Deleterious mutations in the flavin-containing monooxygenase 3 (FMO3) gene causing trimethylaminuria

The primary genetic form of trimethylaminuria (TMAU) is caused by inherited defects in the flavin-containing monooxygenase 3 (FMO3) gene. Defective FMO3 has a decreased ability to catalyze the N-oxygenation of the dietary-derived malodourous amine, trimethylamine. We report two novel deleterious mutations identified in two unrelated individuals affected by the disorder. Sequence analysis of the FMO3 coding exons amplified from genomic DNA revealed that the mutation from individual 1 was heterozygous for a G>A missense mutation in exon 2 of the FMO3 gene. The mutation changed a GAG encoding Glu at codon 32 to AAG encoding Lys. Wild-type and mutant E32K FMO3 were expressed in Escherichia coli as maltose binding-fusion proteins and assayed for their ability to catalyze oxygenation of various FMO3 substrates. The results showed that the E32K mutation abrogated the catalytic activity of the enzyme. Individual 2 was identified as heterozygous for the P153L mutation. In addition, individual 2 was also heterozygous for a novel single nucleotide deletion of A191 in exon 3 at codon 64. The deletion resulted in a frame shift and caused premature termination of the FMO3 gene immediately after codon 65. Family pedigree analysis revealed that the P153L and the deletion mutation were carried on different alleles for this individual. Therefore, both alleles of the FMO3 gene for individual 2 were affected by mutations abolishing the catalytic activity of the enzyme, explaining the severe TMAU condition. The two deleterious mutations reported herein were rare mutations with estimated allelic frequencies of less than 1%.     

Population-specific polymorphisms of the human FMO3 gene: significance for detoxication.

Flavin-containing monooxygenase form 3 (FMO3) is one of the major enzyme systems that protect humans from the potentially toxic properties of drugs and chemicals. FMO3 converts nucleophilic heteroatom-containing chemicals and endogenous materials to polar metabolites, which facilitates their elimination. For example, the tertiary amine trimethylamine is N-oxygenated by human FMO3 to trimethylamine N-oxide, and trimethylamine N-oxide is excreted in a detoxication and deoderation process. In normal humans, virtually all trimethylamine is metabolized to trimethylamine N-oxide. In a few humans, trimethylamine is not efficiently metabolized to trimethylamine N-oxide, and those individuals suffer from trimethylaminuria, or fishlike odor syndrome. Previously, we identified mutations of the FMO3 gene that cause trimethylaminuria. We now report two prevalent polymorphisms of this gene (K158E and V257M) that modulate the activity of human FMO3. These polymorphisms are widely distributed in Canadian and Australian white populations. In vitro analysis of wild-type and variant human FMO3 proteins expressed from the cDNA for the two naturally occurring polymorphisms showed differences in substrate affinities for nitrogen-containing substrates. Thus, for polymorphic forms of human FMO3, lower k(cat)/K(m) values for N-oxygenation of 10-(N, N-dimethylaminopentyl)-2-(trifluoromethyl) phenothiazine, trimethylamine, and tyramine were observed. On the basis of in vitro kinetic parameters, human FMO1 does not significantly contribute to human metabolism of trimethylamine or tyramine. The results imply that prevalent polymorphisms of the human FMO3 gene may contribute to low penetrance predispositions to diseases associated with adverse environmental exposures to heteroatom-containing chemicals, drugs, and endogenous amines.     

Two novel single nucleotide polymorphisms (SNPs) of the FMO3 gene in Japanese

We sequenced all exons and exon-intron junctions of the flavin-containing monooxygenase 3 (FMO3) gene from 27 Japanese individuals who are trimethylaminuria volunteers judged by self-reported analysis. We found two novel single nucleotide polymorphisms (SNPs) (21246 T>A and 21265 C>T) causing amino acid substitutions (Asp(198)Glu and Arg(205)Cys in exon 5), respectively. The Asp(198)Glu allele also presented together with known SNPs (20852 C>T in exon4, 20960_20962 CTT deletion, 21115 G>A in intron 4, and 21243_21244 TG deletion in exon 5) in the same allele of the FMO3 gene to form a novel haplotype.These sequences are as follows:1) SNP, 030609Fujieda019; GENE NAME, FMO3; ACCESSION NUMBER, AL021026; LENGTH, 25 base; 5'-TTCGGGCTG(TG/-)AT/AATTGCCACAGAA-3'.2) SNP, 030609Fujieda020; GENE NAME, FMO3; ACCESSION NUMBER, AL021026; LENGTH, 25 base; 5'-ACAGAACTCAGCC/TGCACAGCAGAAC-3'.     

A novel mutation in the flavin-containing monooxygenase 3 gene, FM03, that causes fish-odour syndrome: activity of the mutant enzyme assessed by proton NMR spectroscopy

We have previously shown that primary trimethylaminuria, or fish-odour syndrome, is caused by an inherited defect in the flavin-containing monooxygenase 3 (FMO3) catalysed N-oxidation of the dietary-derived malodorous amine, trimethylamine (TMA). We now report a novel causative mutation for the disorder identified in a young girl diagnosed by proton nuclear magnetic resonance (NMR) spectroscopy of her urine. Sequence analysis of genomic DNA amplified from the patient revealed that she was homozygous for a T to C missense mutation in exon 3 of the FMO3 gene. The mutation changes an ATG triplet, encoding methionine, at codon 82 to an ACG triplet, encoding threonine. A polymerase chain reaction/restriction enzyme-based assay was devised to genotype individuals for the FMO3Thr82 allele. Wild-type and mutant FMO3, heterologously expressed in a baculovirus-insect cell system, were assayed by ultraviolet spectrophotometry and NMR spectroscopy for their ability to catalyse the N-oxidation of TMA. The latter technique has the advantage of enabling the simultaneous, direct and semi-continuous measurement of both of the products, TMA N-oxide and NADP, and of one of the reactants, NADPH. Results obtained from both techniques demonstrate that the Met82Thr mutation abolishes the catalytic activity of the enzyme and thus represents the genetic basis of the disorder in this individual. The combination of NMR spectroscopy with gene sequence and expression technology provides a powerful means of determining genotype-phenotype relationships in trimethylaminuria.     

Interindividual differences of human flavin-containing monooxygenase 3: genetic polymorphisms and functional variation.

The human flavin-containing monooxygenase (form 3) (FMO3) participates in the oxygenation of nucleophilic heteroatom-containing drugs, xenobiotics, and endogenous materials. Currently, six forms of the FMO gene are known, but it is FMO3 that is the major form in adult human liver that is likely responsible for the majority of FMO-mediated metabolism. The substrate structural feature requirements for human FMO3 is beginning to become known to a greater extent and a few chemicals extensively metabolized by FMO3 have been reported. Expression of FMO3 is species- and tissue-specific, but unlike human cytochrome p450, mammalian FMO3 does not appear to be inducible. Interindividual variation in FMO3-dependent metabolism of drugs, chemicals, and endogenous material is therefore more likely due to genetic effects and not environmental ones. Examples of such interindividual variation come from the study of very rare mutations of the human FMO3 gene that have been associated with deficient N-oxygenation of dietary trimethylamine. Defective trimethylamine N-oxygenation causes trimethylaminuria or "fish-like odor syndrome". Information on human FMO3 mutations from individuals suffering from the condition of trimethylaminuria has provided knowledge about the underlying molecular mechanism(s) for trimethylaminuria. A number of common variants of human FMO3 have been reported. Diversification of the FMO3 gene may have led to selective advantages and new functions. As more examples of human FMO3-mediated metabolism of drugs or new chemical entities are discovered in the future, it is possible that FMO3 allelic variation may be shown to contribute to interindividual and interethnic variability of FMO-mediated metabolism. Human FMO3 may be another example of an environmental gene that participates in a protective mechanism to help humans ward off potentially toxic exposure of chemicals.     

A family study of compound variants of flavin-containing monooxygenase 3 (FMO3) in Japanese subjects found by urinary phenotyping for trimethylaminuria

Phenotype–gene analyses and the increasing availability of mega-databases have revealed the impaired human flavin-containing monooxygenase 3 (FMO3) variants associated with the metabolic disorder trimethylaminuria. In this study, a novel compound variant of FMO3, p.[(Val58Ile; Tyr229His)], was identified in a 1-year-old Japanese girl who had impaired FMO3 metabolic capacity (70%) in terms of urinary trimethylamine N-oxide excretion levels divided by total levels of trimethylamine and its N-oxide. One cousin in the family had the same p.[(Val58Ile); (Tyr229His)]; [(Glu158Lys; Glu308Gly)] FMO3 haplotype and had a similar FMO3 metabolic capacity (69%). In a family study, the novel p.[(Val58Ile); (Tyr229His)] compound FMO3 variant was also detected in the proband 1's mother and aunt. Another novel compound FMO3 variant p.[(Glu158Lys; Met260Lys; Glu308Gly; Ile426Thr)] was identified in a 7-year-old girl, proband 2. This novel compound FMO3 variant was inherited from her mother. Recombinant FMO3 Val58Ile; Tyr229His variant and Glu158Lys; Met260Lys; Glu308Gly; Ile426Thr variant showed moderately decreased capacities for trimethylamine N-oxygenation compared to wild-type FMO3. Analysis of trimethylaminuria phenotypes in family studies has revealed compound missense FMO3 variants that impair FMO3-mediated N-oxygenation in Japanese subjects; moreover, these variants could result in modified drug clearances.     

Genetic polymorphism of the flavin-containing monooxygenase 3 (FMO3) associated with trimethylaminuria (fish odor syndrome): observations from Japanese patients

Trimethylaminuria (fish odor syndrome) is a metabolic disorder characterized by the inability to convert malodorous dietary-derived trimethylamine (TMA) to odorless TMA N-oxide by the flavin-containing monooxygenase 3 (FMO3). Mutations of the FMO3 gene were investigated in Japanese trimethylaminuria that showed low FMO3 metabolic capacity. Novel polymorphisms in the FMO3 gene causing stop codons at Cys197, Trp388, Gln470 or Arg500 of FMO3 were discovered in self-reported trimethylaminuria Japanese volunteers. Different metabolic capacities of FMO3 were observed for Asn114Ser, Thr201Lys, Arg205Cys or Met260Val FMO3 variants in addition to common Glu158Lys, Val257Met, and Glu308Gly FMO3. Estimated allelic frequencies for these novel mutated FMO3 genes for the Japanese population examined was approximately 1-4 % in this Japanese cohort. Recombinant Arg500stop (94% of the whole FMO3 structure) and several missense FMO3 variants showed no detectable activity and different effects on N- and S-oxygenation activities, respectively. The family members of Japanese probands who were heterozygous for these nonsense mutants generally showed moderate TMA N-oxygenation metabolic capacity, suggesting that heterozygotes for the nonsense mutations will exhibit trimethylaminuria symptoms only if they have, on the other chromosome, a mutation that substantially impairs enzyme activity. In addition, other causal factors for decreased FMO3 metabolic capacity such as liver damage or menstruation and treatment with copper chlorophyllin are also included in this minireview. The present article provides fundamental information for the importance of future investigations of the human FMO3 gene associated with trimethylaminuria (fish odor syndrome).     

Genetic polymorphisms of flavin-containing monooxygenase (FMO)

Mammalian flavin-containing monooxygenase (FMO) exists as six gene families and metabolizes a plethora of drugs and xenobiotics. The major FMO in adult human liver, FMO3, is responsible for trimethylamine (TMA) N-oxygenation. A number of FMO3 mutant alleles have been described and associated with a disease termed trimethylaminuria (TMAU). The TMAU patient excretes large amounts of TMA in urine and sweat. A more recent ethnically related polymorphism in expression of the major FMO in lung, FMO2, has been described. All Caucasians and Asians genotyped to date are homozygous for a CAG --> TAG amber mutation resulting in a premature stop codon and a nonfunctional protein truncated at AA 472 (wildtype FMO2 is 535 AA). This allele has been designated hFMO2*2A. Twenty-six percent of individuals of African descent and 5% of Hispanics genotyped to date carry at least one allele coding for full-length FMO2 (hFMO2*1 allele). Preliminary evidence indicates that FMO2.1 is very active toward the S-oxygenation of low MW thioureas, including the lung toxicant ethylene thiourea. Polymorphic expression of functional FMO2 in the individuals of African and Hispanic descent may markedly influence drug metabolism and/or xenobiotic toxicity in the lung.     

Missense and nonsense mutations of the flavin-containing monooxygenase 3 gene in a Japanese cohort

We sequenced all exons and exon-intron junctions of the flavin-containing monooxygenase 3 (FMO3) gene from 3 Japanese individuals and their family members, who were case subjects that showed low FMO3 metabolic capacity among a population of self-reported trimethylaminuria Japanese volunteers (n=50). We found three novel single nucleotide polymorphisms (SNPs) (g. 20752 A>G, g. 27400 G>A, and g. 30308 C>T) causing an amino acid substitution and stop codons, Asn114Ser in exon 4, Trp388Stop in exon 7, and Gln470Stop in exon 9, respectively. The Trp388Stop and Gln470Stop also presented together with known SNPs, Val257Met and Glu158Lys, respectively, in the same alleles of the FMO3 gene to form novel haplotypes. These sequences are as follows: 1) SNP, 060825Shimizu004; GENE NAME, FMO3; ACCESSION NUMBER, AL021026; LENGTH, 25 base; 5'-TATCCAGTGTAAA/GTAAACATCCTGA-3'. 2) SNP, 060825Shimizu005; GENE NAME, FMO3; ACCESSION NUMBER, AL021026; LENGTH, 25 base; 5'-CCAGTCCCGCTGG/AGCAGCACAAGTA-3'. 3) SNP, 060825Shimizu006; GENE NAME, FMO3; ACCESSION NUMBER, AL021026; LENGTH, 25 base; 5'-TGTAGTCCCTACC/TAGTTTAGGCTGG-3'.     

Three novel single nucleotide polymorphisms of the FMO3 gene in a Japanese population

We sequenced all exons and exon-intron junctions of the flavin-containing monooxygenase 3 (FMO3) gene from 2 Japanese individuals and their family members, who were case subjects that showed low FMO3 metabolic capacity among a population of self-reported trimethylaminuria Japanese volunteers. We found two novel single nucleotide polymorphisms (SNPs) (21,254 C>A and 24,006 A>G) causing amino acid substitutions, Thr(201)Lys in exon 5 and Met(260)Val in exon 6, respectively. The Thr(201)Lys and Met(260)Val also presented together with known SNPs (Glu(158)Lys-Glu(308)Gly and Val(257)Met, respectively) in the same alleles of the FMO3 gene to form novel haplotypes. A SNP (30,398 C>T) in the FMO3 gene causing a stop codon at Arg(500) in exon 9 was also discovered. These sequences are as follows: 1) SNP, 060116Shimizu001; GENE NAME, FMO3; ACCESSION NUMBER, AL021026; LENGTH, 25 base; 5'-GTGATATTGCCAC/AAGAACTCAGCCG-3'. 2) SNP, 060116Shimizu002; GENE NAME, FMO3; ACCESSION NUMBER, AL021026; LENGTH, 25 base; 5'-TAC(G/A)TGAAGCAGA/GTGAATGCAAGAT-3'. 3) SNP, 060116Shimizu003; GENE NAME, FMO3; ACCESSION NUMBER, AL021026; LENGTH, 25 base; 5'-CCCATGCAGACAC/TGAGTGGTCGGGA-3'.     

Novel variants in outer protein surface of flavin-containing monooxygenase 3 found in an Argentinian case with impaired capacity for trimethylamine N-oxygenation

Flavin-containing monooxygenase 3 (FMO3) is a polymorphic drug metabolizing enzyme associated with the genetic disorder trimethylaminuria. We phenotyped a white Argentinian 11-year-old girl by medical sensory evaluation. After pedigree analysis with her brother and parents, this proband showed to harbor a new allele p.(P73L; E158K; E308G) FMO3 in trans configuration with the second new one p.(F140S) FMO3. Recombinant FMO3 proteins of the wild-type and the novel two variants underwent kinetic analyses of their trimethylamine N-oxygenation activities. P73L; E158K; E308G and F140S FMO3 proteins exhibited moderately and severely decreased trimethylamine N-oxygenation capacities (~50% and ~10% of wild-type FMO3, respectively). Amino acids P73 and F140 were located on the outer surface region in a crystallographic structure recently reported of a FMO3 analog. Changes in these positions would indirectly impact on key FAD-binding residues. This is the first report and characterization of a patient of fish odor syndrome caused by genetic aberrations leading to impaired FMO3-dependent N-oxygenation of trimethylamine found in the Argentinian population. We found novel structural determinants of FAD-binding domains, expanding the list of known disease-causing mutations of FMO3. Our results suggest that individuals homozygous for any of these new variants would develop a severe form of this disorder.     

Novel variants and haplotypes of human flavin-containing monooxygenase 3 gene associated with Japanese subjects suffering from trimethylaminuria

Abstract

1. Flavin-containing monooxygenase 3 (FMO3) in humans is polymorphic in several ethnic groups, including Caucasians, Africans and Asians. Some FMO3 variants are associated with a disorder trimethylaminuria.

2. In the current study, we used the results from urinary phenotyping assays to identify 63 subjects with <85% FMO3 metabolic capacity with respect to trimethylamine N-oxidation among 787 Japanese volunteers with self-reported trimethylaminuria. The 63 subjects with reduced FMO3 activity were screened and investigated in detail to identify novel FMO3 variants.

3. Homozygous or heterozygous individuals for new single nucleotide substitution variants/haplotypes p.(Pro282Leu), p.[(Glu158Lys; Glu308Gly; Thr329Ala)], p.[(Glu158Lys; Glu308Gly; Asp429Gly)], p.[(Val257Met; Leu473Pro)], p.[(Glu158Lys; Glu308Gly; Ile441Thr)], and p.[(Arg205Cys; Gly503Arg)] were identified in six proband subjects and their family members after pedigree analyses.

4. These variant FMO3 proteins recombinantly expressed in Escherichia coli membranes exhibited decreased N-oxygenation activities toward trimethylamine (V_max/K_m < 40% that of the wild-type).

5. Although the allele frequencies of the six new variants and/or haplotypes were low, the present results indicated that individuals homozygous or heterozygous for any of these novel missense FMO3 variants or known nonsense mutations such as p.(Cys197Ter) or p.(Arg205Cys) highly found in this self-reported Japanese trimethylaminuria cohort may have reduced FMO3 activity with respect to the N-oxygenation of trimethylamine.     

Population distribution of human flavin-containing monooxygenase form 3: gene polymorphisms.

The N-oxygenation of amines by the human flavin-containing monooxygenase (form 3) (FMO3) represents an important means for the conversion of lipophilic nucleophilic heteroatom-containing compounds into more polar and readily excreted products. Certain mutations of the human FMO3 gene have been linked to abnormal drug or chemical metabolism. For example, abnormal N-oxygenation of trimethylamine has been shown to segregate with mutations of human FMO3. To date, however, it is not known whether there is a pharmacogenetic basis for abnormal drug metabolism by human FMO3. The objective of this study was to estimate the allele and genotype frequencies at three variable DNA sites in the FMO3 gene in male and female blood bank donors representative of non-Hispanic Caucasians, non-Hispanic African Americans, Hispanics, and Asians sampled from the United States. The common polymorphisms at variable sites 158, 257, and 308 were experimentally determined using a high-throughput chip-based genotype variation detection method combining MassEXTEND and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. We also compared the genetic variation of nonhuman primate FMO3 with the human FMO3 gene. Exon sequence analysis of the monkey FMO3 gene sequence showed that it was similar to the human gene sequence but differed from the human consensus sequence at 31 fixed positions. Compared with that of human, the chimpanzee exon sequence had one polymorphism that induced an amino acid change. The evolutionary history of the FMO3 gene was inferred from the pattern of haplotype relationships across different populations and species. Statistically significant heterogeneity in the relative frequencies of single and multiple site alleles, haplotypes, and genotypes of the human FMO3 among ethnic subdivisions suggests that population differences in the susceptibility of humans to abnormal metabolism or adverse drug reactions for chemicals metabolized by human FMO3 could exist.     

Genetic variants of flavin-containing monooxygenase 3 (FMO3) derived from Japanese subjects with the trimethylaminuria phenotype and whole-genome sequence data from a large Japanese database

Flavin-containing monooxygenase 3 (FMO3) is a polymorphic xenobiotic- and dietary compound-metabolizing enzyme associated with the genetic disorder trimethylaminuria. We phenotyped 428 Japanese subjects using traditional urinary phenotyping assays and identified two subjects with <20% FMO3 metabolic capacity. Both subjects had novel frameshift mutations. Proband 1 harbored a novel CC deletion resulting in p.[(Pro153Gln fs; Phe166Ter)] FMO3, which was in trans configuration with p.(Cys197Ter). Proband 2 harbored a novel T deletion resulting in p.[(Met211Arg fs; Val220Ter)] FMO3, which was in trans configuration with p.[(Val257Met; Met260Val)]. We also analyzed a new large Japanese database for novel single nucleotide substitutions of FMO3 and identified the following variants with very low frequencies (<∼0.1%): p.(Lys56Glu), p.(Ser112Asn), p.(Asn164Lys), p.(Gly191Cys), p.(Ile199Ser), p.(Pro248Thr), p.(Pro248Leu), p.(Asp286Tyr), and p.(Ala311Pro). Recombinant FMO3 proteins of the above and unanalyzed variants underwent kinetic analysis of their trimethylamine/benzydamine N-oxygenation activities. Gly191Cys, Ile199Ser, Asp286Tyr, and Ala311Pro variant FMO3 proteins exhibited severely decreased activities (V_max/K_m <5% of wild-type). Although these new variants were rare alleles in Japanese self-reported trimethylaminuria sufferers and in the large genomic database, we found that most Japanese individuals compound heterozygous or homozygous for any of these missense FMO3 variants or known severe mutations [e.g., p.(Cys197Ter)] had impaired FMO3-dependent N-oxygenation of malodorous trimethylamine.     

Human flavin-containing monooxygenase: substrate specificity and role in drug metabolism

The human flavin-containing monooxygenase (FMO3) is a prominent enzyme system that converts nucleophilic heteroatom-containing chemicals, drugs and xenobiotics to more polar materials that are more efficiently excreted in the urine. The substrate specificity for FMO 3 is distinct from that of FMO1. Human FMO3 N-oxygenates primary, secondary and tertiary amines whereas human FMO1 is only highly efficient at N-oxygenating tertiary amines. Both human FMO1 and FMO3 S-oxygenate a number of nucleophilic sulfur-containing substrates and in some cases, does so with great stereoselectivity. Human FMO3 is sensitive to steric features of the substrate and aliphatic amines with linkages between the nitrogen atom and a large aromatic group such as a phenothiazine of at least five carbons are N-oxygenated significantly more efficiently than those substrates with two or three carbons. For amines with smaller aromatic substituents such as phenethylamines, often these compounds are efficiently N-oxygenated by human FMO3. Currently, the most promising non-invasive probe of in vivo human FMO3 functional activity is the formation of trimethylamine N-oxide from trimethylamine that comes from dietary choline. (S)-Nicotine N-1'-oxide formation can also be used as a highly stereoselective probe of human FMO3 function for adult humans that smoke cigarettes. Finally, cimetidine S-oxygenation or ranitidine N-oxidation can also be used as a functional probe of human FMO3. With the recent observation of human FMO3 genetic polymorphism and poor metabolism phenotype in certain human populations, variant human FMO3 may contribute to adverse drug reactions or exaggerated clinical response to certain medications. Knowledge of the substrate specificity for human FMO3 may aid in the future design of more efficacious and less toxic drugs.     

Effect of total parenteral nutrition and choline on hepatic flavin-containing and cytochrome P-450 monooxygenase activity in rats.

Total parenteral nutrition provides nutrition by infusion into the systemic circulation. Bypassing the intestine and processes associated with absorption can cause additional pathophysiological changes to occur. For example, in rats, normal gut and pancreatic cell function may change, absorptive capacity may be altered, and enzyme functional activity including drug metabolism may be affected. The objective of this study was to examine the effects of a control diet or a diet of total parenteral nutrition in the presence or absence of choline on urinary biomarkers and hepatic microsome functional activity from rats. Selective functional markers of cytochrome P-4502E1 (CYP2E1) and flavin-containing monooxygenase (FMO) were examined in vitro. The N-oxygenation of trimethylamine was used as an in vivo selective functional marker for FMO. After the administration of total parenteral nutrition plus choline for 5 days, the urinary excretion of trimethylamine and trimethylamine N-oxide declined approximately 7- and 3-fold, respectively, compared with rats treated with control diet. The concentration of urinary biogenic amines was also significantly affected by total parenteral nutrition. Compared with control animals, rats administered total parenteral nutrition plus choline for 5 days showed a decrease of approximately 5- and 2-fold in urinary dopamine and norepinephrine concentration, respectively. To examine a molecular basis for the influence of total parenteral nutrition +/- choline on monooxygenase regulation, hepatic microsomal activity of the FMO and CYP2E1 was examined. Compared with animals treated with a control diet, total parenteral nutrition plus choline in rats caused a 3-fold increase in hepatic microsomal FMO and a 2-fold increase in hepatic cytochrome CYP2E1 functional activity, respectively. Although the data did not reach statistical significance, selective immunoblot studies using hepatic microsomes from rats treated with total parenteral nutrition + choline showed that compared with controls, FMO1 protein was decreased 1.4-fold and FMO3 increased 1.3-fold, respectively. In hepatic microsomes from rats treated with total parenteral nutrition + choline, compared with control animals, FMO4 immunoreactivity was increased 1.6-fold. The data suggest that total parenteral nutrition has a detectable effect on modulating rat FMO3, FMO4, and CYP2E1 monooxygenase functional activity. The clinical relevance of these results is unknown but may be of significance for individuals receiving total parenteral nutrition and those afflicted with trimethylaminuria.     

Further survey of genetic variants of flavin-containing monooxygenase 3 (FMO3) in Japanese subjects found in an updated database of genome resources and identified by phenotyping for trimethylaminuria

The number of single-nucleotide substitutions of human flavin-containing monooxygenase 3 (FMO3) recorded in mega-databases is increasing. Moreover, phenotype–gene analyses have revealed impaired FMO3 variants associated with the metabolic disorder trimethylaminuria. In this study, four novel amino-acid substituted FMO3 variants, namely p.(Gly191Asp), p.(Glu414Gln), p.(Phe510Ser), and p.(Val530CysfsTer1), were identified in the whole-genome sequences in the Japanese population reference panel (8.3K JPN) of the Tohoku Medical Megabank Organization. Additionally, four variants, namely p.(Ile369Thr), p.(Phe463Val), p.(Arg500Gln), and p.(Ala526Thr) FMO3, were found in the 8.3K JPN database but were already recorded in the National Center for Biotechnology Information database. Novel FMO3 variants p.[(Met1Leu)] and p.[(Trp231Ter)] were also identified in phenotype–gene analyses of 290 unrelated subjects with self-reported malodor. Among the eight recombinant FMO3 variants tested (except for p.[(Met1Leu)] and p.[(Trp231Ter)]), Arg500Gln and Gly191Asp FMO3, respectively, had lower and much lower capacities for trimethylamine and/or benzydamine N-oxygenation activities than wild-type FMO3. Because another FMO3 mutation p.[(Gly191Cys)] with diminished recombinant protein activity was previously detected in two independent probands, Gly191 would appear to be important for FMO3 catalytic function. Analysis of whole-genome sequence data and trimethylaminuria phenotypes revealed missense FMO3 variants that severely impaired FMO3-mediated N-oxygenations in Japanese subjects that could be susceptible to low drug clearances.     

Phenotyping of flavin-containing monooxygenase using caffeine metabolism and genotyping of FMO3 gene in a Korean population.

Flavin-containing monooxygenase (FMO) activity was determined in 82 Korean volunteers by taking molar concentration ratio of theobromine and caffeine present in the 1 h urine (between 4 and 5 h) samples collected after administration of a cup of coffee containing 110 mg of caffeine. Among 82 volunteers, there were 19 women and 63 men (30 smokers and 52 non-smokers). Volunteers were divided into two groups comprising low (0.53-2.99) and high (3.18-11.95) FMO activities separated by an antimode of 3.18. Peripheral bloods were sampled from these volunteers and their genomic DNAs were amplified by polymerase chain reaction with oligonucleotides designed from intronic sequences of human FMO3 gene. Comparing nucleotide sequences of the amplified FMO3 gene originating from randomly selected individuals with low and high FMO activities, nine point mutations were identified in the open reading frame sequences. Among these nine mutations, three FMO3 mutant types (FMO3/Stop148, Lys158 and Gly308) were selected and correlated with FMO activities observed in our Korean population. A rare FMO3/Stop148 mutant allele originating from FMO3/Gly148 occurred by substitution of G442T in exon 4 and yielded a premature TGA stop codon. The stop codon was detected in one individual having the second lowest FMO activity and he had the mutation in heterozygous state. In a pedigree study, he was found to have inherited the mutation from his mother who also had a heterozygous stop codon and equally low FMO activity. In our volunteers, two other common mutations were detected in exons 4 and 7. The one in exon 4 resulted from a G472A change eliminating a HinfI restriction site and produced an amino acid substitution from Glu158 to Lys. The other mutation in exon 7 resulted from an A923G change generating a DraII restriction site and produced a non-conservative replacement of Glu308 to Gly. Based on the secondary structure maps of FMO3 enzyme proteins for these two mutant types, FMO3/Gly308 mutation transformed the helix structure into a sheet shape and indicated that dysfunctional FMO3 may be produced. FMO3/Lys158 mutation did not alter the secondary structure. Approximately 80% of volunteers with homozygous and/or heterozygous mutations on either one or two of these mutations had low FMO activities. Thus, individuals with these FMO3 gene mutations may have defective metabolic activity for many clinically used drugs and dietary plant alkaloids which are oxidized primarily by hepatic FMO3.