3) is associated with the inherited disorder trimethylaminuria. Several FMO3 variants have been observed in a variety of ethnic groups, including a Japanese cohort suffering from trimethylaminuria. The aim of this study was to screen another self-reported Japanese trimethylaminuria cohort for novel FMO3 variants and to investigate these new variants. Subjects with low FMO3 metabolic capacities were identified by measuring the urinary trimethylamine and trimethylamine N-oxide concentrationsin171 Japanese volunteers. The FMO3 genes from these subjects and their family members were then sequenced. Heterozygotes or homozygotes for novel single-nucleotide polymorphisms c.20 T > C p.(Ile7Thr), c.122 G > A p.(Trp41Ter), c.127T > A p.(Phe43Ile), c.488 T > C p.(Leu163Pro), and c.1127G > A p.(Gly376Glu) and a heterozygote for the novel duplication c.850_860dupTTTAACGATGA p.(Glu287AspfsTer17) were identified. In addition, the known (but as yet uncharacterized) single-nucleotide polymorphism c.929 C > T p.(Ser310Leu) was found. Pedigree analysis revealed the p.(Ser310Leu) FMO3 allele in cis configuration with c.929 C > T p.(Glu158Lys). These variant FMO3 proteins recombinantly expressed in Escherichia coli membranes exhibited decreased N-oxygenation activities toward trimethylamine and benzydamine. Although the allele frequencies of these seven variants were low, the present results suggest that individuals homozygous or heterozygous for any of these novel missense or duplicationFMO3 variants or known nonsense mutations such as p.(Cys197Ter) may possess abnormal activities toward trimethylamine N-oxygenation.