Rare but impaired flavin-containing monooxygenase 3 (FMO3) variants reported in a recently updated Japanese mega-databank of genome resources.

Genetic variants of human flavin-containing monooxygenase 3 (FMO3) were investigated using an updated Japanese population panel containing 54,000 subjects (the previous panel contained 38,000 subjects). One stop codon mutation and six amino acid-substituted FMO3 variants were newly identified in the updated databank. Of these, two substituted variants (p.Thr329Ala and p.Arg492Trp) were previously identified in compound haplotypes with p.[(Glu158Lys; Glu308Gly)] and were associated with the metabolic disorder trimethylaminuria. Three recombinant FMO3 protein variants (p.Ser137Leu, p.Ala334Val, and p.Ile426Val) expressed in bacterial membranes had similar activities toward trimethylamine N-oxygenation (∼75-125 %) as wild-type FMO3 (117 min-1); however, the recombinant novel FMO3 variant Phe313Ile showed moderately decreased FMO3 catalytic activity (∼20 % of wild-type). Because of the known deleterious effects of FMO3 C-terminal stop codons, the novel truncated FMO3 Gly184Ter variant was suspected to be inactive. To easily identify the four impaired FMO3 variants (one stop codon mutation and three amino-acid substitutions) in the clinical setting, simple confirmation methods for these FMO3 variants are proposed using polymerase chain reaction/restriction fragment length polymorphism or allele-specific PCR methods. The updated whole-genome sequence data and kinetic analyses revealed that four of the seven single-nucleotide nonsense or missense FMO3 variants had moderately or severely impaired activity toward trimethylamine N-oxygenation.

Molecular and functional characterization of flavin-containing monooxygenases (FMO1-6) in tree shrews.

Flavin-containing monooxygenases (FMOs) are a family of important drug oxygenation enzymes that, in humans, consist of five functional enzymes (FMO1-5) and a pseudogene (FMO6P). The tree shrew is a non-rodent primate-like species that is used in various biomedical studies, but its usefulness in drug metabolism research has not yet been investigated. In this study, tree shrew FMO1-6 cDNAs were isolated and characterized by sequence analysis, tissue expression, and metabolic function. Compared with human FMOs, tree shrew FMOs showed sequence identities of 85-90 % and 81-89 %, respectively, for cDNA and amino acids. Phylogenetic analysis showed that each tree shrew and human FMO were closely clustered. The genomic and genetic structures of the FMO genes were conserved in tree shrews and humans. Among the five tissue types analyzed (lung, heart, kidney, small intestine, and liver), FMO3 and FMO1 mRNAs were most abundant in liver and kidney, respectively. Recombinant tree shrew FMO1-6 proteins expressed in bacterial membranes all mediated benzydamine and trimethylamine N-oxygenations and methyl p-tolyl sulfide S-oxygenation. The selective human FMO3 substrate trimethylamine was predominantly metabolized by tree shrew FMO3. Additionally, tree shrew FMO6 was active toward trimethylamine, as is cynomolgus macaque FMO6, in contrast with the absence of activity of the human FMO6P pseudogene product. Tree shrew FMO1-6, which are orthologous to human FMOs (FMO1-5 and FMO6P) were identified, and tree shrew FMO3 has functional and molecular features generally comparable to those of human FMO3 as the predominant FMO in liver.

Simple confirmation methods for rare but impaired variants of human flavin-containing monooxygenase 3 (FMO3) found in an updated genome resource databank.

Forty-seven new nonsense or missense human flavin-containing monooxygenase 3 (FMO3) variants were recently identified in an updated Japanese population reference panel. Of these, 20 rare single-nucleotide substitutions resulted in moderately or severely impaired FMO3 activity. To easily identify these 20 FMO3 variants (2 stop codon mutations, 2 frameshifts, and 16 amino-acid substitutions) in the clinical setting, simple confirmation methods for impaired FMO3 variants are proposed using polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) or allele-specific PCR methods. Using PCR-RFLP, FMO3 variants p.Arg51Gly, p.Met66Lys, p.Asn80Lys, p.Val151Glu, p.Val187fsTer25, p.Gly193Arg, p.Val283Ala, p.Asp286His, p.Val382Ala, and p.Phe451Leu were digested by the designated restriction enzymes and confirmed using reference cDNAs. In contrast, the FMO3 variants p.Gly39Val, p.Arg238Ter, p.Arg387Cys, p.Arg387His, p.Leu457Trp, and p.Met497Arg were not digested, whereas the wild type was digested. FMO3 variants p.Gly11Asp, p.Lys416fsTer72, p.Gln427Ter, and p.Thr453Pro were confirmed using allele-specific PCR systems. The previously identified FMO3 p.Arg500Ter variant has a relatively high frequency and was differentiated from p.Arg500Gln in two steps, i.e., enzyme restriction followed by allele-specific PCR, similar to the method for p.Arg387Cys and p.Arg387His. These systems should facilitate easy detection in the clinical setting of FMO3 variants in Japanese subjects susceptible to low drug clearance possibly caused by impaired FMO3 function.

Synovial fluid and plasma concentrations of tedizolid in patients with osteoarthritis infected with Staphylococcus aureus effectively determined with fluorescence detection.

BACKGROUND: Tedizolid is a new oxazolidinone antibiotic with high potency for the treatment of infections caused by methicillin-resistant Staphylococcus aureus and other species. CASE PRESENTATION: Two patients with osteoarthritis (women aged 79 and 73 years, cases 1 and 2, respectively) infected with S. aureus were successfully treated with tedizolid after administration of 200 mg once daily via intravenous infusion. The synovial fluid and plasma concentrations of tedizolid during surgery in case 1 at day 7 were 2.1 and 1.6 µg/mL, respectively, yielding a ratio of synovial fluid/plasma of 130%. Those in case 2 at day 2 were 2.9 and 3.3 µg/mL, respectively, corresponding to a ratio of synovial fluid/plasma of 88%. CONCLUSIONS: These results imply very similar concentrations of tedizolid in the synovial fluid and plasma of osteoarthritis patients with acute S. aureus infection.

Variants of Flavin-Containing Monooxygenase 3 Found in Subjects in an Updated Database of Genome Resources.

Single-nucleotide substitutions of human flavin-containing monooxygenase 3 (FMO3) identified in the whole-genome sequences of the updated Japanese population reference panel (now containing 38,000 subjects) were investigated. In this study, two stop codon mutations, two frameshifts, and 43 amino-acid-substituted FMO3 variants were identified. Among these 47 variants, one stop codon mutation, one frameshift, and 24 substituted variants were already recorded in the National Center for Biotechnology Information database. Functionally impaired FMO3 variants are known to be associated with the metabolic disorder trimethylaminuria; consequently, the enzymatic activities of the 43 substituted FMO3 variants were investigated. Twenty-seven recombinant FMO3 variants expressed in bacterial membranes had similar activities toward trimethylamine N-oxygenation (∼75%-125%) to that of wild-type FMO3 (98 minutes-1). However, six recombinant FMO3 variants (Arg51Gly, Val283Ala, Asp286His, Val382Ala, Arg387His, and Phe451Leu) had moderately decreased (∼50%) activities toward trimethylamine N-oxygenation, and 10 recombinant FMO3 variants (Gly11Asp, Gly39Val, Met66Lys, Asn80Lys, Val151Glu, Gly193Arg, Arg387Cys, Thr453Pro, Leu457Trp, and Met497Arg) showed severely decreased FMO3 catalytic activity (<10%). Because of the known deleterious effects of FMO3 C-terminal stop codons, the four truncated FMO3 variants (Val187SerfsTer25, Arg238Ter, Lys416SerfsTer72, and Gln427Ter) were suspected to be inactive with respect to trimethylamine N-oxygenation. The FMO3 p.Gly11Asp and p.Gly193Arg variants were located within the conserved sequences of flavin adenine dinucleotide (positions 9-14) and NADPH (positions 191-196) binding sites, which are important for FMO3 catalytic function. Whole-genome sequence data and kinetic analyses revealed that 20 of the 47 nonsense or missense FMO3 variants had moderately or severely impaired activity toward N-oxygenation of trimethylaminuria. SIGNIFICANCE STATEMENT: The number of single-nucleotide substitutions in human flavin-containing monooxygenase 3 (FMO3) recorded in the expanded Japanese population reference panel database was updated. One stop mutation, FMO3 p.Gln427Ter; one frameshift (p.Lys416SerfsTer72); and 19 novel amino-acid-substituted FMO3 variants were identified, along with p.Arg238Ter, p.Val187SerfsTer25, and 24 amino-acid-substituted variants already recorded with reference SNP (rs) numbers. Recombinant FMO3 Gly11Asp, Gly39Val, Met66Lys, Asn80Lys, Val151Glu, Gly193Arg, Arg387Cys, Thr453Pro, Leu457Trp, and Met497Arg variants showed severely decreased FMO3 catalytic activity, possibly associated with the trimethylaminuria.

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.

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.

Plasma and synovial fluid concentrations of linezolid in patients with knee osteoarthritis infected with Staphylococcus aureus.

BACKGROUND: Linezolid is a new oxazolidinone antibiotic used for infections caused by methicillin-resistant Staphylococcus and other species. CASE PRESENTATION: Two cases of knee osteoarthritis with acute infection were successfully treated using linezolid. The plasma and synovial fluid concentrations of linezolid in two patients [women aged 69 and 73 years (cases 1 and 2)] with knee osteoarthritis infected with Staphylococcus aureus were measured after they were administered 600 mg twice daily by intravenous infusion. The plasma linezolid concentrations during knee surgery in case 1 at day 5 and in case 2 at day 2 were 19.6 and 15.6 µg/mL, respectively. The synovial fluid concentrations of linezolid in samples taken during surgery in case 1 and case 2 were 14.9 and 17.0 µg/mL, respectively; these values corresponded to ratios of synovial fluid/plasma of 76 and 109%. Possible metabolite 2-hydroxylated linezolid potentially mediated by cytochrome P450 2 J2 was not detected in the plasma or synovial fluid samples under the current clinical setting after multiple doses. CONCLUSIONS: These results implied nearly equivalent concentrations of linezolid in plasma and synovial fluid of clinical patients with knee osteoarthritis acutely infected with Staphylococcus aureus.

Molecular and functional characterization of flavin-containing monooxygenases in pigs, dogs, and cats.

Flavin-containing monooxygenases (FMOs) are drug-oxygenating enzymes that are present in the human genome as FMO1-5 and FMO6P. Among pig, dog, and cat FMOs, pig and dog FMO1 and FMO3 have been partly characterized, but other FMOs have not been systematically identified. In this study, orthologous FMO cDNAs were isolated from pig, dog, and cat livers and evaluated by sequence and phylogenetic analyses, tissue expression, and catalytic function. The amino acid sequences of pig, dog, and cat FMO1-5 shared high sequence identities (83-89%) with human FMO1-5 and were closely clustered in a phylogenetic tree. The gene structure and genomic organization of FMO1-5 were conserved across these species. Dog and pig FMO6P contained insertions of 1 and 83 bases, respectively, and are possibly pseudogenes similar to human FMO6P. Among the tissue types analyzed, pig FMO1 mRNA was abundant in liver, kidney, and lung; dog FMO3, FMO2, and FMO5 mRNAs were abundant in liver, lung, and kidney, respectively; cat FMO1 and FMO3 mRNAs were abundant in kidney and liver, respectively. Recombinant pig and dog FMO1-5 and cat FMO1-6 all mediated benzydamine and trimethylamine N-oxygenations and methyl p-tolyl sulfoxide S-oxygenation. The selective human FMO3 substrate trimethylamine was predominantly metabolized by pig FMO1, dog FMO3, and cat FMO3. Cat FMO6 was also active toward trimethylamine. These results suggest some similarities in the drug-metabolizing capabilities of FMO3 in dogs, cats, and humans and that dog and cat FMO3 generally have molecular and functional characteristics similar to human FMO3, being the major FMO in human liver.