Ultrarapid metabolism of sparteine: frequency of alleles with duplicated CYP2D6 genes in a Danish population as determined by restriction fragment length polymorphism and long polymerase chain reaction

CYP2D6 is a polymorphically expressed enzyme with two phenotypes. Poor metabolizers lack the enzyme caused by inactivating mutations in the CYP2D6 gene and extensive metabolizers have at least one active CYP2D6 gene. Extensive metabolizers with very high capacity for CYP2D6 dependent drug metabolisms are termed ultrarapid metabolizers and carry alleles with duplicated, multi duplicated or amplified CYP2D6 genes. In the present study, we examined the frequency of CYP2D6 gene duplications in a Danish population and validated a long polymerase chain reaction method for identification of ultrarapid metabolizers. Sixty individuals having a metabolic ratio for sparteine at or below 0.15 were selected and a control group of 53 individuals with a metabolic ratio between 0.16 and 12.4 was used. Based on EcoRI restriction fragment length polymorphism analysis, eight individuals were found with a duplicated CYP2D6 gene, whereas using a long polymerase chain reaction method, nine individuals with a 3.6 kb fragment indicative of two CYP2D6 genes in tandem were found among the 60 individuals with a low metabolic ratio. No gene duplication was found in the control group or in any individuals with a metabolic ratio > 0.14. Based on these results, we estimate the frequency of individuals with CYP2D6 duplication in the Danish population to be 0.8%, which is comparable to the frequency in the Swedish and the German populations, but considerably lower than in Spanish or African populations. We conclude that the long polymerase chain reaction assay is simple and reliable for detection of duplications of the CYP2D6 gene.     

Rapid detection of CYP2C18 genotypes by real-time fluorescence polymerase chain reaction

In man, CYP2C19, a liver enzyme, plays an important role in the metabolism of several drugs. Mutation of the CYP2C19 gene results in a poor metaboliser phenotype. S-Mephenytoin hydroxylation genetic polymorphism is due to two mutations of the CYP2C19 gene, namely CYP2C19*2, located in exon 5, and CYP2C19*3, located in exon 4. CYP2C18 is also polymorphically expressed. The mutant alleles of this enzyme are CYP2C18m1, located in exon 2 and CYP2C18m2, located in the 5'-flanking region. We have developed an allele-specific TaqMan polymerase chain reaction (PCR) assay with which to detect CYP2C18 mutant alleles. This assay combines hybridization of the TaqMan probe and allele-specific amplification primers to the target DNA. The TaqMan probe is labelled with 6-carboxyfluorescein at the 5' end and 6-carboxytetramethylrhodamine together with a phosphate at the 3' end. Genotypes are separated according to the different threshold cycles of the wild type and mutant primers. We applied this procedure to DNA extracted from the blood or saliva of 144 healthy Japanese volunteers. The wt/wt, wt/m1, wt/m2, m1/m1, m1/m2 and m2/m2 genotypes of the CYP2C18 alleles detected by the assay were consistent with the results obtained from restriction enzyme cleavage. In accordance with a previous report, the genotypes of CYP2C18m1 and CYP2C18m2 coincided with those of CYP2C19*3 and CYP2C19*2, respectively. Therefore, detection of CYP2C18 mutant alleles also allows that of CYP2C19 mutant alleles. Among 19 poor metabolisers, eight showed the homozygous CYP2C19*2/CYP2C19*2, two the homozygous CYP2C19*3/CYP2C19*3 and nine the compound heterozygous CYP2C19*2/CYP2C19*3 genotype. We found the allele-specific TaqMan PCR assay rapid, simple and cost-effective, as well as suitable for high-throughput applications in a routine laboratory. This assay allows the fast and reliable detection of inherited disorders that might influence diagnosis and treatment.     

Real-time polymerase chain reaction analysis of CYP1B1 gene expression in human liver.

Procarcinogen-activating cytochrome P450 (CYP) enzymes such as CYP1B1, CYP1A1, and CYP1A2 are considered to play an important role in chemical carcinogenesis. However, conflicting data exist with respect to CYP1B1 expression in human liver. In the present study, we measured CYP1B1 mRNA and protein expression in liver samples from 12 individuals (7 nonsmokers, 4 smokers, and 1 ex-smoker) and compared the levels to those of CYP1A1 and CYP1A2. As analyzed by real-time polymerase chain reaction, CYP1B1 mRNA was present in all samples and the inter-individual variability was 16-fold. The group mean level was 5-fold greater in smokers than nonsmokers (121 +/- 46 vs. 26 +/- 5 molecules/ng double-stranded DNA, p < 0.05). By comparison, CYP1A1 mRNA was detectable in samples from 4 of 7 nonsmokers, 3 of 4 smokers, and one ex-smoker, whereas CYP1A2 mRNA was detectable in samples from 5 nonsmokers, 4 smokers, and the ex-smoker. The mean levels of CYP1A1 and CYP1A2 mRNA were 4-fold and 9-fold greater, respectively, in smokers than nonsmokers, but the differences were not statistically significant. The inter-individual variability in CYP1A1 and CYP1A2 mRNA expression was 26-fold and 500-fold, respectively. Immunoblot analysis using several antibodies and with a larger panel (n = 27) of liver microsomes showed that CYP1A1 and CYP1B1 proteins were undetectable, whereas CYP1A2 was detectable in all samples and quantifiable in 24 of 27 samples. In summary, our novel finding indicates that CYP1B1 mRNA is expressed in human liver and the levels are increased in smokers, but the protein is undetectable.     

检测CYP2A6基因多态性的PCR技术的基因型方法

综述检测CYP2A6基因多态性的PCR技术的基因型方法。CYP2A6基因多态性存在于白种人、东方人及非洲裔美国人群中 ,包括CYP2A6 1至CYP2A6 12 ,总共 12个变异等位基因。本文列举了检测CYP2A6基因型的不同的DNA来源、引物和限制性内切酶。已开发PCR技术结合诊断性限制性内切酶分析 (RFLP)如两步PCR和一步PCR RFLP方法检测CYP2A6基因型。此外 ,尚有PCR SSCP +RFLP及直接DNA测序方法。     

Cytochrome P450 polymorphism--molecular, metabolic, and pharmacogenetic aspects. IV. Application of long polymerase chain reaction for identification of CYP2D6 gene duplication

In the human organism 58 cytochrome P450 (CYP) isoenzymes belonging to 18 families have been described. Isoenzyme CYP2D6 is an important human xenobiotic-metabolizing enzyme. CYP2D6 biotransforms a significant number of drugs, widely used in clinical practice, such as antidepressants, neuroleptics, antiarrhythmics, analgesics, antiemetics and anticancer agents. The occurrence of polymorphic variants of the enzyme results in different metabolic capacity ranging from poor to ultrarapid. Ultrarapid metabolizer phenotype explains lack of response and decreased levels of drugs which are metabolized by CYP2D6. Therefore, the identification of ultrarapid metabolizers as potential non-compliance cases requiring dose adjustment, has serious clinical importance. In this study we evaluate a long-PCR procedure for detecting CYP2D6 gene duplication.     

Characterization of a genotype previously designated as CYP2A6 D-type: CYP2A6*4B,another entire gene deletion allele of the CYP2A6 gene in Japanese

CYP2A6 is known as an enzyme responsible for the metabolism of several clincally used drugs such as tegafur. Previously, we found two novel genotypes of the CYP2A6 gene, D-type and E-type, and the E-type was clarified to be homozygous for the CYP2A6*4A allele. On the other hand, since the D-type was reported to lack regions from at least intron 5 to a part of exon 9 of the CYP2A6 gene, it caused a misunderstanding that the D-type would be a partial CYP2A6 gene-deleted allele. In this paper, we demonstrate that the D-type is a genotype heterozygous for the CYP2A6*4A and another novel entire CYP2A6 gene-deleted allele, CYP2A6*4B, by analyzing a Japanese family including parents genotyped as the CYP2A6*4A/4A and CYP2A6*1A/*4B, respectively.     

Ethnic variation in CYP2A6*7, CYP2A6*8 and CYP2A6*10 as assessed with a novel haplotyping method

Cytochrome P450 2A6 is the main human nicotine metabolizing enzyme coded for by a highly polymorphic gene, CYP2A6. CYP2A6*7, CYP2A6*8 and CYP2A6*10 are variant alleles common to Asian ethnicities. The CYP2A6*7 and CYP2A6*8 alleles each contain a non-synonymous single nucleotide polymorphism (SNP) 6558T>C and 6600G>T, respectively, whereas the CYP2A6*10 haplotype allele contains both. We have developed the first haplotyping assay; it can unambiguously distinguish the CYP2A6*7, CYP2A6*8 and CYP2A6*10 alleles. The allele frequencies of these three variants were assessed using the novel haplotyping assay in Chinese-Canadian (n=112), Chinese-American (n=221), Taiwanese (n=319), Korean-American (n=207) and Japanese-Canadian (n=64) populations, as well as in Caucasian (n=110) and African-Canadian (n=113) populations. Our new method demonstrated higher frequencies of CYP2A6*7 and CYP2A6*10, and a lower frequency of CYP2A6*8 in Asian populations, but no significant change of allele frequencies in Caucasian or African-Canadian populations.     

大肠癌组织中环氧化酶2基因表达的实时定量PCR检测

目的探讨环氧化酶2(COX-2)在大肠癌发生和发展中的意义。方法建立实时定量RT-PCR方法,采用LightCycler PCR仪检测了28例大肠癌COX-2 mRNA及内参GAPDH mRNA的表达,以COX-2N=(COX-2拷贝数/GAPDH拷贝数)×103表示COX-2 mRNA表达水平。结果肿瘤组织COX-2 mRNA与正常组织表达水平有极明显差异(P<0.05),转移组织较正常组织COX-2mRNA表达有明显差异(P<0.01)。结论COX-2参与了大肠癌的发生和发展,并起到了重要作用。     

Structural characterization of a new variant of the CYP2A6 gene (CYP2A6*1B) apparently diagnosed as heterozygotes of CYP2A6*1A and CYP2A6*4C

During the course of investigating the frequency of a CYP2A6 whole deletion-type polymorphism (CYP2A6*4C) in Japanese, an unexpectedly large population of heterozygotes for CYP2A6*4C and the wild-type (CYP2A6*1A) was found. Cloning of a cDNA encoding CYP2A6 from the liver of individuals judged as heterozygotes for CYP2A6*4C and the CYP2A6*1A was carried out to identify the causal allele(s) responsible for a possible overestimation. A clone isolated from the liver cDNA library possessed 58 bp sequences in the 3'-untranslated region, which was replaced with the corresponding region of the CYP2A7 gene. The same gene conversion existed in the genomic DNA, indicating that the replacement was not a cloning artifact. Based on the gene structure of the allele (CYP2A6*1B), this variant was thought to be one of the causal alleles responsible for overestimation of heterozygotes for CYP2A6*4C and CYP2A6* A. To investigate this further, we developed a genotyping method which could distinguish the CYP2A6*A, CYP2A6*1B and CYP2A6*4C alleles from each other. The results clearly showed that CYP2A6*1B was the sole allele responsible for the overestimation. We conclude that the new genotyping method allows determination of six genotypes of the CYP2A6 gene, simultaneously and precisely, in both Oriental and Caucasian populations.     

Association of CYP2D6 and CYP1A2 gene polymorphism with tardive dyskinesia in Chinese schizophrenic patients

AIM: To investigate the possible association of the CYP2D6 gene C100T polymorphism and the CYP1A2 gene C163A polymorphism with tardive dyskinesia (TD) in Chinese patients with schizophrenia. METHODS: The recruited schizophrenic patients were assessed with the Abnormal Involuntary Movement Scale (AIMS), and divided into groups with TD (n=91) and without TD (n=91) according to the AIMS score. Polymorphisms of the CYP2D6 and CYP1A2 genes were determined by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). RESULTS: No allele frequencies deviated from Hardy-Weinberg equilibrium. No significant differences in genotypes frequencies of the CYP2D6 C100T polymorphism were observed between patients with TD and without TD (Chi2=4.078, P>0.05), but patients with TD had a significant excess of the T allele compared with those without TD (Chi2=4.28, P<0.05). Moreover, the frequency of the CYP1A2 C allele in patients with TD was significantly higher than that in those without TD (Chi2=6.38, P<0.05). An association between TD and the CYP2D6 100T and CYP1A2 163C alleles was observed. Additionally, there were no differences in the mean AIMS scores among different genotypes in TD patients as a group or in smokers. The results of logistic regression analysis demonstrated that mean age and duration of illness were risk factors for TD, but not sex, cumulative exposure to neuroleptic drugs in years, CYP2D6 or CYP1A2 genotype. CONCLUSION: The C100T polymorphism of the CYP2D6 gene and the C163A polymorphism of the CYP1A2 gene may be associated with neuroleptic drug-induced tardive dyskinesia in Chinese patients with schizophrenia. However, genetic factors have a weaker association with susceptibility to TD compared with mean age and duration of illness.     

Phenotypic polymorphism of CYP2A6 activity in a Chinese population

AIMS: To investigate the distribution characteristics of CYP2A6 activity in a Chinese population and to examine the sex-related differences in CYP2A6 activities. METHODS: One hundred and twenty healthy volunteers, 63 men and 57 women, were included in the study. Cytochrome P450 (CYP) 2A6 activity was measured using the ratio of urinary 7-hydroxycoumarin (7-OHC) excreted in 8 h after a coumarin dose. The concentrations of 7-OHC in urine were determined using high performance liquid chromatography. RESULTS: A 300-fold interindividual variation of CYP2A6 activity was shown in the studied Chinese population. The coefficient of variation of CYP2A6 activity was 27.2%. A Kolmogorov-Smirnov test indicated a non-normal distribution of CYP2A6 activity ( P<0.001). Probit plots of CYP2A6 activity revealed a bimodal distribution with breakpoint of activity index near 0.47. The percentage of poor metabolizers (PMs) was 13.3% (95% confidence interval 7.3%-19.4%) in this population. Residual analysis also supported bimodality ( P<0.01). The CYP2A6 activities of females were obviously higher than those of males when the activity index was less than 0.74, although no statistically significant difference in the activity index of CYP2A6 between males and females was found. However, there was no sex-related difference in the incidence of PMs ( P>0.5). CONCLUSIONS: There are pronounced interindividual variations and phenotypic polymorphism of CYP2A6 activities in the Chinese population.     

Impact of CYP2A6 gene polymorphism on the pharmacokinetics of dexmedetomidine for premedication

Background: Dexmedetomidine is a widely used sedative in clinic, which is mainly metabolized by cytochrome P450 2A6 (CYP2A6). Dexmedetomidine was rarely reported for off-label usage of premedication, but lacking relevant pharmacokinetic investigations. Therefore, our study determined the dexmedetomidine pharmacokinetics of CYP2A6*4 allele in Chinese patients pretreated with dexmedetomidine whose mutation frequency of CYP2A6*4 are high, in order to provide clinical references.

Methods: Thirty-one elective surgery patients received premedication with 0.5 μg/kg dexmedetomidine via intravenous pump. Their plasma concentrations at multiple time-points and polymorphism of CYP2A6*4 were determined and statistically analyzed.

Results: 9 patients were *1/*4 or *4/*4, and 22 patients were *1/*1. The main pharmacokinetic parameters were area under curve (AUC) 1396.19 ± 332.47h· ng· l^?1, peak blood concentration (C_max) 495.50 ± 104.90ng· l^?1, distribution volume (V) 0.68 ± 0.20 L/kg, clearance (CL) 0.38 ± 0.11 L/h/kg, distribution half-life (t_1/2α) 0.05 ± 0.01h, elimination half-life (t_1/2β) 2.53 ± 0.04h. No significant pharmacokinetic differences were found among CYP2A6*1/*1, *1/*4, and *4/*4 patients.

Conclusions: In Chinese patients pretreated with dexmedetomidine, T_1/2β was consistent with that published, but T_1/2α, V and Cl were lower. It was unnecessary to consider the mutation when developing the precision regimen of dexmedetomidine.     

Comparison of cytochrome P450 2A6 polymorphism frequencies in Caucasians and African-Americans using a new one-step PCR-RFLP genotyping method.

CYP2A6 (cytochrome P450 2A6), which was first identified as the human coumarin 7-hydroxylase, is the most important enzyme in nicotine C-oxidation. The enzyme also metabolically activates the tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in vitro. Polymorphisms in the CYP2A6 gene may thus impact on both smoking behavior and lung cancer susceptibility. Several different genotyping methods have been reported with conflicting results in the frequencies of CYP2A6 polymorphic variants. Thus we decided to perform a sequence analysis of the entire CYP2A6 gene. Sequencing confirmed the published CYP2A6 cDNA sequence. However, intron sequences differed considerably from the reported sequence of the CYP2A6*3 (v2) variant. Our analyses revealed that parts of introns shared homologies with the published sequence of CYP2A13. Based on our sequence data we developed a one step protocol for specific amplification of exon 3 of CYP2A6. The resulting PCR product can be used directly for restriction endonuclease digestion with XcmI and DdeI to determine the frequencies of the reported variant alleles CYP2A6*2 and CYP2A6*3. In a population of 305 African-Americans and 145 Caucasians, we found allele frequencies of 0.003 (2/610) for CYP2A6*2 and 0 (0/610) for CYP2A6*3 in African-Americans and allele frequencies of 0.014 (4/290) and 0 (0/290) in Caucasians. We conclude that both alleles are considerably less frequent in populations than previously reported.     

Evaluation of a real-time polymerase chain reaction method for the quantification of CYP1B1 gene expression in MCF-7 human breast carcinoma cells

INTRODUCTION: Cytochrome P450 1B1 (CYP1B1) catalyzes the bioactivation of numerous procarcinogens and it is expressed in tumor cells, including human breast cancer cells. To study CYP1B1 gene expression, it is important to have an accurate, precise, reproducible, specific, and quantitative method. METHODS: MCF-7 human breast carcinoma cells were treated with beta-naphthoflavone (BNF; 50 microM), emodin (0.1-3 microM), trans-resveratrol (2.5-20 microM), or 0.1% dimethylsulfoxide (DMSO; vehicle control). Total cellular RNA was isolated and reverse transcribed. cDNA samples were quantified by a fluorescence assay and a constant amount (1 ng) was amplified in a real-time DNA thermal cycler (LightCycler). RESULTS: Melting curve analysis and agarose gel electrophoresis of the amplicons resulted in a single peak and a single band, respectively. The identity of the amplicon was confirmed to be CYP1B1 by sequencing analysis. The standard curve for the real-time PCR amplification of CYP1B1 cDNA was log-linear for at least four orders of magnitude. The limit of quantitation (LOQ) of the assay was 100 copies. At the LOQ, the assay had an accuracy of 8% and a precision of 10%. The intraday (n=4) variability (expressed as percent coefficient of variation) was 9% for a sample with low CYP1B1 mRNA expression (cells treated with 0.1% DMSO; i.e., Sample A) and 3% for a sample with elevated CYP1B1 mRNA expression (cells treated with BNF; i.e., Sample B). The interday (n=4) variability was 16% for Sample A and 15% for Sample B. Emodin increased CYP1B1 mRNA expression in cultured MCF-7 cells (maximal effect of ninefold induction achieved at 1 microM), whereas trans-resveratrol suppressed it (IC(50)=6.6+/-1.0 microM, mean+/-S.E.M., n=3). DISCUSSION: An accurate, precise, reproducible, and specific method is described for the real-time PCR quantification of CYP1B1 gene expression in MCF-7 human breast carcinoma cells.