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。     

CYP2D6*11 and challenges in clinical genotyping of the highly polymorphic CYP2D6 gene

CYP2D6 is genotyped clinically for prediction of response to tamoxifen, psychotropic drugs and other medications. Phenotype prediction is dependent upon accurate genotyping. The CYP Allele Nomenclature Committee maintains the allelic nomenclature for CYP2D6; however, in some cases, the list of polymorphisms associated with a given allele is incomplete. Clinical laboratories and in vitro diagnostic manufacturers rely upon this nomenclature, in addition to the literature, to infer allelic function and haplotypes and when they design CYP2D6-testing platforms. This article provides more complete sequencing data for the CYP2D6*11 allele and describes the difficulties encountered in genotyping CYP2D6 when incomplete data are available. The CYP Allele Nomenclature Committee should provide clear information about the completeness of the original data used to define each allele.     

A novel CYP2A6 allele, CYP2A6*23, impairs enzyme function in vitro and in vivo and decreases smoking in a population of Black-African descent

OBJECTIVES: CYP2A6 is the main enzyme involved in nicotine metabolism in humans. We have identified a novel allele, CYP2A6*23 (2161C>T, R203C), in individuals of Black-African descent and investigated its impact on enzyme activity and association with smoking status. METHODS: Wild-type and variant enzymes containing amino acid changes R203C (CYP2A6*23), R203S (CYP2A6*16) and V365M (CYP2A6*17) were expressed in Escherichia coli. The effect of CYP2A6*23 in vivo was examined in individuals of Black-African descent given 4 mg oral nicotine. RESULTS: CYP2A6*23 occurred at an allele frequency of 2.0% in individuals of Black-African descent (N=560 alleles, 95% confidence interval, 0.8-3.1%) and was not detected in Caucasians (N=334 alleles), Chinese (N=288 alleles) or Japanese (N=104 alleles). In vitro, CYP2A6.23 had greatly reduced activity toward nicotine C-oxidation similar to CYP2A6.17, as well as reduced coumarin 7-hydroxylation. Conversely, CYP2A6.16 did not differ in activity compared with the wild-type enzyme. The trans-3'-hydroxycotinine to cotinine ratio, a phenotypic measure of CYP2A6 activity in vivo, was lower in CYP2A6*1/*23 and CYP2A6*23/*23 individuals (mean adjusted ratio of 0.60, n=5) compared with CYP2A6*1/*1 individuals (mean adjusted ratio of 1.21, n=150) (P<0.04). CYP2A6*23 trended toward a higher allele frequency in nonsmokers (3.1%, N=9/286 alleles) compared with smokers (0.7%, N=2/274 alleles) (P=0.06). CONCLUSION: These results suggest the novel CYP2A6*23 allele impairs enzyme function in vitro and in vivo and trends toward an association with lower risk of smoking.     

Novel structure of the CYP2D6 gene that confuses genotyping for the CYP2D6*5 allele

We encountered DNA samples which showed a positive product using a long PCR-based method for the detection of CYP2D6*5, indicating deletion of the entire CYP2D6 gene, but the samples did not show a band related to CYP2D6*5 in either XbaI- or EcoRI-RFLP analysis. To achieve genotyping with accuracy, we performed a further genetic analysis to clarify the discrepancy. An unknown 1.6-kb insert was identified in a region downstream from the CYP2D6 stop codon where a specific primer was designed for long-PCR analysis for CYP2D6*5 genotyping. This finding suggested that the CYP2D6 gene might not be deleted in the samples even if a positive product was detected by the long-PCR method. Furthermore, the allelic frequency of this type was found to be approximately 0.3% (4 heterozygous/771 samples) in a Japanese population. In conclusion, we found a novel structure of the CYP2D6 gene, which might lead to incorrect genotyping for CYP2D6*5. Although the long PCR-based strategy for the detection of CYP2D6*5 has been widely used due to its usefulness and convenience, we recommend caution when adopting this method and propose re-evaluating the method for detecting CYP2D6*5.     

Isolation, sequence and genotyping of the drug metabolizer CYP2D6 gene in the Colombian population

Cytochrome P450 2D6 monooxygenase metabolizes several commonly used drugs, particularly psychotropics and cardiovascular agents. The gene that encodes this isoenzyme is highly polymorphic, with 1-10% of the population carrying mutations that produce an inactive enzyme, and 1-29% of individuals who possess additional copies of functional CYP2D6 genes. The genotypic features of the CYP2D6 gene have already been studied in many ethnic groups; however, the genetic characteristics of this enzyme are unknown in the Colombian population. The allelic variants and mutations of this polymorphic isoenzyme are the main cause of interindividual and interethnic differences in the therapeutic efficacy and adverse effects at standard doses of drugs metabolized by the products of the CYP2D6 gene. In the present study we have isolated, sequenced and genotyped the CYP2D6 gene in the Colombian population. The distribution of allelic frequencies of 10 alleles associated with normal, diminished or increased CYP2D6 activity has been studied in 121 healthy volunteers. The commonest alleles detected in the Colombian people were the functional alleles *1 (38.8%) and *2 (37%). Among the seven nonfunctional alleles studied in our sample, we found frequencies of 19.4%, 1.6%, 1.2% and 0.8%, for the *4, *17, *3 and *5 alleles, respectively. The alleles *6, *7 and *8 could not be identified in any of the subjects studied. The frequency of the duplicate allele was 1.2%. In this Colombian sample, 91.7% of the individuals were normal metabolizers (EM), 6.6% were poor metabolizers (PM), and 1.7% were ultrarapid metabolizers (UM). These results show that the allelic distribution of the CYP2D6 gene in the Colombian population of mestizo-prevalent subjects is compatible with the genomic assembly of the constitutive tri-ethnic origin of this Latin American country.     

CYP2A6 gene polymorphism and personality traits for NEO-FFI on the smoking behavior of youths

We performed a survey on the relationship between nicotine dependence and psychological (the personality traits using neuroticism extroversion openess-five factor inventory (NEO-FFI)/nicotine metabolism (the CYP2A6 gene polymorphism) factors among Japanese young students to elucidate the mechanism of the development of nicotine dependence. As a result, the frequency of the CYP2A6*4C gene (enzyme activity deficit) was significantly (p<0.05) higher among nonsmokers than smokers, and the Openness score by NEO-FFI was higher among smokers than nonsmokers. However, there were no significant differences in the Neuroticism, Extraversion, Agreeableness, and Conscientiousness scores by NEO-FFI between smokers and nonsmokers. We conclude that the CYP2A6*4C gene and the Openness personality trait may affect the development of the smoking behavior of youth.     

AmpliChip CYP450 GeneChip: a new gene chip that allows rapid and accurate CYP2D6 genotyping

Methods for Cytochrome P450-2D6 (CYP2D6) genotyping are often time-consuming and laborious, which can restrict their use in pretherapeutic screening programs. Gene chip technology could overcome this problem. The aim of this study was to evaluate CYP2D6 genotyping by a new improved gene chip compared to a PCR-RFLP method. AmpliChip CYP450 GeneChip(R) (AmpliChip) is a microarray hybridization method for genotyping CYP2D6 and CYP2C19. One hundred fifty-nine DNA samples were genotyped both by AmpliChip as well as by PCR-RFLP and, where applicable, by a SNaPshot technique which detects single nucleotide polymorphisms based on the single base extension principle. In 152 of the 159 samples, CYP2D6 genotypes determined with the AmpliChip were in accordance with the results of PCR-RFLP. All seven discrepant samples had gene duplications and were subjected to SNaPshot analysis. SNaPshot results concurred with those of the AmpliChip for six out of seven samples. In the one divergent result, DNA sequencing confirmed that the AmpliChip had assigned the correct genotype. In conclusion, AmpliChip is a highly reliable method for CYP2D6 genotyping that allows the correct determination of all relevant CYP2D6 alleles in one single run. It therefore represents a very efficient and fast method, offering new perspectives for the application of pharmacogenetics in clinical medicine.     

Pharmacogenetic Screening of the Gene Deletion and Duplications of CYP2D6

Cytochrome P450 (CYP) 2D6 is one of the most important enzymes involved in the metabolism of drugs. Multiple, clinically relevant, genetic variants of this gene have been identified and, among them, a gene deletion as well as multiplications of the gene. These large structural mutations in CYP2D6 occur at a relatively high frequency in several populations. Genotyping of CYP2D6 could therefore be applied to individualize drug therapy to improve therapeutic efficacy and decrease adverse effects in patients. However, a prerequisite for the pharmacogenetic screening of CYP2D6 in a clinical setting is the development of fast, reliable and cost-effective techniques for the routine genotyping of patients. In the case of CYP2D6, besides the general problems that arise in the detection of large gene deletions and multiplications, the presence of two highly homologous pseudogenes, CYP2D7 and CYP2D8, forms an extra challenge.

This review provides an overview of the techniques that have been described to detect the CYP2D6 gene deletion and multiplication: Southern-blotting RFLP, long-template PCR, and real-time PCR. Of these techniques, real-time PCR is the only technique giving quantitative information about the exact copy number of the gene. Considering all of the other advantages of this method over other methods, such as cost-effectiveness and suitability for high throughput screening, real-time PCR is the most promising method for the genotyping of large structural alterations in the CYP2D6 gene in a routine clinical setting.     

Sequence-based CYP2D6 genotyping in the Korean population

For clinical application of pharmacogenetic tests, quantitative prediction of enzyme activity based on accurate determination of genotype is essential. There has been limited information available on the genetic polymorphism of CYP2D6 in the Korean population. In this study, CYP2D6 genotypes were assessed in 400 Korean subjects. Twenty-eight different CYP2D6 alleles and 35 genotypes were detected. On the basis of the genotype determined, the frequency of poor metabolizers and ultrarapid metabolizers were 0.22% and 1.25%, respectively. The CYP2D6 activity expected in regard to different allele combinations varies widely within the extensive and intermediate metabolizer groups. The frequencies of CYP2D6*10 and CYP2D6*5 were 45.00% and 6.13%, respectively. CYP2D6*10xN was found in 4 out of 9 cases with a CYP2D6 duplication. Fifteen heterozygotes for *41 were noted. In addition, the authors measured plasma concentrations of 16 healthy volunteers after administration of nortriptyline and identified the impact of the CYP2D6 genotype on nortriptyline metabolism. This is the first large-scale study to examine the genetic polymorphism of CYP2D6 using sequence-based genotyping in an Asian population. Our results further the understanding of CYP2D6 pharmacogenetics and could be helpful for future clinical studies in the Asian population.     

Lack of association between smoking and CYP2A6 gene polymorphisms in A Japanese population.

This study determined the genotypes of the CYP2A6 gene in 96 smokers and 141 non-smokers in a Japanese population. The frequencies of wild-type of the CYP2A6* 1 and those with a whole deletion of the CYP2A6 gene were 93 (96.9%) and 3 (3.1%) in 96 smokers, and 134 (95.0%) and 7 (5.0%) in non-smokers, respectively. In addition, neither the CYP2A6* 2 nor CYP2A6* 3 alleles were observed in the population studied. There were no significant differences in the CYP2A6 genotype frequencies between smokers and non-smokers, as well as in the number of cigarettes smoked and the nicotine amounts inhaled per day between the CYP2A6* 1 and the deletion of CYP2A6. These results suggest that either the deletion or non-deletion of the CYP2A6 gene shows no significant effect on smoking behavior for the Japanese population examined.     

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.     

丹参注射液对家兔体内CYP1A2、CYP2D6和CYP3A4活性的影响

目的：用Cocktail探针药物法研究丹参注射液对家兔细胞色素P450亚型酶活性的影响。方法：将家兔随机分成四组,耳缘静脉注射给予丹参注射液,分低、中、高剂量三个实验组,空白对照组给予生理盐水。用HPLC法测定各组Cock-tail探针药物（咖啡因、美托洛尔和氨苯砜）的血药浓度,比较药时曲线及药代动力学参数的变化,评价各组细胞色素P450亚型酶的活性。结果：丹参注射液低、中、高剂量组CYP2D6的活性与对照组相比降低,具有统计学意义（P〈0.05或P〈0.01）;CYP1A2和CYP3A4的活性与对照组相比差异均无统计学意义。结论：丹参注射液对家兔CYP2D6有抑制作用,对CYP1A2和CYP3A4的影响不显著。提示临床应用丹参注射液时,应关注其对CYP2D6的抑制作用,避免联合用药引发CYP2D6介导的药物相互作用,促进临床安全合理用药。     

Pharmacogenomics of CYP2A6, CYP2B6, CYP2C19, CYP2D6, CYP3A4, CYP3A5 and MDR1 in Vietnam

Aim  The aim of this study was to obtain pharmacogenetic data in a Vietnamese population on genes coding for proteins involved in the elimination of drugs currently used for the treatment of malaria and human immunodeficiency virus/acquired immunodeficiency syndrome. Method  The main polymorphisms on the cytochrome P450 (CYP) genes, CYP2A6, CYP2B6, CYP2C19, CYP2D6, CYP3A4 and CYP3A5, and the multi-drug resistance 1 gene (MDR1) were genotyped in 78 healthy Vietnamese subjects. Pharmacokinetic metrics were available for CYP2A6 (coumarin), CYP2C19 (mephenytoin), CYP2D6 (metoprolol) and CYP3As (midazolam), allowing correlations with the determined genotype. Results  In the CYP2 family, we detected alleles CYP2A6*4 (12%) and *5 (15%); CYP2B6*4 (8%), *6 (27%); CYP2C19*2 (31%) and *3 (6%); CYP2D6*4, *5, *10 (1, 8 and 44%, respectively). In the CYP3A family, CYP3A4*1B was detected at a low frequency (2%), whereas CYP3A5 *3 was detected at a frequency of 67%. The MDR1 3435T allele was present with a prevalence of 40%. Allele proportions in our cohort were compared with those reported for other Asian populations. CYP2C19 genotypes were associated to the S-4′-OH-mephenytoin/S-mephenytoin ratio quantified in plasma 4 h after intake of 100 mg mephenytoin. While CYP2D6 genotypes were partially reflected by the α-OH-metroprolol/metoprolol ratio in plasma 4 h after dosing, no correlation existed between midazolam plasma concentrations 4 h post-dose and CYP3A genotypes. Conclusions  The Vietnamese subjects of our study cohort presented allele prevalences in drug-metabolising enzymes that were generally comparable with those reported in other Asian populations. Deviations were found for CYP2A6*4 compared to a Chinese population (12 vs. 5%, respectively; P = 0.023), CYP2A6*5 compared with a Korean population (15 vs. <1%, respectively; P < 0.0001), a Malaysian population (1%; P < 0.0001) and a Chinese population (1%; P < 0.0001); CYP2B6*6 compared with a Korean population (27 vs. 12%; P = 0.002) and a Japanese population (16%; P = 0.021). Pharmacokinetic metrics versus genotype analysis reinforces the view that the predictive value of certain globally common variants (e.g. CYP2D6 single nucleotide polymorphisms) should be evaluated in a population-specific manner.     

Decreasing smoking behaviour and risk through CYP2A6 inhibition

The consequences of tobacco dependence are both health related and economic. Novel treatment approaches are needed to offer alternatives to patients and to improve treatment outcomes. We review concepts and selected recent discoveries in the area of treatment, with a specific orientation towards drug development. Current treatments are outlined and we highlight new strategies that are based on the manipulation of cytochrome P450 2A6 (CYP2A6) activity, which is responsible for the metabolism of nicotine. The clinical implications of CYP2A6 polymorphisms have been linked to a decreased risk of tobacco dependence, a decrease in number of cigarettes smoked and reduced risk of tobacco-related cancers. Further, we discuss a range of models for proof-of-concept studies for new treatments to alleviate tobacco dependence     

An optimized methodology for combined phenotyping and genotyping on CYP2D6 and CYP2C19

A method for simultaneous phenotyping and genotyping for CYP2D6 and CYP2C19 was tested. Six healthy volunteers were selected (three extensive and three poor metabolisers for CYP2D6). CYP2D6 was probed with dextromethorphan and metoprolol and CYP2C19 was probed with omeprazole. Blood samples were collected and analysed for dextromethorphan, dextrorphan, metoprolol, alpha-hydroxymetoprol, omeprazole and 5-hydroxyomeprazole by HPLC. Genotyping was performed for both CYP2D6 and CYP2C19. Generally, plasma levels could be measured up to 8 h post-dose except for alpha-hydroxymetoprolol in poor metabolizers (PMs) and dextromethorphan in extensive metabolizers (EMs) (35% below quantification limit). The correlation between the metabolic ratio based on timed individual measurements and the metabolic ratio based on the AUC0-12 values was significant at 3 h post-dose for all probes. In conclusion, the following procedure is suggested: administer metoprolol (100 mg) and omeprazole (40 mg); after 3 h, take a blood sample to assess the genotype and the metabolic ratio for CYP2D6 (metoprolol over alpha-hydroxymetoprolol) and CYP2C19 (omeprazole over 5-hydroxyomeprazole) in plasma. With this procedure, all necessary information on the individual CYP2D6 and CYP2C19 metabolising capacity can be obtained in a practical, single-sample approach.     

Use of CYP2D6 genotyping in practice: tamoxifen dose adjustment

Tamoxifen is a selective estrogen-receptor modulator that is commonly utilized in the treatment and prevention of endocrine receptor-positive breast cancer. Ultimate conversion of the parent drug by the enzyme CYP2D6 to the active metabolite, endoxifen, is required for tamoxifen to exert its anticancer effects. CYP2D6 exists in varying concentrations across individuals due, in part, to genetic variation. Lower concentrations of endoxifen have been associated with inferior breast cancer outcomes in numerous retrospective trials. In an effort to increase the endoxifen concentrations, three prospective trials have assessed different methods of increasing tamoxifen dose based on patient CYP2D6 genotypes. All three demonstrated the ability to increase endoxifen concentrations using tamoxifen at a dose of 30 or 40 mg daily. These positive findings support future investigations to determine, not only the clinical benefit of genotype-guided therapy, but also the optimal dose needed for individual patients.     

Short communication. The effects of drinking and smoking on the CYP2D6 metabolic capacity.

We studied the influence of drinking and smoking habits on CYP2D6 metabolic capacity measured by the use of debrisoquine as a substance test. We did not find any significant differences in the frequency of subjects with CYP2D6 deficiency (poor metabolizers) among four groups of healthy individuals: nonsmokers/nondrinkers, smokers/drinkers, nondrinkers/smokers, and nonsmokers/drinkers. We demonstrated that, among poor metabolizers, alcohol and tobacco consumption was associated with higher metabolic ratios than it was with the control group, but the differences were not statistically significant. Among extensive metabolizers, the lowest metabolic ratio (highest enzyme activity) was detected for nondrinkers/smokers, intermediate values for smokers/drinkers, and the highest metabolic ratio (lowest enzyme activity) for nonsmokers/drinkers. These variations were slight but statistically significant when logarithmic ratio values were applied. These results show that smoking and drinking habits do not need to be taken into account when humans are phenotyped for CYP2D6.     

CYP2D6 PCR基因型与DXT表型和基因芯片检测的比较

目的：为了评价CYP2D6的基因型和表型的联系以及基因芯片在CYP2D6多基因分析中的应用。方法：242健康志愿者，口服dextromethorphan后收集认测定其代谢率，收集20ml血提取DNA，并通过基因特异性PCR和／（或）基因芯片分析CYP2D6＊2－＊11，＊17和多拷贝CYP2D6基因，其中5个基因（＊3，＊4，＊6，＊7和＊9）用PCR和CYD450基因芯片同时分析。结果：CYP2D6基因型比表型更富有信息和更能反映CYP2D6酶的表达。CYP2D6＊3，＊4，＊6，＊6和＊9的基因检测在CYP450基因芯片和基因特异性PCR中显示高度的一致性。结论：基因芯片在检测基因多位点的多基因中是一个有发展前途和可靠的方法。