Novel variants of the human flavin-containing monooxygenase 3 (FMO3) gene associated with trimethylaminuria

doi:10.1016/j.ymgme.2009.02.006

Molecular Genetics and Metabolism

Volume 97, Issue 2, June 2009, Pages 128-135

https://doi.org/10.1016/j.ymgme.2009.02.006 Get rights and content

Abstract

The disorder trimethylaminuria (TMAu) often manifests itself in a body odor for individuals affected. TMAu is due to decreased metabolism of dietary-derived trimethylamine (TMA). In a healthy individual, 95% or more of TMA is converted by the flavin-containing monooxygenase 3 (FMO3, EC 1.14.13.8) to non-odorous trimethylamine N-oxide (TMA N-oxide). Several single nucleotide polymorphisms (SNPs) of the FMO3 gene have been described and result in an enzyme with decreased or abolished functional activity for TMA N-oxygenation thus leading to TMAu. Herein, we report two novel mutations observed from phenotyping and genotyping two self-reporting individuals. Sequence analysis of the exon regions of the FMO3 gene of a young woman with severe TMAu revealed heterozygous mutations at positions 187 (V187A), 158 (E158K), 308 (E308G), and 305 (E305X). Familial genetic analysis showed that the E158K/V187A/E308G derived from the same allele from the mother, and the E305X was derived from the father. FMO3 variants V187A and V187A/E158K were characterized for oxygenation of several common FMO3 substrates (i.e., 5- and 8-DPT, mercaptoimidazole (MMI), TMA, and sulindac sulfide) and for its thermal stability. Our findings show that with the combination of V187A/E158K mutations in FMO3, the enzyme activity is severely affected and possibly contributes to the TMAu observed. In another study, genotyping analysis of a 17 year old female revealed a mutation that caused a frame shift after K415 and resulted in a protein variant with only 486 amino acid residues that was associated with severe TMAu.

Introduction

The family of flavin-containing monooxygenases (FMOs, EC 1.14.13.8) represents, after the cytochrome P450s, the most important drug-metabolizing monooxygenase enzymes in adult human liver. FMOs catalyze the oxygenation of various nucleophilic nitrogen-, sulfur-, and phosphorous-containing xenobiotics [1]. In humans, five isoforms (FMO1–5) and 6 pseudogenes (FMO6P–11P) have been described [2], [3], and FMO3 is considered to be the major drug-metabolizing FMO isozyme in adult human liver.

The disorder trimethylaminuria (TMAu) often manifests itself in a body odor for the individual affected. TMAu is caused by the accumulation and excretion of unmetabolized trimethylamine (TMA), a substance derived from foodstuffs including choline. In a healthy individual, 95% or more of TMA is converted by FMO3 to non odorous TMA N-oxide. Two different major forms of TMAu have been described [4]: a primary genetic form that causes decreased FMO3 enzyme function and a secondary form that is due to TMA or a TMA-precursor overload. The two forms could be associated with each other, because individuals with a slightly decreased enzyme activity (primary TMAu) might not exhibit TMAu symptoms until the affected individual is challenged with increased amounts of TMA as a result of diet, liver disease, or bacterial overgrowth (secondary form). In addition, minor forms of TMAu including an acquired TMAu with no obvious genetic background, a transient childhood form, and a transient form in women associated with menstruation have been described [5], [6], [7], [8], [9], [10], [11]. Nevertheless, in general, the majority of TMAu cases reported are associated with single nucleotide polymorphisms (SNPs) of the FMO3 and therefore categorized as the primary genetic form of TMAu. More than 300 SNPs of the human FMO3 have been reported (http://www.ncbi.nlm.nih.gov/projects/SNP/) and over 40 of these polymorphisms have been linked to TMAu due to decreased or abolished TMA N-oxygenation ability of the FMO3 variant [4]. Depending on the FMO3 SNP, the incidence and severity of the disorder varies [12]. By themselves, several common polymorphic variants do not significantly decrease TMA N-oxygenation activity, but in combination with other SNPs may have a more deleterious impact (e.g., the common SNPs E158K and E308G) [6], [13], [14]. For the affected individual, accurate diagnosis of TMAu can relieve concerns and provide an impetus to obtain medical advice on dietary restriction to limit the intake of TMA precursors and thereby decrease the TMAu condition [15]. Diagnosis includes phenotype analysis (i.e., measurement of the urinary percentage of oxygenated TMA (TMA N-oxide) compared with total TMA (TMA plus TMA N-oxide) that in healthy individuals should be ⩾95%), and genotyping of the affected individuals in order to identify FMO3 gene mutations that cause TMAu.

In genotyping studies of two individuals, we identified two novel mutations associated with TMAu. In a 33 year old woman, in addition to the common polymorphisms E158K and E308G, we observed a SNP at position 187 (i.e., V187A) that had not been described to date and a truncation mutation E305X reported previously. Examination of both biological parents showed that the biological mother carried the E158K/V187A/E308G allele, and the biological father carried the E305X allele. While it is known that E305X abolish the FMO3 function, the V187A mutation has not been reported nor previously characterized. In order to characterize this new and unusual variant, we cloned, expressed, and purified the V187A and the V187A/E158K variants of FMO3 as maltose-binding fusion proteins. The triple mutant E158K/V187A/E308G reflecting the genotype of one allele of the affected individual examined was not studied because of the difficulty in expression and characterization of the enzyme. The expressed and purified variants were tested in vitro for the oxygenation of selective functional substrates for the FMO3 enzyme (i.e., 5- and 8-DPT, mercaptoimidazole (MMI), TMA, and sulindac sulfide). The thermal stability of the variant FMO3s were also examined and compared to wild-type enzyme. In a separate study, genotype analysis of a 17-year-old female revealed a mutation that caused a frame shift after K415 and resulted in a variant protein with only 486 amino acid residues.

Section snippets

Reagents

Chemicals and reagents used in this study were purchased from Sigma–Aldrich Chemical Co. (St. Louis, MO) in the highest purity commercially available. Buffers and other reagents were purchased from VWR Scientific, Inc. (San Diego, CA). The synthesis of the phenothiazines 5-DPT and 8-DPT has been previously described [16], [17], [18].

Genomic DNA preparation and polymerase chain reaction (PCR) amplification

Genomic DNA preparation and PCR amplification was done as described before [19]. Briefly, blood samples from individuals with self-reported TMAu symptoms were

Phenotyping and genotyping results

Diagnosis of TMAu included measurement of the urinary ratios of TMA N-oxide to TMA and genotyping of the affected individuals. Genotyping of the 33-year-old female showed several mutations at a number of different loci. One heterozygous missense mutation identified was Val (GTT) to Ala (GCT) at position 187 in exon 4 of the FMO3 gene. This is the first time we have detected this mutation after genotyping over 100 individuals with self-reported body odor. To our knowledge, the V187A mutation has

Discussion

Comprehensive biochemical characterization of recombinant variant FMO3 enzymes based on data from genotype and phenotype analysis of individuals with self-reported symptoms of TMAu can potentially reveal important new information about structure and function of human FMO3 [5], [11], [13], [25], [26], [27], [28]. Findings from the studies herein provide important new information to our understanding of factors contributing to the primary genetic form of TMAu, and identify functionally important

Acknowledgments

The authors thank Rob Reddy for contributing to the TMAu genotyping effort, Anisa Bora for protein purification technical assistance, and Dr. Erik Ralph for helpful discussions. The financial support of NIH Grant DK 59618 is greatly acknowledged.

References (42)

S.C. Mitchell
Trimethylaminuria: susceptibility of heterozygotes
Lancet
(1999)
A.Q. Zhang et al.
Exacerbation of symptoms of fish-odour syndrome during menstruation
Lancet
(1996)
H. Yamazaki et al.
Effects of the dietary supplements, activated charcoal and copper chlorophyllin, on urinary excretion of trimethylamine in Japanese trimethylaminuria patients
Life Sci.
(2004)
B.R. Akerman et al.
Trimethylaminuria is caused by mutations of the FMO3 gene in a North American cohort
Mol. Genet. Metab.
(1999)
J. Zschocke et al.
Mild trimethylaminuria caused by common variants in FMO3 gene
Lancet
(1999)
M.A. Hamman et al.
Stereoselective sulfoxidation of sulindac sulfide by flavin-containing monooxygenases. Comparison of human liver and kidney microsomes and mammalian enzymes
Biochem. Pharmacol.
(2000)
C.K. Yeung et al.
Functional characterization of genetic variants of human FMO3 associated with trimethylaminuria
Arch. Biochem. Biophys.
(2007)
N.B. Beaty et al.
The oxidative half-reaction of liver microsomal FAD-containing monooxygenase
J. Biol. Chem.
(1981)
N.B. Beaty et al.
The reductive half-reaction of liver microsomal FAD-containing monooxygenase
J. Biol. Chem.
(1981)
K.C. Jones et al.
Reactions of the 4a-hydroperoxide of liver microsomal flavin-containing monooxygenase with nucleophilic and electrophilic substrates
J. Biol. Chem.
(1986)

L.L. Poulsen et al.

The liver microsomal FAD-containing monooxygenase. Spectral characterization and kinetic studies

J. Biol. Chem.

(1979)

H. Yamazaki et al.

Stop codon mutations in the flavin-containing monooxygenase 3 (FMO3) gene responsible for trimethylaminuria in a Japanese population

Mol. Genet. Metab.

(2007)

M. Fujieda et al.

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

Drug Metab. Pharmacokinet.

(2003)

D.M. Ziegler et al.

Microsomal oxidase. IV. Properties of a mixed-function amine oxidase isolated from pig liver microsomes

Arch. Biochem. Biophys.

(1972)

D.M. Lambert et al.

In vivo variability of TMA oxidation is partially mediated by polymorphisms of the FMO3 gene

Mol. Genet. Metab.

(2001)

J.R. Cashman et al.

Human flavin-containing monooxygenases

Annu. Rev. Pharmacol. Toxicol.

(2006)

R.N. Hines et al.

Alternative processing of the human FMO6 gene renders transcripts incapable of encoding a functional flavin-containing monooxygenase

Mol. Pharmacol.

(2002)

D. Hernandez et al.

Organization and evolution of the flavin-containing monooxygenase genes of human and mouse: identification of novel gene and pseudogene clusters

Pharmacogenetics

(2004)

M.S. Motika et al.

Flavin-containing monooxygenase 3 and human disease

Exp. Opin. Drug Metab. Toxicol.

(2007)

J.R. Cashman et al.

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

Drug Metab. Dispos.

(2002)

J.R. Cashman et al.

Biochemical and clinical aspects of the human flavin-containing monooxygenase form 3 (FMO3) related to trimethylaminuria

Curr. Drug Metab.

(2003)

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Increasing numbers of single-nucleotide substitutions of the human flavin-containing monooxygenase 3 (FMO3) gene are being recorded in mega-databases. Phenotype–gene analyses revealed impaired FMO3 variants associated with the metabolic disorder trimethylaminuria. Here, a series of reliable FMO3 genotyping confirmation methods was assembled and developed for 45 impaired FMO3 variants, mainly found in Japanese populations, using singleplex or duplex polymerase chain reaction (PCR)–restriction fragment length polymorphism (RFLP) methods and singleplex, duplex, or tetraplex allele-specific PCR methods. Nine PCR-RFLP procedures with single restriction enzymes and fourteen duplex PCR-RFLP procedures (for p.Trp41Ter and p.Thr329Ala, p.Met66Val and p.Leu163Pro, p.Pro70Leu and p.Glu308Gly, p.Asn114Ser and p.Ser195Leu, p.Glu158Lys and p.Ile441Thr, p.Cys197Ter and p.Trp388Ter, p.Arg205Cys and p.Val257Met, p.Arg205His and p.Cys397Ser, p.Met211ArgfsTer10 and p.Arg492Trp, p.Arg223Gln and p.Leu473Pro, p.Met260Val and p.Thr488Ala, p.Tyr269His and p.Ala311Pro, p.Ser310Leu and p.Gly376Glu, and p.Gln470Ter and p.Arg500Ter) were newly established along with eight singleplex (for p.Pro153GlnfsTer14, p.Gly191Cys, p.Pro248Thr, p.Ile486Met, and p.Pro496Ser, among others), one duplex (p.Ile199Ser and p.Asp286Tyr), and one tetraplex (p.Ile7Thr, p.Val58Ile, p.Thr201Lys, and p.Gly421Val) allele-specific PCR systems. This series of systems should facilitate the easy detection in a clinical setting of FMO3 variants in Japanese subjects susceptible to low drug clearances or drug reactions possibly caused by impaired FMO3 function.
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More than 30 different mutations and around 40 polymorphisms of hFMO3 have been reported so far [13]. Many of these have been associated with a metabolic disorder known as trimethylaminuria (TMAU) [14,15]. TMAU or “fish odor syndrome” is a non-life-threatening condition arising from the altered or incomplete expression of the FMO3 isoform.
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.
Monoamine Oxidases and Flavin-Containing Monooxygenases
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Progress in structural biology and related disciplines has advanced our knowledge of the physical and mechanistic properties of flavoproteins. This article will focus on two classes of flavoproteins (i.e., monoamine oxidase A and B (MAO A and B) and the flavin-containing monooxygenases (FMOs)) that are involved in the biotransformation of chemicals, drugs, pesticides, and xenobiotics in both small and large animals. Emphasis will be placed on the human enzymes, but comparison with animal forms will highlight the differences. MAO and FMO enzymes have largely been associated with detoxication of chemicals to more polar and generally less toxic metabolites that are usually efficiently excreted. Sometimes, however, MAOs or FMOs convert less toxic chemicals to more toxic materials. This article will attempt to present a view of the balance between these dual roles of MAO and FMO flavoenzymes. Before a detailed discussion of each enzyme is considered, however, a few general comments about the manner that MAO A and B and the FMOs fit into the overall view of the family of flavoproteins that exist in nature will be presented.
Analysis of six novel flavin-containing monooxygenase 3 (FMO3) gene variants found in a Japanese population suffering from trimethylaminuria
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Decreased or abolished functional activities with respect to trimethylamine N-oxygenation, resulting in trimethylaminuria, are caused mainly by single nucleotide polymorphisms of the FMO3 gene. Such FMO3 polymorphisms have been reported in the literature [5–9]. FMO3 mutations resulting in the amino acid substitutions p.(Glu158Lys), p.(Val257Met), and p.(Glu308Gly) have been reported as common FMO3 gene variants in the International HapMap project (http://www.hapmap.org) in multiple ethnic populations.
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.
Trimethylaminuria (fish odor syndrome): Genotype characterization among Portuguese patients
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Citation Excerpt :
Trimethylaminuria [TMAu; MIM 602079] (Cashman et al., 2001; Christodoulou, 2012; Mitchell and Smith, 2005; Motika et al., 2009; Treacy et al., 1998), also known as “fish odor syndrome”, is an autosomal recessive metabolic disorder (Cashman and Zhang, 2002; Chalmers et al., 2006; Mackay et al., 2011) caused by a defect in the normal production of flavin containing monooxygenase 3 (FMO3; EC 1.14.13.8) enzyme.
Trimethylaminuria (TMAu) or “fish odor syndrome” is a metabolic disorder characterized by the inability to convert malodorous dietarily-derived trimethylamine (TMA) to odorless TMA N-oxide by the flavin-containing monooxygenase 3 (FMO3). Affected individuals unable to complete this reaction exude a “fishy” body odor due to the secretion of TMA in their corporal fluids leading to a variety of psychosocial problems. Interindividual variability in the expression of FMO3 gene may affect drug and foreign chemical metabolism in the liver and other tissues. Therefore, it is important to screen for common TMAu mutations but also extend the search to other genetic variants in order to correlate genotype and disease-associated phenotypes.
In this study, 25 Portuguese patients with phenotype suggestive of TMAu were evaluated for molecular screening of the FMO3 gene. Herein, we found 16 variants in the FMO3 coding region, some of which had not been previously documented (Gly38Trp, Asp232Val, Thr307Pro, Ser310Leu). Whenever common variants (Glu158Lys, Glu308Gly) were considered in combination a distinct pattern between the control population and patients was observed, mainly in what concerns the presence of Lys158 and Gly308 in homozygous state.
Further studies are necessary to clarify the pathogenicity of novel variants identified in this study, as well as the effect of the common single nucleotide polymorphisms, which may play an important role in disease presentation and/or protective mechanism to xenobiotics drugs or environment.
Survey of variants of human flavin-containing monooxygenase 3 (FMO3) and their drug oxidation activities
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Human flavin-containing monooxygenase 3 (FMO3, EC 1.14.13.8) in liver catalyzes a variety of oxygenations of nitrogen- and sulfur-containing medicines and xenobiotic substances. Loss-of-function mutations of the FMO3 gene, the enzyme responsible for trimethylamine N-oxygenation, cause the inherited disorder trimethylaminuria (also known as fish odor syndrome). In this mini-review, mutations of the FMO3 gene reported in the literature and in the National Center for Biotechnology Information single nucleotide polymorphism database were surveyed. Then, the activities of FMO3 variants in human liver microsomes and the activities of recombinantly expressed FMO3 variant proteins with respect to the oxygenation of nitrogen- and sulfur-containing drugs were summarized and the potential for drug interactions was demonstrated. Individual differences in FMO3 function were seen in subjects genotyped for homozygous FMO3 variants. Specific regions of the FMO3 C-terminus are required for functional activity. Naturally truncated FMO3 is believed to have barely detectable function, thereby explaining the relationship with severe impaired phenotypes. The present article provides fundamental, up-to-date information on the importance of human FMO3 in individual xenobiotic oxygenations, including those of new medicines and dietary-derived trimethylamine.

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Novel variants of the human flavin-containing monooxygenase 3 (FMO3) gene associated with trimethylaminuria

Abstract

Introduction

Section snippets

Reagents

Genomic DNA preparation and polymerase chain reaction (PCR) amplification

Phenotyping and genotyping results

Discussion

Acknowledgments

Lancet

Lancet

Life Sci.

Mol. Genet. Metab.

Lancet

Biochem. Pharmacol.

Arch. Biochem. Biophys.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Mol. Genet. Metab.

Drug Metab. Pharmacokinet.

Arch. Biochem. Biophys.

Mol. Genet. Metab.

Human flavin-containing monooxygenases

Annu. Rev. Pharmacol. Toxicol.

Alternative processing of the human FMO6 gene renders transcripts incapable of encoding a functional flavin-containing monooxygenase

Mol. Pharmacol.

Organization and evolution of the flavin-containing monooxygenase genes of human and mouse: identification of novel gene and pseudogene clusters

Pharmacogenetics

Flavin-containing monooxygenase 3 and human disease

Exp. Opin. Drug Metab. Toxicol.

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

Drug Metab. Dispos.

Biochemical and clinical aspects of the human flavin-containing monooxygenase form 3 (FMO3) related to trimethylaminuria

Curr. Drug Metab.