Genetic polymorphisms and haplotypes of por, encoding cytochrome p450 oxidoreductase, in a Japanese population

Cytochrome P450 oxidoreductase (POR) transfers electrons from NADPH to all microsomal cytochrome P450 (CYP) enzymes and is necessary for microsomal CYP activities. In this study, to find genetic variations and to elucidate the haplotype structures of POR, we comprehensively screened the genetic variations in the 5'-flanking region, all the exons and their flanking introns of POR for 235 Japanese subjects. Seventy-five genetic variations including 26 novel ones were found: 7 were in the 5'-flanking region, 2 in the 5'-untranslated region (5'-UTR, non-coding exon 1), 16 in the coding exons (10 nonsynonymous and 6 synonymous), 45 in the introns, 4 in the 3'-UTR and 1 in the 3'-flanking region. Of these, 4 novel nonsynonymous variations, 86C>T (T29M), 1648C>T (R550W), 1708C>T (R570C) and 1975G>A (A659T), were detected with allele frequencies of 0.002. We also detected known nonsynonymous SNPs 683C>T (P228L), 1237G>A (G413S), 1453G>A (A485T), 1508C>T (A503V), 1510G>A (G504R) and 1738G>C (E580Q) with frequencies of 0.002, 0.009, 0.002, 0.434, 0.002 and 0.002, respectively. Based on the linkage disequilibrium (LD) profiles, the analyzed region could be divided into two LD blocks. For Blocks 1 and 2, 14 and 46 haplotypes were inferred, respectively, and 2 and 6 common haplotypes found in more than 0.03 frequencies accounted for more than 81% of the inferred haplotypes. This study provides fundamental and useful information for the pharmacogenetic studies of drugs metabolized by CYPs in the Japanese population.     

Identification and functional characterization of novel CYP2J2 variants: G312R variant causes loss of enzyme catalytic activity

CYP2J2 plays important roles in the metabolism of therapeutic drugs, such as astemizole and ebastine, as well as endogenous fatty acids. This study aimed to identify CYP2J2 genetic variants in Koreans and to characterize their functional consequences. From direct sequencing of the CYP2J2 gene, 12 genetic variations, including the two novel nonsynonymous mutations G312R and P351L, were identified from 93 Korean subjects. The two novel CYP2J2 variants were co-expressed with NADPH-cytochrome P450 reductase in Sf9 cells and their catalytic activities were quantified. The recombinant CYP2J2 G312R variant showed almost complete loss of enzymatic activity, as determined by CYP2J2-catalysed astemizole O-demethylation and ebastine hydroxylation. The CYP2J2 P351L variant showed enzymatic activities that were comparable with the wild-type CYP2J2. The reduced CO spectra of the recombinant CYP2J2 proteins suggested no CO binding to the heme in CYP2J2 G312R. In addition, molecular modelling of the three-dimensional structure consistently predicted that there might be spatial hindrance between heme and the bulky side chain of the R312 residue in CYP2J2 G312R variant. The CYP2J2 G312R variant was not found in 192 Chinese, 99 African-Americans, 100 Caucasians and 159 Vietnamese subjects. Two of the 192 Chinese subjects (0.52%) were heterozygous for CYP2J2 P351L. Twelve CYP2J2 variants, including two novel nonsynonymous variants, were identified in a Korean population. The G312R variant is the first nonfunctional CYP2J2 allele to be identified, and is expected to influence the disposition of its substrate therapeutics, as well as endogenous compounds.     

Genetic variation of Cytochrome P450 1B1 (CYP1B1) and risk of breast cancer among Polish women

Four single nucleotide polymorphisms (SNPs) in CYP1B1 (Ex2 + 143 C > G, Ex2 + 356 G > T, Ex3 + 251 G > C, Ex3 + 315 A > G) cause amino acid changes (R48G, A119S, L432V and N453S, respectively) and are associated with increased formation of catechol estrogens; however, epidemiologic evidence only weakly supports an association between these variants and breast cancer risk. Because genetic variability conferring increased susceptibility could exist beyond these putative functional variants, we comprehensively examined the common genetic variability within CYP1B1. A total of eight haplotype-tagging (ht)SNPs (including Ex3 + 315 A > G), in addition to two putatively functional SNPs (Ex2 + 143 C > G and Ex3 + 251 G > C), were selected and genotyped in a large case-control study of Polish women (1995 cases and 2296 controls). Haplotypes were estimated using the expectation-maximization algorithm, and overall differences in the haplotype distribution between cases and controls were assessed using a global score test. We also evaluated levels of tumor CYP1B1 protein expression in a subset of 841 cases by immunohistochemistry, and their association with genetic variants. In the Polish population, we observed two linkage disequilibrium (LD)-defined blocks. Neither haplotypes (global P-value of 0.99 and 0.67 for each block of LD, respectively), nor individual SNPs (including three putatively functional SNPs) were associated with breast cancer risk. CYP1B1 was expressed in most tumor tissues (98%), and the level of expression was not related to the studied genetic variants. We found little evidence for modification of the estimated effect of haplotypes or individual SNPs by age, family history of breast cancer, or tumor hormone receptor status. The present study provides strong evidence against the existence of a substantial overall association between common genetic variation in CYP1B1 and breast cancer risk.     

Metabolism of styrene in the human liver in vitro: interindividual variation and enantioselectivity.

1. The interindividual variation and enantioselectivity of the in vitro styrene oxidation by cytochrome P450 have been investigated in 20 human microsomal liver samples. Liver samples were genotyped for the CYP2E1*6 and CYP2E1*5B alleles. 2. Kinetic analysis indicated the presence of at least two forms of styrene-metabolizing cytochrome P450. The enzyme constants for the high-affinity component were subject to appreciable interindividual variation, i.e. Vmax1 ranged from 0.39 to 3.20 nmol mg protein(-1) min(-1) (0.96+/-0.63) and Km1 ranged from 0.005 to 0.03 mM (0.011+/-0.006). Inhibition studies with chemical inhibitors of CYP2E1, CYP1A2, CYP2C8/9 and CYP3A4 demonstrated that CYP2E1 was the primary enzyme involved in the high-affinity component of styrene oxidation. No relationship between the interindividual variation in Vmax1 and Km1 and the genetic polymorphisms of the CYP2E1 gene was found. 3. Cytochrome P450-mediated oxidation of styrene demonstrated a moderate enantioselectivity, with an enantiomeric excess (ee) of (S)-styrene oxide of 15% (range 4-27%) at low styrene concentration and an ee of (R)-styrene oxide of 7% (range -11 to +22%) at high styrene concentration. This points towards the involvement of at least two cytochrome P450, with different enantioselectivities. 4. The data indicate that cytochrome P450-mediated styrene oxidation is subject to considerable interindividual variation, but only to a moderate product enantioselectivity.     

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.     

Genetic variations and haplotype structures of the ABC transporter gene ABCC1 in a Japanese population

Multidrug resistance-related protein 1 (MRP1), an ATP-binding cassette transporter encoded by the ABCC1 gene, is expressed in many tissues, and functions as an efflux transporter for glutathione-, glucuronate- and sulfate-conjugates as well as unconjugated substrates. In this study, the 31 exons and their flanking introns of ABCC1 were comprehensively screened for genetic variations in 153 Japanese subjects to elucidate the linkage disequilibrium (LD) profiles and haplotype structures of ABCC1 that is necessary for pharmacogenetic studies of the substrate drugs. Eighty-six genetic variations including 31 novel ones were found: 1 in the 5'-flanking region, 1 in the 5'-untranslated region (UTR), 20 in the coding exons (9 synonymous and 11 nonsynonymous variations), 4 in the 3'-UTR, and 60 in the introns. Of these, eight novel nonsynonymous variations, 726G>T (Trp242Cys), 1199T>C (Ile400Thr), 1967G>C (Ser656Thr), 2530G>A (Gly844Ser), 3490G>A (Val1164Ile), 3550G>A (Glu1184Lys), 3901C>T (Arg1301Cys), and 4502A>G (Asp1501Gly), were detected with an allele frequency of 0.003. Based on the LD profiles, the analyzed regions of the gene were divided into five LD blocks (Blocks -1 and 1 to 4). The multiallelic repeat polymorphism in the 5'-UTR was defined as Block -1. For Blocks 1, 2, 3 and 4, 32, 23, 23 and 13 haplotypes were inferred, and 9, 7, 7 and 6 haplotypes commonly found on > or = 10 chromosomes accounted for > or = 91% of the inferred haplotypes in each block. Haplotype-tagging single nucleotide polymorphisms for each block were identified to capture the common haplotypes. This study would provide fundamental and useful information for the pharmacogenetic studies of MRP1-dependently effluxed drugs in Japanese.     

CYP1A1 and CYP1B1 genotypes, haplotypes, and TCDD-induced gene expression in subjects from Seveso, Italy

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is highly toxic in experimental animals, and is known to induce cytochrome P450 (CYP) gene expression. We investigated the effect of CYP1A1 and CYP1B1 variant genotypes and haplotypes on CYP1A1 and CYP1B1 mRNA expression and ethoxyresorufin-O-deethylase (EROD) activity in lymphocytes from 121 subjects from the Seveso population, Italy, accidentally exposed to TCDD in 1976. The 3'UTR 3801T>C and I462V variants of CYP1A1 were present in 16% and 6% of the subjects, respectively. The frequency of CYP1B1 variants was 85.2% for L432V, 49.6% for R48G and A119S, and 28.7% for N453S. There was complete linkage disequilibrium (LD) among the CYP1B1 variant loci (D'=-1) and high LD among the CYP1A1 loci (D'=0.86). Gene expression measured by RT-PCR did not vary by CYP1B1 genotype in uncultured lymphocytes. However, when lymphocytes were treated in vitro with 10 nM TCDD, CYP1B1 and CYP1A1 mRNA expression was strongly induced and modified by CYP variant alleles. Specifically, the CYP1B1*3 haplotype (L432V) was associated with increased CYP1B1 mRNA expression (P=0.03), following an additive model; the CYP1A1 I462V polymorphism was positively, although not significantly, associated with CYP1A1 expression. The CYP1B1*3 variant may have affected CYP1B1 expression in subjects highly and acutely exposed to dioxin at the time of the accident. Although based on small number of subjects, a slight increase in eczema (P=0.05, n=8) and urticaria (P=0.02, n=2) was observed 20 years after the accident in subjects carrying the CYP1B1*3 allele. Genetic variation in cytochrome P450 induction may identify subjects with variable responsiveness to TCDD and potentially increased risk of disease.     

Genetic polymorphisms of FCGRT encoding FcRn in a Japanese population and their functional analysis

Neonatal Fc receptor (FcRn) plays an important role in regulating IgG homeostasis in the body. Changes in FcRn expression levels or activity caused by genetic polymorphisms of FCGRT, which encodes FcRn, may lead to interindividual differences in pharmacokinetics of therapeutic antibodies. In this study, we sequenced the 5'-flanking region, all exons and their flanking regions of FCGRT from 126 Japanese subjects. Thirty-three genetic variations, including 17 novel ones, were found. Of these, two novel non-synonymous variations, 629G>A (R210Q) and 889T>A (S297T), were found as heterozygous variations. We next assessed the functional significance of the two novel non-synonymous variations by expressing wild-type and variant proteins in HeLa cells. Both variant proteins showed similar intracellular localization as well as antibody recycling efficiencies. These results suggested that at least no common functional polymorphic site with amino acid change was present in the FCGRT of our Japanese population.     

CYP2C8 polymorphisms in Caucasians and their relationship with paclitaxel 6alpha-hydroxylase activity in human liver microsomes

Published cDNA sequences suggest the existence of non-synonymous single nucleotide polymorphisms in the cytochrome P450 CYP2C8. To determine whether these polymorphisms could be confirmed in a Caucasian population and to investigate whether additional polymorphisms occur in the coding and upstream regions of this gene, we screened for previously described and for novel polymorphisms using PCR-RFLP and SSCP analysis. We confirmed the existence of two of the previously detected polymorphisms which give rise to the amino acid substitutions I264M and K399R, respectively, but failed to detect three others in our population. We also confirmed that a recently identified polymorphism (R139K) is linked to K399R (CYP2C8*3) in our study population. The allele frequencies for the I264M (CYP2C8*4 allele) and the CYP2C8*3 allele were 0.075 and 0.15, respectively. Three novel polymorphisms (T-370G, C-271A and T1196C/L390S) were also detected with the upstream polymorphisms showing allele frequencies of 0.061 and 0.196, respectively, but the L390S polymorphism detected only in a single subject. An additional single subject was heterozygous for a polymorphism recently described in African-Americans (A805T; CYP2C8*2 allele). The functional significance of the two upstream polymorphisms and the CYP2C8*3 and CYP2C8*4 alleles was investigated in human liver microsomes. Samples heterozygous for CYP2C8*3 showed significantly lower paclitaxel 6alpha-hydroxylase activity compared with wild-type samples. Median activity associated with CYP2C8*4 also appeared lower than the wild-type but the difference was not significant. There was no evidence that either upstream polymorphism gave rise to altered CYP2C8 expression.     

Genetic polymorphisms of CYP2C8 in Japanese population.

CYP2C8 plays important roles in metabolizing therapeutic drugs and endogenous compounds. Although genetic polymorphisms of CYP2C8 were reported, there is little information on CYP2C8 polymorphisms in the Japanese population. In the present study, we screened for previously described polymorphisms in the coding region of this gene using polymerase chain reaction (PCR)-restriction fragment length polymorphism or allele specific-PCR analyses. Eleven polymorphisms of CYP2C8*2 (I269F), CYP2C8*3 (R139K, K399R), CYP2C8*4 (I264M), CYP2C8*5 (frameshift), T130N, E154D, N193K, K249R, L390S, P404A, and H411L have been comprehensively investigated in at least 200 Japanese individuals. A single subject was heterozygous for CYP2C8*5, and the allele frequency was calculated as 0.0025. The other single nucleotide polymorphisms (SNPs) were not found in the Japanese subjects in the present study. Thus, it appears that the frequencies of these alleles in Japanese are extremely low. In addition, concerning the SNPs of T130N, E154D, N193K, K249R, and H411L, it remains clear that these alleles exist as polymorphisms or represent sequence errors or cloning artifacts. Although several SNPs such as CYP2C8*2, CYP2C8*3, CYP2C8*4, and P404A have been reported to reduce the enzymatic activity, pharmacokinetic abnormalities of drugs metabolized by polymorphic CYP2C8 might be rare in Japanese.     

Novel CYP2C9 promoter variants and assessment of their impact on gene expression

There are a considerable number of reports identifying and characterizing genetic variants within the CYP2C9 coding region. Much less is known about polymorphic promoter sequences that also might contribute to interindividual differences in CYP2C9 expression. To address this problem, approximately 10,000 base pairs of CYP2C9 upstream information were resequenced using 24 DNA samples from the Coriell Polymorphism Discovery Resource. Thirty-one single-nucleotide polymorphisms (SNPs) were identified; nine SNPs were novel, whereas 22 were reported previously. Using both sequencing and multiplex single-base extension, individual SNP frequencies were determined in 193 DNA samples obtained from unrelated, self-reported Hispanic Americans of Mexican descent, and they were compared with similar data obtained from a non-Latino white cohort. Significant interethnic differences were observed in several SNP frequencies, some of which seemed unique to the Hispanic population. Analysis using PHASE 2.1 inferred nine common (>1%) variant haplotypes, two of which included the g.3608C>T (R144C) CYP2C9(*)2 and two the g.42614A>C (I359L) CYP2C9(*)3 SNPs. Haplotype variants were introduced into a CYP2C9/luciferase reporter plasmid using site-directed mutagenesis, and the impact of the variants on promoter activity assessed by transient expression in HepG2 cells. Both constitutive and pregnane X receptor-mediated inducible activities were measured. Haplotypes 1B, 3A, and 3B each exhibited a 65% decrease in constitutive promoter activity relative to the reference haplotype. Haplotypes 1D and 3B exhibited a 50% decrease and a 40% increase in induced promoter activity, respectively. These data suggest that genetic variation within CYP2C9 regulatory sequences is likely to contribute to differences in CYP2C9 phenotype both within and among different populations.     

Search for an association between the human CYP1A2 genotype and CYP1A2 metabolic phenotype

The genotype responsible for more than 60-fold interindividual differences in human hepatic CYP1A2 constitutive expression is not understood. Resequencing the human CYP1A1_CYP1A2 locus (39.6 kb) in five major geographically isolated subgroups recently led to the identification of 85 single nucleotide polymorphisms (SNPs), 57 of which were double-hit SNPs. Here, we attempted to correlate the CYP1A2 genotype with a metabolic phenotype. We chose 16 SNPs (all having a minor allele frequency > or =0.05 in Caucasians) to genotype 32 DNA samples (26 Caucasians, six Ethiopians) in which CYP1A2 metabolism had previously been determined. From 280 subjects (five locations worldwide) that had been CYP1A2-phenotyped, we genotyped the 10 highest, 14 lowest and eight intermediate DNA samples. Although no SNP was significant (P<0.05), possibly due to the small sample size, we found a trend for several of the six SNPs across the CYP1A2 linkage disequilibrium block associated with the trait. Five CYP1A2 haplotypes were inferred, two of which had not previously been reported; haplotype 1A2H10 showed the greatest association with CYP1A2 activity. Regulatory sequences responsible for the large interindividual differences in hepatic CYP1A2 gene basal expression might reside, in part, with some of these CYP1A2 SNPS but, in large part, might be located either cis (in nearby sequences not yet haplotyped) or trans in that they are not linked to the gene. We conclude that no SNP or haplotype in the CYP1A2 gene has yet been identified that can unequivocally be used to predict the metabolic phenotype in any individual patient.     

Pharmacogenetics of CYP1A2, novel polymorphisms and haplotypes in three distinct Asian populations

CYP1A2 play an important role in the metabolism of many carcinogens and clinically important drugs. CYP1A2 activity has been found to be influenced by the presence of polymorphic variants which were reported to display wide interethnic variation. This study investigates the frequency distribution and linkage disequilibrium patterns of CYP1A2 genetic polymorphisms, and characterize their haplotype structures in three healthy Asian populations in Singapore (Chinese, Malay, and Indian). The entire CYP1A2 gene was screened in 126 healthy subjects from all three ethnic groups (N=42 each). A total of 25 polymorphisms was identified, of which nine were novel. The polymorphisms, -2467delT and -163C>A were detected at high frequencies in all Asian ethnic groups. Significant interethnic differences were observed in the genotypic frequency distribution of IVS2-99G>A (P<0.01) and 1548C>T (P=0.05) across the three ethnic groups while -163C>A (P=0.02) was found to differ between Chinese and Malays. Haplotype analyses revealed four to six major haplotypes in each ethnic population which accounted for more than 60% of the cumulative haplotype frequencies. Future studies should be done to investigate the functional roles of these haplotypes.     

Gene-gene interactions between CYP2B6 and CYP2A6 in nicotine metabolism

OBJECTIVES: CYP2A6 is the major enzyme involved in nicotine metabolism, yet large interindividual variations in the rate of nicotine metabolism exist within groups of individuals having the same CYP2A6 genotype. We investigated the influence of genetic variation in another potential nicotine-metabolizing enzyme, CYP2B6, and its interaction with CYP2A6, on the metabolism of nicotine. METHODS: Two hundred and twelve healthy Caucasian adult twin volunteers underwent an intravenous infusion of stable isotope-labeled nicotine and its major metabolite, cotinine, for characterization of pharmacokinetic and metabolism phenotypes. Five CYP2B6 genetic polymorphisms causing amino acid substitutions (R22C, Q172 H, S259R, K262R, and R487C) were analyzed. RESULTS: We observed that the CYP2B6*6 haplotype (defined as having both Q172 H and K262R variants) was associated with faster nicotine and cotinine clearance, and that such associations were more prominent among individuals having decreased-activity CYP2A6 genotypes. Statistically significant interactions between CYP2B6 and CYP2A6 genotypes were observed (P<0.003 for nicotine clearance and P<0.002 for cotinine clearance). CONCLUSIONS: Our results indicate that CYP2B6 genetic variation is associated with the metabolism of nicotine and cotinine among individuals with decreased CYP2A6 activity. Further investigation of the roles of CYP2B6 and the interaction between CYP2B6 and CYP2A6 genotypes in mediating nicotine dependence and tobacco-related diseases is merited.     

A novel duplication type of CYP2A6 gene in African-American population.

Human CYP2A6 is responsible for the metabolism of nicotine and its genetic polymorphisms affect smoking behavior and risk of lung cancer. In the present study, we identified a novel type of CYP2A6 gene duplication that is created through an unequal crossover event with the CYP2A7 gene at 5.2 to 5.6 kilobases downstream from the stop codon. The novel duplication type of CYP2A6 was found in African Americans (n = 176) at an allele frequency of 1.7%, but was not found in European-American (n = 187), Korean (n = 209), or Japanese (n = 184) populations. The plasma cotinine/nicotine ratio in subjects possessing the novel CYP2A6 gene duplication with the CYP2A6*1 allele (10.8 +/- 7.0, n = 4) was 1.4-fold higher than that in homozygotes of the wild type (8.0 +/- 5.0, n = 87), although the difference was not statistically significant. The findings in the present study suggested that the novel duplicated CYP2A6 allele, which is specific for African Americans, would increase nicotine metabolism and may affect smoking behavior.     

CYP1A2 F21L and F186L polymorphisms in an Italian population sample

P450 cytochromes (CYPs) enzymes play a major role in variability of drug response and cancer susceptibility. In particular, up to 60-fold interindividual variation has been detected in the activity of CYP1A2, which is involved in the metabolism of caffeine, several drugs and various toxic and carcinogenic compounds. Aim of this study is to assess the frequency of CYP1A2 F21L and F186L polymorphisms (formerly CYP1A2(*)2 and (*)11 alleles), up to now found in Asiatic populations only. These variants were absent in 500 Italian healthy subjects. Therefore it can be suggested that the variation of CYP1A2-dependent metabolism in the Caucasian population is not related to these two CYP1A2 polymorphisms. Thus, this study supports the view that ethnicity is a relevant factor to be carefully considered in pharmacogenetic studies.