CYP2A13 genetic polymorphism in French Caucasian, Gabonese and Tunisian populations

Since human CYP2A13 is expressed in the respiratory tract and is involved in the activation of tobacco-specific nitrosamines, some of the previously reported sequence variations may contribute to inter-individual and inter-ethnic differences in the susceptibility of tobacco-related tumorigenesis. The aim was to compare the frequencies of the 578C > T (Arg101Stop), 3375C > T (Arg257Cys) and 7520C > G (3'-untranslated region) mutations in several populations. The frequencies of the 578C > T polymorphism were 3.8, 0 and 1.0% in French Caucasians, Gabonese and Tunisians, respectively. In the same populations, the frequencies of the 3375C > T mutation were 0, 15.3 and 4.2%, respectively, whereas the frequencies of the 7520C > G mutation were 1.0, 20.8 and 7.3%, respectively. Marked inter-ethnic variations in CYP2A13 were identified and confirmed. These findings provide data for further studies that associate CYP2A13 haplotypes with an incidence of smoking-related tumours in respect of ethnicity.     

Eighteen novel polymorphisms of the CYP2A13 gene in Japanese

We sequenced all nine exons, exon-intron junctions including a part of introns, 5'-flanking and 3'-untranslated regions of the cytochrome P450 (CYP) 2A13 gene from 192 Japanese individuals. We found eighteen novel genetic polymorphisms including five single nucleotide polymorphisms (SNP) and one three base pair insertion causing amino acid substitution and one amino acid insertion, respectively, one silent SNP in exon 4, four SNPs in a 5'-flanking region, and seven SNPs in introns. The five SNPs (74G>A in exon 1, 579G>A in exon 2, 1706C>G in exon 3, and 7343T>A and 7465C>T in exon 9) causing amino acid substitutions (Arg(25)Gln, Arg(101)Gln, Asp(158)Glu, Phe(453)Tyr, and Arg(494)Cys), respectively. The one three base pair insertion (1634_1635 ACC insertion in exon 3) caused one amino acid insertion ((133_134)Thr ins). These sequences are as follows:SNP, 021125Fujieda005; GENE NAME, CYP2A13; ACCESSION NUMBER, NG_000008; LENGTH, 25 base;5'-TGTCAGTCTGGCG/AGCAGAGGAAGAG-3'.SNP, 021125Fujieda007; GENE NAME, CYP2A13; ACCESSION NUMBER, NG_000008; LENGTH, 25 base; 5'-AGTTCAGCGGGCG/AAGGCGAGCAGGC-3'.SNP, 021125Fujieda009; GENE NAME, CYP2A13; ACCESSION NUMBER, NG_000008; LENGTH, 25 base; 5'-CTTCCTCATCGAC/GGCCCTCCGGGGC-3'.SNP, 021125Fujieda017; GENE NAME, CYP2A13; ACCESSION NUMBER, NG_000008; LENGTH, 25 base; 5'-TCTTTCTCTTCTT/ACACCACCATCAT-3'.SNP, 021125Fujieda018; GENE NAME, CYP2A13; ACCESSION NUMBER, NG_000008; LENGTH, 25 base; 5'-AGCTTCCTGCCCC/TGCTGAGCGAGGG-3'.SNP, 021125Fujieda008; GENE NAME, CYP2A13; ACCESSION NUMBER, NG_000008; LENGTH, 25 base; 5'-CTCCATCGCCACC-/ACCCTAAGGGGTTTT-3'.     

Extensive genetic polymorphism in the human CYP2B6 gene with impact on expression and function in human liver

The human cytochrome P450, CYP2B6, is involved in the metabolism of several therapeutically important drugs and environmental or abused toxicants. In this study, we present the first systematic investigation of genetic polymorphism in the CYP2B6 gene on chromosome 19. A specific direct sequencing strategy was developed based on CYP2B6 and CYP2B7 genomic sequence information and DNA from 35 subjects was completely analysed for mutations throughout all nine exons and their exon-intron boundaries. A total of nine novel point mutations were identified, of which five result in amino acid substitutions in exon 1 (C64T, Arg22Cys), exon 4 (G516T, Gln172His), exon 5 (C777A, Ser259Arg and A785G, Lys262Arg) and exon 9 (C1459T, Arg487Cys) and four are silent mutations (C78T, G216C, G714A and C732T). Polymerase chain reaction-restriction fragment length polymorphism tests were developed to detect each of the five nonsynonymous mutations in genomic DNA. By screening a population of 215 subjects the C64T, G516T, C777A, A785G and C1459T mutations were found at frequencies of 5.3%, 28.6%, 0.5%, 32.6% and 14.0%, respectively. Haplotype analysis revealed six different mutant alleles termed CYP2B6*2 (C64T), *3 (C777A), *4 (A785G), *5 (C1459T), *6 (G516T and A785G) and *7 (G516T, A785G and C1459T). By analysing a large number of human liver samples, significantly reduced CYP2B6 protein expression and S-mephenytoin N-demethylase activity were found in carriers of the C1459T (R487C) mutation (alleles *5 and *7). These data demonstrate that the extensive interindividual variability of CYP2B6 expression and function is not only due to regulatory phenomena, but also caused by a common genetic polymorphism.     

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'.     

CYP2A13 genetic polymorphism in French Caucasian,Gabonese and Tunisian populations

Since human CYP2A13 is expressed in the respiratory tract and is involved in the activation of tobacco-specific nitrosamines, some of the previously reported sequence variations may contribute to inter-individual and inter-ethnic differences in the susceptibility of tobacco-related tumorigenesis. The aim was to compare the frequencies of the 578C?>?T (Arg¹⁰¹Stop), 3375C?>?T (Arg²⁵⁷Cys) and 7520C?>?G (3′-untranslated region) mutations in several populations. The frequencies of the 578C?>?T polymorphism were 3.8, 0 and 1.0% in French Caucasians, Gabonese and Tunisians, respectively. In the same populations, the frequencies of the 3375C?>?T mutation were 0, 15.3 and 4.2%, respectively, whereas the frequencies of the 7520C?>?G mutation were 1.0, 20.8 and 7.3%, respectively. Marked inter-ethnic variations in CYP2A13 were identified and confirmed. These findings provide data for further studies that associate CYP2A13 haplotypes with an incidence of smoking-related tumours in respect of ethnicity.     

The missense genetic polymorphisms of human CYP2A13: functional significance in carcinogen activation and identification of a null allelic variant.

Cytochrome P450 2A13 (CYP2A13), an enzyme predominantly expressed in human respiratory tissues, is highly efficient for the metabolic activation of two suspected human lung carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and aflatoxin B1 (AFB1). Functional genetic polymorphisms of CYP2A13 may therefore be an important factor in human susceptibility to related lung cancers. Among the reported CYP2A13 polymorphisms with missense variations, only CYP2A13*2 variant (containing either a single or double variation of R25Q and R257C) was studied for its NNK-metabolizing activity. The present study demonstrated that there was no remarkable difference in AFB1- and NNK-induced toxicity between the Flp-In Chinese Hamster Ovary (CHO) cells stably expressing wild-type CYP2A13 and the cells expressing the individual polymorphic variants R25Q, D158E, R257C, R25Q/R257C, V323L, F453Y, and R494C. In contrast, cells transfected with R101Q variant complementary DNA (cDNA), same as the vector control cells, showed no significant death even at highest concentrations of AFB1 (10microM) and NNK (200microM). This result correlated with the lack of CYP2A13 protein in the R101Q-CHO cells, although the genomic integration of transfected R101Q cDNA and the expression of R101Q messenger RNA were clearly demonstrated in these stable transfectants. Consistent with the possibility that the variation might reduce the protein stability, R101Q variant protein expressed in insect cells showed a loss of P450 peak and coumarin 7-hydroxylase activity as well as an increased susceptibility to limited protein digestion. Thus, the R101Q polymorphic change results in a null allelic variant of CYP2A13. Our results should be useful in designing and interpreting molecular epidemiological studies related to CYP2A13 genetic polymorphisms.     

Four novel defective alleles and comprehensive haplotype analysis of CYP2C9 in Japanese

Genetic variations in cytochrome P450 2C9 (CYP2C9) are known to contribute to interindividual and interethnic variability in response to clinical drugs such as warfarin. In the present study, CYP2C9 from 263 Japanese subjects was resequenced, resulting in the discovery of 62 variations including 32 novel ones. In addition to the two known non-synonymous single nucleotide polymorphisms (SNPs), Ile359Leu (*3; allele frequency=0.030) and Leu90Pro (*13; 0.002), seven novel non-synonymous SNPs, Leu17Ile (0.002), Lys118ArgfsX9 (*25; 0.002), Thr130Arg (*26; 0.002), Arg150Leu (*27; 0.004), Gln214Leu (*28; 0.002), Pro279Thr (*29; 0.002) and Ala477Thr (*30; 0.002), were found. Functional characterization of novel alleles using a mammalian cell expression system in vitro revealed that *25 was a null allele and that *26, *28 and *30 were defective alleles. The *26 product showed a 90% decrease in the Vmax value but little change in the Km value towards diclofenac. Both *28 and *30 products showed two-fold higher Km values and three-fold lower Vmax values than the *1 allele, suggesting the importance of Gln214 and Ala477 for substrate recognition. Linkage disequilibrium and haplotype analyses were performed using the detected variations. Only five haplotypes (frequency >0.02) accounted for most (>87%) of the inferred haplotypes, and they were closely associated with the haplotypes of CYP2C19 in Japanese. Although the haplotype structure of CYP2C9 was rather simple in Japanese, the haplotype distribution was quite different from those previously reported in Caucasians and Africans. Taken together, novel defective alleles and detailed haplotype structures would be useful for determining metabolic phenotypes of CYP2C9 substrate drugs in Japanese and probably Asians.     

Analysis of CYP2A contributions to metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in human peripheral lung microsomes.

The objectives of this study were to determine the contributions of CYP2A13 and CYP2A6 to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) metabolism in human peripheral lung microsomes and to determine the influence of the genetic polymorphism, CYP2A13 Arg257Cys, on NNK metabolism. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), the keto-reduced metabolite of NNK, was the major metabolite produced, ranging from 0.28 to 0.9%/mg protein/min. Based on total bioactivation of NNK and NNAL by alpha-carbon hydroxylation, subjects could be classified as either high (17 subjects) or low (12 subjects) bioactivators [(5.26 +/- 1.23) x 10(-2) and (6.49 +/- 5.90) x 10(-3)% total alpha-hydroxylation/mg protein/min, P < 0.05]. Similarly, for detoxification, subjects could be grouped into high (9 subjects) and low (20 subjects) categories [(2.03 +/- 1.65) x 10(-3) and (2.50 +/- 3.04) x 10(-4)% total N-oxidation/mg protein/min, P < 0.05]. When examining data from all individuals, no significant correlations were found between levels of CYP2A mRNA, CYP2A enzyme activity, or CYP2A immunoinhibition and the degree of total NNK bioactivation or detoxification (P > 0.05). However, subgroups of individuals were identified for whom CYP2A13 mRNA correlated with total NNK and NNAL alpha-hydroxylation and NNAL-N-oxide formation (P < 0.05). The degree of NNAL formation and CYP2A13 mRNA was also correlated (P < 0.05). Subjects (n = 84) were genotyped for the CYP2A13 Arg257Cys polymorphism, and NNK metabolism for the one variant (Arg/Cys) was similar to that for other subjects. Although results do not support CYP2A13 or CYP2A6 as predominant contributors to NNK bioactivation and detoxification in peripheral lung of all individuals, CYP2A13 may be important in some.     

Novel mutations of CYP3A4 in Chinese.

Human cytochrome P450 3A4 is a major P450 enzyme in the liver and gastrointestinal tract. It plays important roles in the metabolism of a wide variety of drugs, some endogenous steroids, and harmful environmental contaminants. CYP3A4 exhibits a remarkable interindividual activity variation as high as 20-fold. To investigate whether the interindividual variation in CYP3A4 levels can be partly explained by genetic polymorphism, we analyzed DNA samples from 102 Chinese subjects by polymerase chain reaction (PCR)-single-strand conformation polymorphism analysis for novel point mutation in the CYP3A4 coding sequence and promoter region. Using PCR and directed sequencing method to establish the complete intron sequence of CYP3A4 from leukocytes, the complete genomic sequence from exon 1 through 13 of CYP3A4 was determined and published in the GenBank database (accession no. AF209389). CYP3A4-specific primers were designed accordingly. After PCR-single-strand conformation polymorphism and restriction fragment length polymorphism screening, we found three novel mutations; two are point mutations and one is insertion. The first variant allele (CYP3A4*4), an Ile118Val change, was found in 3 of 102 Chinese subjects. The next allele (CYP3A4*5), which causes a Pro218Arg amino acid change, was found in 2 of 102 subjects. We found an insertion in A(17776), designated as CYP3A4*6, which causes frame shift and an early stop codon in exon 9, in one heterozygous subject. We also investigated the CYP3A4 activity in these mutant subjects by measuring the morning spot urinary 6beta-hydroxycortisol to free cortisol ratio with the enzyme-linked immunosorbent assay method. When compared with healthy Chinese population data, the 6beta-hydroxycortisol to free cortisol ratio data suggested that these alleles (CYP3A4*4, CYP3A4*5, and CYP3A4*6) may decrease the CYP3A4 activity. Incidences of these mutations in Chinese subjects are rare. The prevalence of these point mutations in other ethnic groups and its effect on the metabolic activity of CYP3A4 remain to be further evaluated.     

Twenty one novel single nucleotide polymorphisms (SNPs) of the CYP2A6 gene in Japanese and Caucasians

We sequenced all nine exons and exon-intron junctions of the CYP2A6 gene from 33 Japanese and 28 Caucasians. We found twenty one single nucleotide polymorphisms (SNPs) including four SNPs causing amino acid substitutions, one silent SNP in exon 5, one SNP in a 5'-flanking region, four SNPs in a 3'-untranslated region, and eleven SNPs in introns. The four mutations (13G>A and 86G>A in exon 1, and 2134A>G and 2161C>T in exon 4) causing amino acid substitutions (Gly(5)Arg, Ser(29)Asn, Lys(194)Glu, and Arg(203)Ser), respectively, were as follows: SNP, 020719Kiyotani004; GENE NAME, CYP2A6; ACCESSION NUMBER, NG_000008.4; LENGTH, 25 base; 5'-ATGCTGGCCTCAG/AGGATGCTTCTGG-3'. SNP, 020719Kiyotani005; GENE NAME, CYP2A6; ACCESSION NUMBER, NG_000008.4; LENGTH, 25 base; 5'-AGCAGAGGAAGAG/ACAAGGGGAAGCT-3'. SNP, 020719Kiyotani011; GENE NAME, CYP2A6; ACCESSION NUMBER, NG_000008.4; LENGTH, 25 base; 5'-CGCTTTGACTATA/GAGGACAAAGAGT-3'. SNP, 020719Kiyotani012; GENE NAME, CYP2A6; ACCESSION NUMBER, NG_000008.4; LENGTH, 25 base; 5'-CTGTCACTGTTGC/TGCATGATGCTAG-3'. New alleles having these SNPs were designated as CYP2A6( *)13-CYP2A6( *)16.     

Genetic polymorphisms in KCNQ1, HERG, KCNE1 and KCNE2 genes in the Chinese, Malay and Indian populations of Singapore

AIMS: To determine the genetic variability of long QT syndrome (LQTS)-associated genes (KCNQ1, HERG, KCNE1 and KCNE2) among three distinct ethnic groups in the Singapore population. METHODS: Genomic DNA samples from up to 265 normal healthy Chinese, 118 Malay and 139 Indian volunteer subjects were screened for genetic variations in the coding region of the LQTS-associated genes using denaturing high-performance liquid chromatography and sequencing analyses. RESULTS: In total, 37 single nucleotide polymorphisms (SNPs) were identified in the coding exons of the LQTS-associated potassium ion channel genes, seven of which were novel nonsynonymous polymorphisms. SNPs 356G-->A (exon 1 of KCNQ1), 2624C-->T and 2893G-->A (exon 11 of HERG), 3164G-->A, 3322C-->G and 3460G-->A (exon 14 of HERG), and 79C-->T (exon 3 of KCNE2) resulted in Gly119Asp, Thr875Met, Gly965Arg, Arg1055Gln, Leu1108Val, Gly1154Ser and Arg27Cys amino acid substitutions, respectively. In addition, 16 intronic variants were detected. The functional consequence of these variants has not been studied and their association with risk of LQTS is unclear. CONCLUSIONS: There exist multiple genetic polymorphisms of the LQTS-associated genes in the three distinct Asian populations. Though the functional significance of many of these SNPs is unknown, this interindividual and interethnic genetic variability may underlie the different susceptibilities of individuals to developing LQTS.     

An investigation of the catalytic activity of CYP2A13*4 with coumarin and polymorphisms of CYP2A13 in a Chinese Han population

CYP2A13 has been identified as an efficient catalyst for the metabolisms of coumarin, aflatoxin B(1) (AFB(1)), and several tobacco-specific carcinogens. The reported CYP2A13 polymorphisms with missense variations have been studied for their functional consequences, and CYP2A13*4 (R101Q) variant was found to be a null enzyme in metabolizing 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), AFB(1), and 5-methoxypsoralen. In the present study, CYP2A13*4 was expressed in Sf9 cells and evaluated for coumarin 7-hydroxylation activity. Our results demonstrated that CYP2A13*4 showed no activity in coumarin 7-hydroxylation. Furthermore, computer modeling studies were conducted to probe the mechanisms underlying the loss of catalytic activity of CYP2A13*4. The results suggested that the R101Q alteration may result in the absence of several hydrogen bonds involved in heme binding and thus lead to the loss of function in CYP2A13*4. In addition, for the first time, the distribution frequencies of all eight known CYP2A13 missense alleles were examined in a Chinese Han population. The distribution frequencies of CYP2A13*3 allele and CYP2A13*4 allele in the Chinese Han population were statistically significantly different from the reported values in Japanese. Considering that the two variants of CYP2A13 are incapable of metabolic activation of NNK and AFB(1), the susceptibility to NNK or AFB(1) exposure between the Chinese Han population and Japanese can be different.     

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).     

Polymorphisms in human soluble epoxide hydrolase

Human soluble epoxide hydrolase (hsEH) metabolizes a variety of epoxides to the corresponding vicinal diols. Arachidonic and linoleic acid epoxides are thought to be endogenous substrates for hsEH. Enzyme activity in humans shows high interindividual variation (e.g., 500-fold in liver) suggesting the existence of regulatory and/or structural gene polymorphisms. We resequenced each of the 19 exons of the hsEH gene (EPHX2) from 72 persons representing black, Asian, and white populations. A variety of polymorphisms was found, six of which result in amino acid substitutions. Amino acid variants were localized on the crystal structure of the mouse sEH, resulting in the prediction that at least two of these (Arg287Gln and Arg103Cys) might significantly affect enzyme function. The six variants of the hsEH cDNA corresponding to each single polymorphism and one corresponding to a double polymorphism were then constructed by site-directed mutagenesis and expressed in insect cells. As predicted, Arg287Gln and the double mutant Arg287Gln/Arg103Cys showed decreased enzyme activity using trans-stilbene oxide, trans-diphenylpropene oxide, and 14,15-epoxyeicosatrienoic acid as substrates. Lys55Arg and Cys154Tyr mutants had elevated activity for all three substrates. Detailed kinetic studies revealed that the double mutant Arg287Gln/Arg103Cys showed significant differences in Km and Vmax. In addition, stability studies showed that the double mutant was less stable than wild-type protein when incubated at 37 degrees C. These results suggest that at least six hsEH variants exist in the human population and that at least four of these may influence hsEH-mediated metabolism of exogenous and endogenous epoxide substrates in vivo.     

Functional analysis of three CYP1A2 variants found in a Japanese population.

Human cytochrome P450 1A2 (CYP1A2) catalyzes the metabolism of many important drugs and environmental chemicals. We previously reported three naturally occurring genetic polymorphisms (125C>G, Pro42Arg, CYP1A2*15; 1130G>A, Arg377Gln, *16; and 1367G>A, Arg456His, *8) found in a Japanese population. In this study, these variant enzymes were expressed in Chinese hamster V79 cells, and their mRNA and protein expression levels as well as catalytic activities were determined. All three variant enzymes showed reduced protein expression levels (66% for Pro42Arg and approximately 30% for Arg377Gln and Arg456His) compared with that of the wild type (WT) without any change in mRNA expression levels. Kinetic analysis for 7-ethoxyresorufin O-deethylation revealed that V(max) and V(max)/K(m) of all three variants were less than 3 and 1% of the WT, respectively, although the K(m) value was significantly increased only in the Arg377Gln variant (approximately a 9-fold increase). Markedly reduced activities of the three variants were also observed for phenacetin O-deethylation. In the reduced CO difference spectral analysis using recombinant proteins produced in the Sf21/baculovirus system, the peak at 450 nm seen in the WT protein was hardly observed in the three variants, suggesting marked reductions in their hemoprotein formation. These results suggest that Pro42, Arg377, and Arg456 are critical amino acids for the production of catalytically active CYP1A2 holoenzyme.     

Genetic polymorphism of the human cytochrome P450 CYP4B1: evidence for a non-functional allelic variant

In the present study, we report the first systematic investigation of polymorphism in the human CYP4B1 gene. Using a strategy based on single-strand conformation polymorphism analysis of PCR products (PCR-SSCP), we analyzed the twelve exons of the gene, as well as their 5'- and 3'- proximal flanking sequences, in DNA samples from 190 French Caucasians. In addition to the wild-type CYP4B1* allele (CYP4B1*1), four variants, namely CYP4B1*2, *3, *4 and *5, were characterized. The CYP4B1*3, *4 and *5 alleles encode missense mutations Arg173Trp, Ser322Gly and Met331Ile, respectively. The fourth variant, CYP4B1*2, harbors three missense mutations (Met331Ile, Arg340Cys and Arg375Cys) and a double nucleotide deletion (AT881-882del) that causes a frameshift and premature stop codon in the second third of the coding sequence of the gene. This latter mutation can be assumed to lead to the synthesis of a severely truncated protein and, therefore, probably contributes to interindividual variability of CYP4B1 expression and enzymatic activity. In order to investigate the extent of the CYP4B1*2 allele in a large population, a rapid genotyping test, based on restriction analysis of PCR products, was developed and applied to 2082 French Caucasians. Forty-two subjects were found homozygous for the AT881-882 deletion, which suggests that about 2% of individuals should be unable to develop metabolic reactions mediated by CYP4B1. Given the relatively high frequency and the functional consequences of the CYP4B1*2 allele, associations between CYP4B1 polymorphism and certain pathological processes should be considered.     

Identification of genetic variations of the human organic cation transporter hOCT1 and their functional consequences

By systematic mutation screening of the polyspecific organic cation transporter hOCT1 (SLC22A1) in 57 Caucasians, 25 genetic variations were identified and further analysed for population frequency. Five mutations resulting in the amino acid changes Arg61Cys, Cys88Arg, Phe160Leu, Gly401Ser, and Met420del, with respective allele frequencies of 9.1, 0.6, 22, 3.2, and 16%, were functionally characterized upon expression in Xenopus oocytes. Phe160Leu and Met420del exhibited substrate affinities and selectivites identical to hOCT1 wild-type. In contrast, uptake of 0.1 microm [3H]1-methyl-4-phenylpyridinium ([3H]MPP) by Arg61Cys, Cys88Arg and Gly401Ser were reduced to 30, 1.4 and 0.9% compared to wild-type, respectively. Since transport of 1 microm [3H]serotonin by Cys88Arg and Gly401Ser was reduced to only 13 and 12% of wild-type, these mutants exhibit a changed substrate selectivity. The data show that the mutants Arg61Cys, Cys88Arg and Gly401Ser could affect the disposition of OCT1 substrates and as a consequence may alter the duration and intensity of effects of drugs and neurotransmitters which are substrates for hOCT1.     

Functional characterization of non-synonymous single nucleotide polymorphisms in the gene encoding human multidrug resistance protein 1 (MRP1/ABCC1)

The 190-kDa ATP-binding cassette (ABC) multidrug resistance protein 1 (MRP1) encoded by the MRP1/ABCC1 gene mediates the active cellular efflux of glucuronide, glutathione and sulfate conjugates. It can also confer resistance to a diverse spectrum of chemotherapeutic agents and transport a variety of toxicants. In the present study, we examined 10 MRP1/ABCC1 missense genetic variants [non-synonymous single nucleotide polymorphisms (SNPs)] to determine whether or not they affect expression or function of the transporter. Variants 218C>T (Thr73Ile), 257C>T (Ser92Phe), 350C>T (Thr117Met), 689G>A (Arg230Gln), 1898G>A (Arg633Gln), 2168G>A (Arg723Gln), 2965G>A (Ala989Thr), 3140G>C (Cys1047Ser), 3173G>A (Arg1058Gln) and 4535C>T (Ser1512Leu) were recreated using site-directed mutagenesis and transfected into human embryonic kidney cells. Immunoblotting experiments showed that all mutant proteins were expressed at levels comparable to wild-type MRP1. Vesicular transport assays revealed that the Ala989Thr mutation caused a significant decrease in estradiol 17beta-glucuronide transport due to a decrease in apparent affinity (Km) for this organic anion. The transport properties of the other mutants were comparable to wild-type MRP1. When the MRP1/ABCC1 non-synonymous SNPs were evaluated by the SIFT algorithm using subsets of homologs and orthologs of MRP1/ABCC1, Arg230Gln, Val353Met, Arg433Ser, Gly671Val and Arg1058 mutations were predicted to be deleterious, whereas the PolyPhen algorithm predicted Ser92Phe and Gly671Val to be potentially damaging. Thus most predictions of these algorithms were not in accordance with our experimental results. In conclusion, our data suggest that none of the MRP1/ABCC1 variants studied are likely by themselves to have major deleterious effects in healthy individuals, and the SIFT and PolyPhen algorithms appear to be poor predictors of the phenotypic consequences of these MRP1 mutations at least in vitro.     

A novel single nucleotide polymorphism (SNP) of the CYP2C19 gene in a Japanese subject with lowered capacity of mephobarbital 4'-hydroxylation

We sequenced all nine exons and exon-intron junctions of the cytochrome P450 2C19 (CYP2C19) gene from a Japanese subject with a lowered capacity of CYP2C19-mediated 4'-hydroxylation after an oral administration of mephobarbital. We found a novel single nucleotide polymorphism (SNP) of CYP2C19 gene as follows: SNP, 040110MoritaJ001; GENENAME: CYP2C19; ACCESSION NUMBER: NT_030059.8; LENGTH; 25 bases; 5'-GAGGGCCTGGCCC/TGCATGGAGCTGT-3'. The SNP (168946C>T) induced an amino acid alteration (Arg442Cys) located in exon 9 close to the heme-binding region of CYP2C19, which may result in the decrease in the catalytic properties of CYP2C19. A new allele having this SNP was designated as CYP2C19*16.     

Genetic variants of flavin-containing monooxygenase 3 (FMO3) in Japanese subjects identified by phenotyping for trimethylaminuria and found in a database of genome resources

The oxygenation of food-derived trimethylamine to its N-oxide is a representative reaction mediated by human flavin-containing monooxygenase 3 (FMO3). Impaired FMO3 enzymatic activity is associated with trimethylaminuria (accumulation of substrate), whereas trimethylamine N-oxide (metabolite) is associated with arteriosclerosis. We previously reported FMO3 single-nucleotide and/or haplotype variants with low FMO3 metabolic capacity using urinary phenotyping and the whole-genome sequencing of Japanese populations. Here, we further analyze Japanese volunteers with self-reported malodor and interrogate an updated Japanese database for novel FMO3 single-nucleotide and/or haplotype variants. After 3 years of follow up, seven probands were found to harbor the known impaired FMO3 variant p.(Gly191Cys) identified in the database or novel variants/haplotypes including p.(Met66Val), p.(Arg223Gln), p.(Glu158Lys;Glu308Gly;Arg492Trp), and p.(Glu158Lys;Glu308Gly;Pro496Ser). The known severe mutation p.(Cys197Ter) (a TG deletion) and four variants including p.(Tyr269His) and p.(Pro496Ser) were first detected in the updated genome panel. Among previously unanalyzed FMO3 variants, the trimethylamine/benzydamine N-oxygenation activities of recombinant p.(Met66Val), p.(Arg223Gln), p.(Tyr269His), p.(Glu158Lys;Glu308Gly;Arg492Trp), and p.(Glu158Lys;Glu308Gly;Pro496Ser) FMO3 variant proteins were severely decreased (V_max/K_m <10% of wild-type). Although the present novel mutations or alleles were relatively rare, both in self-reported Japanese trimethylaminuria sufferers and in the genomic database panel, three common FMO3 missense or deletion variants severely impaired FMO3-mediated N-oxygenation of trimethylamine.