Identification and Functional Analysis of Common Human Flavin-Containing Monooxygenase 3 Genetic Variants

Abstract

Flavin-containing monooxygenases (FMOs) are important for the disposition of many therapeutics, environmental toxicants, and nutrients. FMO3, the major adult hepatic FMO enzyme, exhibits significant interindividual variation. Eighteen FMO3 single-nucleotide polymorphism (SNP) frequencies were determined in 202 Hispanics (Mexican descent), 201 African Americans, and 200 non-Latino whites. Using expressed recombinant enzyme with methimazole, trimethylamine, sulindac, and ethylenethiourea, the novel structural variants FMO3 E24D and K416N were shown to cause modest changes in catalytic efficiency, whereas a third novel variant, FMO3 N61K, was essentially devoid of activity. The latter variant was present at an allelic frequency of 5.2% in non-Latino whites and 3.5% in African Americans, but it was absent in Hispanics. Inferring haplotypes using PHASE, version 2.1, the greatest haplotype diversity was observed in African Americans followed by nonLatino whites and Hispanics. Haplotype 2A and 2B, consisting of a hypermorphic promoter SNP cluster (-2650CϾG, -2543TϾA, and -2177GϾC) in linkage with synonymous structural variants was inferred at a frequency of 27% in the Hispanic population, but only 5% in non-Latino whites and African Americans. This same promoter SNP cluster in linkage with one or more hypomorphic structural variant also was inferred in multiple haplotypes at a total frequency of 5.6% in the AfricanAmerican study group but less than 1% in the other two groups. The sum frequencies of the hypomorphic haplotypes H3 [15,167GϾA (E158K)], H5B [-2650CϾG, 15,167GϾA (E158K), 21,375CϾT (N285N), 21,443AϾG (E308G)], and H6 [15,167GϾA (E158K), 21,375CϾT (N285N)] was 28% in Hispanics, 23% in non-Latino whites, and 24% in African Americans.The flavin-containing monooxygenases (FMOs; EC 1.14.13.8) are a family of microsomal enzymes that catalyze the NADPH-dependent N-and S-oxidation of a variety of therapeutics, environmental toxicants, carcinogens, and nutrients (Krueger and Williams, 2005). The FMO multigene family consists of a five-gene cluster at 1q24.3 (FMO1-4 and FMO6p), a second cluster of five genes at 1q24.2 (FMO7p-11p), and a single gene, FMO5, at 1q21.1, encoding a total of five active proteins in the human (Hernandez et al., 2004).The most recent common precursor for all placental mammals is predicted to have already had a cluster containing FMO1-6P and a separate FMO5 locus that arose from duplication of an ancestral gene approximately 210 to 275 million years ago (Hernandez et al., 2004). Despite the antiquity of the FMO gene family, a sequence comparison among modern FMO enzymes reveals 76 to 86% sequence identity between orthologous proteins, suggesting these genes have been highly conserved.The individual FMO enzymes exhibit broad but distinct substrate specificities (Krueger and Williams, 2005) as well as species-, sex-, tissue-, and age-dependent differences in expression patterns (for review, see Hines, 2006). In the human, FMO3 is the predominant adult hepatic enzyme with a specific conten...

Flavin-containing monooxygenases: mutations, disease and drug response

“…If the increased activity observed in vitro is reflected in vivo, individuals with this haplotype, particularly homozygotes, would be expected to metabolize FMO3 substrates more rapidly, leading to a potential reduction in drug efficacy. Although this haplotype is present in relatively high frequency, it is often associated with the linked variant E158K/E308G [39]. In this case an increase in the amount of protein produced, via promoter SNPs, might compensate for an enzyme with lower activity due to coding SNPs.…”

“…Individually, most of the SNPs have little or no effect on enzyme activity [1][2][3][4]38]. Three, however, do affect activity: g.11177C>A(N61K) causes a dramatic decrease in enzyme activity towards four different substrates [39], g.21599T>C(L360P) increases catalytic activity 2-to 5-fold [40] and is the only SNP known to increase FMO3 activity and g.15550C>T(R205C) has a moderate effect on enzyme activity but, interestingly, exhibits substrate inhibition of sulindac sulfide Soxygenation [41]. However, each of the three SNPs is present in low frequency and/or is restricted to single population groups [dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP)].…”

“…However, although N61S abolishes TMA N-oxygenation, it does not affect S-oxygenation of methimazole [76]. N61K, a polymorphic variant that severely impairs enzyme activity [39] and, thus, is predicted to cause TMAuria, has not been identified in affected individuals and, therefore, is not included in the figure. Adverse drug reactions or increased drug efficacy amphetamine [68,69] benzydamine [22] ethionamide [1] clozapine [70] deprenyl [69] metamphetamine [68,69] tamoxifen [71] thiacetazone [30] sulindac sulfide [53] Various [72] imipramine [72] itopride [73] olopatidine [74] tamoxifen [71] thiacetazone [30] Promoter SNPs Decrease expression [66] 1…”

In silico analysis of mutations occurring in the protein N-acetylgalactosamine-6-sulfatase (GALNS) and causing mucopolysaccharidosis IVA

“…Protein stability is a key feature to protein function (Ye et al, 2006;Zhang et al, 2012), and a missense mutation may affect, besides the secondary structure, its stability, leading to misfolding (Dobson, 2003;Koukouritaki et al, 2007;Zhang et al, 2012). Our findings showed that, unexpectedly, even in missense mutations, without ID regions, when there are no changes in 19.4% of the secondary structures and in 25.6% of the chemical characteristics of the amino acids, the function of GALNS is still suppressed in patients with MPS IVA.…”

Clinical utility gene card for: Trimethylaminuria

“…7 Of these, only one, p.Asn61Lys, which occurs at low frequency, results in a severe reduction of enzyme activity. 8 However, some polymorphic variants (eg, p.Glu158Lys and p.Glu308Gly) when present in cis can cause a moderate decrease in enzyme activity. 7,9 When present in the homozygous state, they may cause mild or transient trimethylaminuria, particularly in infants and young children, 10,11 who have low expression of FMO3.…”