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.

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.