The effect of the CYP 2C19*2 polymorphism on stroke care

Clopidogrel is an inhibitor of platelet-aggregation used in the prevention of secondary stroke. The molecule is activated by the cytochrome P450 2C19 (CYP2C19) enzyme. The frequent CYP2C19*2 point mutation causes loss of enzyme function, a decreased (heterozygous form) or blocked (homozygous form) formation of the active molecule. Thus, for a patient harboring a mutated allele, clopidogrel does not provide effective protection against stroke. Multiple drugs inhibit the CYP2C19 enzyme and their simultaneous use with clopidogrel is especially hazardous for patients with genetically decreased enzyme activity. Frequency of the CYP2C19*2 is variable in different populations, highest rates were detected in some Asian groups. In our study the CYP2C19 genotype was determined in one Hungarian sample of 354 stroke patients and 221 healthy controls. Frequency of the minor allele was found to be 12.87% (12.85% in stroke patients, 12.89% in healthy controls). The proportion of the homozygous CYP2C19*2 variant causing total loss of gene function was 1.74%, rate of the heterozygous allele causing reduced enzyme activity was 22.26% in the total population. Our results for the allele frequencies of the CYP2C19*2 gene are similar to those found in other Caucasian populations. In conclusion, the homozygous mutation, causing ineffectiveness of clopidogrel is relatively rare. However, the heterozygous form in which interaction of CYP2C19 inhibitors causes further decrease in the genetically impaired enzyme activity is present in every fifth drug-taking patient. Based on our findings, we would like to emphasize that it is important to adjust individually antiplatelet treatment in ischemic stroke patients and to take into consideration genetic factors as well as drugs taken for comorbid conditions.     

Functional Polymorphisms in the CYP2C19 Gene Contribute to Digestive System Cancer Risk: Evidence from 11,042 Subjects

Background

CYP2C19 belongs to the cytochrome P450 superfamily of enzymes involved in activating and detoxifying many carcinogens and endogenous compounds, which has attracted considerable attention as a candidate gene for digestive system cancer. CYP2C19 has two main point mutation sites (CYP2C19*2, CYP2C19*3) leading to poor metabolizer (PM) phenotype. In the past decade, the relationship between CYP2C19 polymorphism and digestive system cancer has been reported in various ethnic groups; however, these studies have yielded contradictory results.

Methods

To clarify this inconsistency, we performed this meta-analysis. Databases including Pubmed, EMBASE, Web of Science and China National Knowledge Infrastructure (CNKI) were searched to find relevant studies. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of association.

Results

In total, 18 studies with 4,414 cases and 6,628 controls were included. Overall, significantly elevated digestive system cancer risk was associated CYP2C19 PM with OR of 1.66 (95%CI: 1.31–2.10, P<10⁻⁵) when all studies were pooled into the meta-analysis. There was strong evidence of heterogeneity (P = 0.006), which largely disappeared after stratification by cancer type. In the stratified analyses according to cancer type, ethnicity, control source and sample size, significantly increased risks were found.

Conclusions

In summary, our meta-analysis suggested that the PM phenotype caused by the variation on CYP2C19 gene is associated with increased risk of digestive system cancer, especially in East Asians.     

Association of poor metabolizers of cytochrome P450 2C19 with head and neck cancer and poor treatment response

A case-control study consisting of 300 patients and an equal number of healthy controls was carried out to investigate the association of polymorphism in cytochrome P450 2C19 (CYP2C19), which results in poor and extensive metabolizers (PMs and EMs) genotypes, with squamous cell carcinoma of head and neck (HNSCC) and treatment response in patients receiving combination of chemo-radiotherapy. A higher frequency of CYP2C19*2 variants was observed in the cases resulting in significantly higher risk to HNSCC (Ad OR 3.36, 95% CI 1.94-5.82, p-value<0.05). The PM genotype of CYP2C19*3 was also found to be slightly increased in the cases, though the increase in risk was not significant when analyzed by multivariate logistic regression model. Tobacco chewing amongst the cases resulted in almost 13-fold increase in the risk with CYP2C19*2 (OR: 12.39) and 3-fold with CYP2C19*3 genotype (OR: 2.90) when compared to the tobacco chewers amongst the controls. Likewise, cigarette smoking in the cases increased the risk approximately 9-fold and 3-fold with CYP2C19*2 (OR: 8.93) and CYP2C19*3 (OR: 2.18) genotypes respectively when compared to smokers amongst the controls. Similar increase in risk was associated with alcohol use amongst the cases carrying variant genotypes of CYP2C19*2 (OR: 7.75) or CYP2C19*3 (OR: 2.60), demonstrating the importance of gene-environment interaction in modifying susceptibility to HNSCC. Interestingly, patients with PMs of CYP2C19 (CYP2C19*2 and CYP2C19*3) exhibited little response to the respective chemotherapy than the patients carrying wild-type genotype demonstrating that functional enzyme deficiencies due to polymorphism in CYPs may not only be important in modifying the susceptibility to HNSCC but also in determining chemotherapeutic response.     

Analysis of CYP2C9*2, CYP2C9*3 and VKORC1 ‐1639 G>A polymorphisms in a population from South‐Eastern Europe

The CYP2C9 enzyme metabolizes a wide range of relevant drugs, among which are oral anticoagulants. VKORC1 is the pharmacodynamic target of the oral anticoagulants. The genetic polymorphisms CYP2C9*2, CYP2C9*3 and VKORC1 ‐1639 G>A are the major determinants of the inter‐individual variability in the dosage requirements of oral anticoagulants. This study provides a first evaluation of these 3 polymorphisms in a Romanian population. A total of 332 Romanian individuals were genotyped for the CYP2C9*2, CYP2C9*3 and VKORC1 ‐1639 G>A polymorphisms using the PCR‐RFLP technique. Sixty‐two individuals (18.7%) were heterozygous for CYP2C9*2, whereas 47 individuals (14.1%) were heterozygous for CYP2C9*3. Fourteen individuals (4.2%) had a CYP2C9*2 homozygous, CYP2C9*3 homozygous or CYP2C9*2/CYP2C9*3 compound heterozygous genotype. These individuals are predicted to have the lowest CYP2C9 enzymatic activity. The allele frequencies of the CYP2C9*2 and CYP2C9*3 polymorphisms were 11.3% and 9.3% respectively. For the VKORC1 ‐1639 G>A polymorphism, there were 170 heterozygotes (51.2%) and 55 (16.6%) homozygotes for the A allele. The frequency of the A allele was 42.2%. Overall, the distribution of the CYP2C9*2, CYP2C9*3 and VKORC1 ‐1639 G>A polymorphisms observed in our cohort is in accordance with other Caucasian populations. A large number of Romanians are expected to harbour at least one CYP2C9 variant allele and/or one VKORC1 ‐1639 G>A allele. This frequency has major implications in the pharmacogenomics of oral anticoagulants in Romanians.     

中国苗族人群中细胞色素P450 2C19基因多态性的研究

为研究细胞色素P45 0 2C19在中国苗族人群中的基因型 ,采用聚合酶链式反应与限制性内切核酸酶片段长度多态性技术分析了 98例无血缘关系苗族人群的基因型。结果显示 5 0人为CYP2C19野生型纯合子 (wt/wt) ;39人为CYP2C19m1杂合子 (wt/m1) ;9人为CYP2C19m1突变型纯合子 (m1/m1)。与国内外相关报道比较 ,本实验得出的CYP2C19m1突变频率与文献报道相符     

Cytochrome P450 2D6 variants in a Caucasian population: allele frequencies and phenotypic consequences.

Cytochrome P450 2D6 (CYP2D6) metabolizes many important drugs. CYP2D6 activity ranges from complete deficiency to ultrafast metabolism, depending on at least 16 different known alleles. Their frequencies were determined in 589 unrelated German volunteers and correlated with enzyme activity measured by phenotyping with dextromethorphan or debrisoquine. For genotyping, nested PCR-RFLP tests from a PCR amplificate of the entire CYP2D6 gene were developed. The frequency of the CYP2D6*1 allele coding for extensive metabolizer (EM) phenotype was .364. The alleles coding for slightly (CYP2D6*2) or moderately (*9 and *10) reduced activity (intermediate metabolizer phenotype [IM]) showed frequencies of .324, .018, and .015, respectively. By use of novel PCR tests for discrimination, CYP2D6 gene duplication alleles were found with frequencies of .005 (*1x2), .013 (*2x2), and .001 (*4x2). Frequencies of alleles with complete deficiency (poor metabolizer phenotype [PM]) were .207 (*4), .020 (*3 and *5), .009 (*6), and .001 (*7, *15, and *16). The defective CYP2D6 alleles *8, *11, *12, *13, and *14 were not found. All 41 PMs (7.0%) in this sample were explained by five mutations detected by four PCR-RFLP tests, which may suffice, together with the gene duplication test, for clinical prediction of CYP2D6 capacity. Three novel variants of known CYP2D6 alleles were discovered: *1C (T1957C), *2B (additional C2558T), and *4E (additional C2938T). Analysis of variance showed significant differences in enzymatic activity measured by the dextromethorphan metabolic ratio (MR) between carriers of EM/PM (mean MR = .006) and IM/PM (mean MR = .014) alleles and between carriers of one (mean MR = .009) and two (mean MR = .003) functional alleles. The results of this study provide a solid basis for prediction of CYP2D6 capacity, as required in drug research and routine drug treatment.     

Prevalence of CYP2C9 and VKORC1 alleles in the Argentine population and implications for prescribing dosages of anticoagulants

Dicumarinic oral anticoagulants have a narrow therapeutic range and a great individual variability in response, which makes calculation of the correct dose difficult and critical. Genetic factors involved in this variability include polymorphisms of genes that encode the metabolic enzyme CYP2C9 and the target enzyme vitamin K epoxide reductase complex 1 (VKORC1); these polymorphisms can be associated with reduced enzymatic expression. We examined the frequency of the most relevant variants encoding CYP2C9 (alleles *1, *2 and *3) and VKORC1 (SNP -1639A>G) in the Argentinian population. Molecular typing was performed by PCR-RFLP on a randomly selected sample of 101 healthy volunteers from the Hospital Italiano de Buenos Aires gene bank. Fifty-seven subjects were identified as homozygous for CYP2C9*1 and 14 for *2, while 24 and 5 were heterozygous for *2 and *3 alleles; one individual was a composite heterozygote (*2/*3). When we examined VKORC1, 21 subjects were AA homozygous, 60 were AG heterozygotes and 20 were GG homozygotes. This is the first analysis of genotypic frequencies for CYP2C9 and VKORC1 performed in an Argentinian population. These allele prevalences are similar to what is known for Caucasian population, reflecting the European ancestor of our patient population, coming mostly from Buenos Aires city and surroundings. Knowledge of this prevalence information is instrumental for cost-effective pharmacogenomic testing in patients undergoing oral anticoagulation treatment.     

Modulation of human dUTPase using small interfering RNA

Analyzing the CYP2A6 gene of subjects who showed a poor metabolic phenotype toward SM-12502, we discovered a novel mutant allele (CYP2A6*4C) lacking the whole CYP2A6 gene. Using genetically engineered Salmonella typhimurium expressing a human CYP, we found that CYP2A6 was involved in the metabolic activation of a variety of nitrosamines such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) contained in tobacco smoke. Taking these results into consideration, we hypothesized that the subjects carrying the CYP2A6*4C allele had lower risk of tobacco-related lung cancer. In accordance with our hypothesis, our epidemiological studies indicated that smokers homozygous for the CYP2A6*4C allele showed much lower odds ratios toward cancer risk. Other mutant alleles reducing the CYP2A6 activity, besides CYP2A6*4C, also reduced the risk of lung cancer in smokers, particularly of squamous-cell carcinoma and small-cell carcinoma, both smoking-related cancers. 8-Methoxypsoralen, an inhibitor of CYP2A6, efficiently prevented the occurrence of adenoma caused by NNK in A/J mice.     

Interaction between CYP 2C19*3 polymorphism and smoking in relation to laryngeal carcinoma in the Chinese Han population

Cytochrome P450 (CYP) 2C19 metabolizes arachidonic acid to biologically active epoxyeicosatrienoic acids, which strongly promote proliferation of cancer cells in vitro and in vivo. Knowing that smoking is the most important risk factor for laryngeal carcinoma, we examined the relationships between CYP2C19*3 polymorphism, smoking and laryngeal carcinoma in the Chinese Han population. In a Chinese Han case-control study of 300 laryngeal carcinoma patients and 300 healthy controls, we investigated polymorphism in the CYP2C19 gene by PCR-RFLP analysis. The CYP2C19*3 AG + AA genotype was significantly more prevalent in laryngeal carcinoma patients (6.67 vs 2.67%; P = 0.02). Multiple logistic regression analysis showed smoking (odds ratio (OR) = 6.353, 95% confidence interval (CI) = 4.413-9.144; P < 0.001) and alcohol consumption (OR = 2.607, 95%CI = 1.130-6.016; P = 0.025) as independent risk factors for laryngeal carcinoma; there was a significant interaction between CYP2C19*3 and smoking (OR = 17.842, 95%CI = 13.32-31.102; P = 0.009). We conclude that CYP2C19*3 polymorphism is significantly associated with laryngeal carcinoma in the Chinese Han population.     

Identification of new alleles and the determination of alleles and genotypes frequencies at the CYP2D6 gene in Emiratis

CYP2D6 belongs to the cytochrome P450 superfamily of enzymes and plays an important role in the metabolism of 20-25% of clinically used drugs including antidepressants. It displays inter-individual and inter-ethnic variability in activity ranging from complete absence to excessive activity which causes adverse drug reactions and toxicity or therapy failure even at normal drug doses. This variability is due to genetic polymorphisms which form poor, intermediate, extensive or ultrarapid metaboliser phenotypes. This study aimed to determine CYP2D6 alleles and their frequencies in the United Arab Emirates (UAE) local population. CYP2D6 alleles and genotypes were determined by direct DNA sequencing in 151 Emiratis with the majority being psychiatric patients on antidepressants. Several new alleles have been identified and in total we identified seventeen alleles and 49 genotypes. CYP2D6*1 (wild type) and CYP2D6*2 alleles (extensive metaboliser phenotype) were found with frequencies of 39.1% and 12.2%, respectively. CYP2D6*41 (intermediate metaboliser) occurred in 15.2%. Homozygous CYP2D6*4 allele (poor metaboliser) was found with a frequency of 2% while homozygous and heterozygous CYP2D6*4 occurred with a frequency of 9%. CYP2D6*2xn, caused by gene duplication (ultrarapid metaboliser) had a frequency of 4.3%. CYP2D6 gene duplication/multiduplication occurred in 16% but only 11.2% who carried more than 2 active functional alleles were considered ultrarapid metabolisers. CYP2D6 gene deletion in one copy occurred in 7.5% of the study group. In conclusion, CYP2D6 gene locus is heterogeneous in the UAE national population and no significant differences have been identified between the psychiatric patients and controls.     

Frequency of single nucleotide polymorphisms of CYP2D6 in the Czech population

CYP2D6 is a member of cytochrome P450 enzymes that metabolise over 25% of commonly used drugs. Genetic polymorphisms can cause insufficient drug efficacy at usually administered doses or can be the cause of adverse drug reaction. CYP2D6 genotyping can be used to predict CYP2D6 phenotype and thereby explain some abnormalities in drug response and thus optimize pharmacotherapy. The aim of this study was to investigate the frequency of functionally important variant alleles of the CYP2D6 gene throughout the Czech population to predict the prevalence of ultra-rapid and poor metabolizer phenotypes. The DNA of 223 unrelated, healthy volunteers was analysed to detect the presence of CYP2D6*6, *5, *4, *3 and gene duplication. The variant allele frequencies in our population were 0.22%, 3.14%, 22.87%, 1.12% and 3.14% for CYP2D6*6, CYP2D6*5, CYP2D6*4, CYP2D6*3 and CYP2D6*MxN, respectively. Fifteen subjects carried two variant alleles leading to predicted poor type of metabolism, 84 subjects were heterozygous extensive metabolizers (het-EM). The full-text contains detailed comparison with European white populations. The distribution of variant alleles complies with the Hardy-Weinberg equilibrium. The frequencies of functional variant alleles of CYP2D6 in Czech population are in concordance with other Caucasian populations.     

中国苗族人群中细胞色素P450 2C19基因多态性的研究

为研究细胞色素P4502C19在中国苗族人群中的基因型,采用聚合酶链式反应与限制性内切核酸酶片段长度多态性技术分析了98例无血缘关系苗族人群的基因型。结果显示50人为CYP2C19野生型纯合子（wt/wt）;39人为CYP2C19m1杂合子（wt/m1）;9人为CYP2C19m1突变型纯合子（m1/m1）。与国内外相关报道比较,本实验得出的CYP2C19m1突变频率与文献报道相符。 Abstract：In order to study the cytochrome P450 2C19 gene polymorphism in Miao population, the genotypes of ninety-eight unrelated Miao subjects were studied by polymera se chain reaction and restricted fragment length polymorphism (RFLP). The result s showed that fifty were homozygous for wild-type (wt/wt); thirty-nine wer e heterozygous for the CYP2C19m1 (wt/m1);nine were homozygou s for CYP2C19m1(m1/m1).The frequency of CYP2C19m1 was in agreement with that of other published data.     

Bisoprolol responses (PK/PD) in hypertensive patients: A cytochrome P450 (CYP) 2D6 targeted polymorphism study

BackgroundBisoprolol is an effective β1-adrenergic blocker, an inter-individual genetic variability was recorded in its response. This study aimed at investigating the association of CYP2D6*2A (rs1080985) and CYP2D6*10 (rs1065852) single-nucleotide polymorphism (SNP) with Bisoprolol response in cardiac patients attending King Abdulaziz University Hospital, Jeddah, Kingdom of Saudi Arabia.Patients and methodsIn the study, 107 patients were enrolled. Five mL of venous blood was collected from each patient and genotyping for CYP2D6*2A and CYP2D6*10 using Vivid® CYP2D6 Green Screening Kit (Life Technologies, USA). Response to Bisoprolol was evaluated through assessment of diastolic and systolic blood pressure and by measuring Bisoprolol plasma level using triple quad mass spectrometer (TQ-MS).ResultsAll patients were found to carry homozygous wild type CYP2D6*10 (GG) and none were carrying heterozygous (GA) or mutant homozygous (AA) genotype. CYP2D6*2A allele was detected in the homozygous wild type (GG) in 70 out of 107 patients, the heterozygous (GC) in 19 patients, and the homozygous mutant (CC) in 18 patients with minor allele frequency (MAF) of 25.7%. The plasma concentrations of Bisoprolol in CC carriers were significantly lower than those in GG & CC carriers by 25%, and 51%; respectively. Higher systolic and diastolic blood pressures were also observed in CC carriers than GG and CC carriers.ConclusionThere is a possible association of CYP2D6*2A genotype with plasma concentration of bisoprolol. This could provide a helpful tool to choose the optimum dose for bisoprolol, depending on the patient’s genotyping, in order to increase effectiveness and ameliorate its toxicity.     

应用HRM技术对CYP2C19*2和CYP2C19*3进行双重SNP分型

氯吡格雷是一种广泛用于预防静脉血栓形成的抗血小板药物。研究表明, 携带有CYP2C19基因功能缺失型等位基因CYP2C19*2、CYP2C19*3的病人, 其体内代谢氯吡格雷成为其活性形式的能力降低, 导致氯吡格雷抑制血小板聚集功能减弱。文章旨在建立一种利用高分辨率熔解曲线分析(High-resolution melting curve analysis,HRM)技术在闭合单管中同时对CYP2C19*2、CYP2C19*3两个多态性位点进行简便、准确分型的方法。本实验针对两个SNP位点分别设计特异性的HRM引物, 并在两个位点引物的5′端分别加上富含AT和GC的序列, 保证两个位点的扩增产物熔解峰无重叠。利用HRM技术, 快速、灵敏地对64例随机DNA样本的CYP2C19*2 、CYP2C19*3两个多态性位点进行了基因分型, 且HRM方法的分型结果与测序验证结果完全一致。因此, 利用HRM技术可以实现在闭合单管中简便、准确地对CYP2C19*2 、CYP2C19*3两个多态性位点同时进行基因分型。该方法有望应用于临床, 指导氯吡格雷的个体化用药。     

Allele and genotype frequencies of the polymorphic cytochrome P450 genes (CYP1A1, CYP3A4, CYP3A5, CYP2C9 and CYP2C19) in the Jordanian population

Drug metabolizing enzymes participate in the neutralizing of xenobiotics and biotransformation of drugs. Human cytochrome P450, particularly CYP1A1, CYP2C9, CYP2C19, CYP3A4 and CYP3A5, play an important role in drug metabolism. The genes encoding the CYP enzymes are polymorphic, and extensive data have shown that certain alleles confer reduced enzymatic function. The goal of this study was to determine the frequencies of important allelic variants of CYP1A1, CYP2C9, CYP2C19, CYP3A4 and CYP3A5 in the Jordanian population and compare them with the frequency in other ethnic groups. Genotyping of CYP1A1(m1 and m2), CYP2C9 (*2 and *3), CYP2C19 (*2 and *3), CYP3A4*5, CYP3A5 (*3 and *6), was carried out on Jordanian subjects. Different variants allele were determined using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). CYP1A1 allele frequencies in 290 subjects were 0.764 for CYP1A1*1, 0.165 for CYP1A1*2A and 0.071 for CYP1A1*2C. CYP2C9 allele frequencies in 263 subjects were 0.797 for CYP2C9*1, 0.135 for CYP2C9*2 and 0.068 for CYP2C9*3. For CYP2C19, the frequencies of the wild type (CYP2C19*1) and the nonfunctional (*2 and *3) alleles were 0.877, 0.123 and 0, respectively. Five subjects (3.16?%) were homozygous for *2/*2. Regarding CYP3A4*1B, only 12 subjects out of 173 subjects (6.9?%) were heterozygote with none were mutant for this polymorphism. With respect to CYP3A5, 229 were analyzed, frequencies of CYP3A5*1,*3 and *6 were 0.071, 0.925 and 0.0022, respectively. Comparing our data with that obtained in several Caucasian, African-American and Asian populations, Jordanians are most similar to Caucasians with regard to allelic frequencies of the tested variants of CYP1A1, CYP2C9, CYP2C19, CYP3A4 and CYP3A5.     

Prevalence of CYP2C19 polymorphisms in the Lebanese population

Clopidogrel is one of the most commonly prescribed drugs, as its combination with low-dose aspirin is the recommended oral anti-platelet therapy, to prevent ischaemic events following coronary syndromes or stent placement. Numerous recent studies have shown that polymorphisms in the gene encoding the cytochrome P450 (CYP450) 2C19 enzyme (CYP2C19) contribute to variability in response to clopidogrel; patients with certain common genetic variants of CYP2C19 (*2, *3) have a reduced metabolism of clopidogrel and have a higher rate of cardiovascular events or stent thrombosis compared to patients with the CYP2C19 (*1) allele. CYP2C19*2 is most common in Caucasians, Africans and Asians while CYP2C19*3 has been found mostly in Asians. Since the prevalence of these variants in the Lebanese population has not yet been reported, our aim was to determine the genotypes of CYP2C19 in our population. CYP2C19 (*1/*2/*3) variants were assessed by Polymerase Chain Reaction-Restriction Length Polymorphism (PCR–RFLP) assays in a representative sample of 161 unrelated healthy Lebanese volunteers. The allele frequencies of CYP2C19 *2 and *3 were 0.13 and 0.03. Carriers of the CYP2C19 *2 or *3 represented 24.2% of the subjects. Our data show no significant difference in the frequency of CYP2C19 allelic variants when compared to Caucasian populations and demonstrate that the application of the recent FDA recommendations would also be beneficial in Lebanon, allowing physicians to identify patients at high risk for atherothrombotic events, and eventually advising them to consider other antiplatelet medications or alternative dosing strategies in poor metabolizers.     

Debrisoquine/sparteine hydroxylation genotype and phenotype: analysis of common mutations and alleles of CYP2D6 in a European population.

Four different mutations of the cytochrome P450 CYP2D6 gene associated with the poor metabolizer phenotype (PM) of the debrisoquine/sparteine polymorphism were analyzed by Xba I restriction fragment length polymorphism (RFLP) analysis and a polymerase chain reaction (PCR)-based DNA amplification method in DNA of 394 healthy European subjects; 341 of these were phenotyped by sparteine or debrisoquine administration and urinary metabolic ratios (MR). Our study demonstrates the efficiency of the PCR-test for phenotype prediction; 96.4% of individuals were correctly predicted, i.e., 100% of the extensive metabolizers (EMs) and 86.0% of the poor metabolizers (PMs). In contrast, Xba I RFLP analysis was far less informative, predicting the phenotype in only 26.8% of PMs. By combining both DNA tests, the prediction rate of the PM phenotype increased to 90.6%. A point mutation at a splice-site consensus sequence termed D6-B represented the most common mutant CYP2D6 gene and accounted for more than 75% of mutant alleles. In addition, other known mutations such as D6-D (14%), D6-A (5%), and the rare D6-C mutation bring the identified mutant alleles to greater than 95% of all mutant PM-alleles. Most of Xba I 44-kb alleles were confirmed as mutant alleles carrying the D6-B mutation. However, 9.7% did not have this mutation and may express a functional CYP2D6 gene. Moreover, all Xba I 16 + 9-kb alleles contained the D6-B mutation. Heterozygous EM individuals had a significantly higher MR when compared to homozygous EMs. Genotyping provides an important advantage for investigations of the influence of CYP2D6 activity on drug therapy and its association with certain diseases.     

Comparison between MassARRAY and pyrosequencing for CYP2C19 and ABCB1 gene variants of clopidogrel efficiency genotyping

Clopidogrel is one of the most frequently used drugs in patients to reduce cardiovascular events. Since patients with different genetic variations respond quite differently to clopidogrel therapy, the related genetic testing plays a vital role in its dosage and genetic testing related to clopidogrel therapy is currently considered as routine test worldwide. In this study, we aim to use two different methods MALDI-TOF mass spectrometry and pyrosequencing to detect gene variant of CYP2C19 and ABCB1. Six single nucleotides polymorphisms (SNP) within CYP2C19 (*2, *3, *4, *5, *17) and ABCB1 C3435T in 458 Chinese Han patients were determined using both MassARRAY and Pyrosequencing. Sanger sequencing was used for verification. Results of both methods were analyzed and compared. Allele frequencies of each SNP and distribution of different genotypes were calculated based on the MassARRAY and Sanger sequencing results. Both methods provided 100% call rates for gene variants, while results of six samples were different with two methods. With Sanger sequencing as the reference results, MassARRAY generated all the same results. The minor allele frequencies of the above six SNPs were 27.1% (CYP2C19*), 5.9% (CYP2C19*3), 0% (CYP2C19*4), 0% (CYP2C19*5), 1.1% (CYP2C19*17), 40.9% (ABCB1), respectively. MassARRAY provides accurate clopidogrel related genotyping with relatively high cost-efficiency, throughput and short time when compared with pyrosequencing.     

Association of CYP2C19*3 gene polymorphism with breast cancer in Chinese women

Cytochrome P450 (CYP) 2C19 metabolizes arachidonic acid to biologically active epoxyeicosatrienoic acids, which significantly promote proliferation of cancer cells in vitro and in vivo. We looked for a possible association between human CYP2C19*3 gene polymorphism and breast cancer in the Chinese Han population. In a Chinese Han case-control study of breast cancer patients (N = 600) and age- and gender-matched healthy controls (N = 600), we investigated polymorphism in the CYP2C19 gene by PCR-RFLP analysis. The CYP2C19*3 AG + AA genotype was significantly more prevalent in breast cancer patients than in control subjects (6.67 vs 3.00%; P = 0.003). The odds ratio for carriers of AG + AA genotype for breast cancer was 2.31 (95% confidence interval = 1.27-4.43). Among patients, estrogen receptor, tumor size, histologic grade, presence of primary lymphonode metastases, progesterone receptor positivity, and age at diagnosis were not found to be significantly associated with CYP2C19*3 genotypes (all P > 0.05). We conclude that the CYP2C19*3 gene polymorphism is associated with breast cancer risk in Chinese Han women.     

Gene-environmental interactions between alcohol-drinking behavior and ALDH2 and CYP2E1 polymorphisms and their impact on micronuclei frequency in human lymphocytes

Ethanol is converted to acetaldehyde by alcohol dehydrogenase (ADH), cytochrome p4502E1 (CYP2E1) and catalase. This metabolite is then detoxified by aldehyde dehydrogenase 2 (ALDH2), a key enzyme in the elimination of acetaldehyde, via further oxidation to acetic acid. The toxic effects of acetaldehyde are well documented and may be partially mediated by genotoxic damage. In the present study, we investigated the effects of alcohol-drinking behavior and genetic polymorphisms in two different genes (ALDH2 and CYP2E1) on the micronuclei (MN) frequency in 248 healthy Japanese men. Genotyping was performed by PCR-RFLP analysis. The ALDH2 variant (deficient type) was significantly associated with an increased MN frequency in subjects drinking more than three times/wk, while habitual drinkers with wild-type CYP2E1 also had a significantly increased MN frequency. Furthermore, when the subjects were divided into eight groups according to their drinking frequency and genotypes of ALDH2 and CYP2E1, we found that habitual drinkers with homozygous CYP2E1*1/*1 and heterozygous ALDH2*1/*2 or homozygous ALDH2*2/*2 showed the highest mean MN frequency. In the present study, we found clear associations among ALDH2 and CYP2E1 gene polymorphisms, alcohol-drinking behavior and genotoxic effects in a healthy Japanese population. Therefore, analysis of the polymorphisms of alcohol-metabolizing enzymes may lead to elucidation of the mechanism(s) for individual susceptibilities to the toxicity of ethanol metabolites.