等位基因特异荧光PCR法检测CYP2C19基因型

目的了解人群CYP2C19基因型的分布,评价等位基因特异荧光PCR法检测CYP2C19基因型。方法等位基因特异荧光PCR法和金标准Sanger DNA直接测序法检测356名供者外周血CYP2C19基因型,实验采用同步盲法。结果 CYP2C19*1/*1型、CYP2C19*1/*2型、CYP2C19*2/*3型、CYP2C19*3/*3型、CYP2C19*2/*2型及CYP2C19*1/*3型的频率分布:使用PCR荧光法检测时分别为46.6%(166/356)、33.2%(118/356)、2.0%(7/356)、0.8%(3/356)、10.7%(38/356)及6.7%(24/356);采用DNA测序法时分别为46.3%(165/356)、33.4%(119/356)、2.0%(7/356)、0.8%(3/356)、11.0%(39/356)及6.5%(23/356)。两种方法检测CYP2C19基因型具有一致性(P=0.392),且一致性较好(Kappa=0.987);两种方法基因型检测结果一致率为99.2%。结论为保障医疗安全,临床需开展CYP2C19基因型检测;等位基因特异荧光PCR法检测CYP2C19基因型简便可靠。     

Performance of commercial platforms for rapid genotyping of polymorphisms affecting warfarin dose

Initiation of warfarin therapy is associated with bleeding owing to its narrow therapeutic window and unpredictable therapeutic dose. Pharmacogenetic-based dosing algorithms can improve accuracy of initial warfarin dosing but require rapid genotyping for cytochrome P-450 2C9 (CYP2C9) *2 and *3 single nucleotide polymorphisms (SNPs) and a vitamin K epoxide reductase (VKORC1) SNP. We evaluated 4 commercial systems: INFINITI analyzer (AutoGenomics, Carlsbad, CA), Invader assay (Third Wave Technologies, Madison, WI), Tag-It Mutation Detection assay (Luminex Molecular Diagnostics, formerly Tm Bioscience, Toronto, Canada), and Pyrosequencing (Biotage, Uppsala, Sweden). We genotyped 112 DNA samples and resolved any discrepancies with bidirectional sequencing. The INFINITI analyzer was 100% accurate for all SNPs and required 8 hours. Invader and Tag-It were 100% accurate for CYP2C9 SNPs, 99% accurate for VKORC1 -1639/3673 SNP, and required 3 hours and 8 hours, respectively. Pyrosequencing was 99% accurate for CYP2C9 *2, 100% accurate for CYP2C9 *3, and 100% accurate for VKORC1 and required 4 hours. Current commercial platforms provide accurate and rapid genotypes for pharmacogenetic dosing during initiation of warfarin therapy.     

Evaluation of single nucleotide polymorphism typing with invader on PCR amplicons and its automation

Large-scale pharmacogenetics and complex disease association studies will require typing of thousands of single-nucleotide polymorphisms (SNPs) in thousands of individuals. Such projects would benefit from a genotyping system with accuracy >99% and a failure rate <5% on a simple, reliable, and flexible platform. However, such a system is not yet available for routine laboratory use. We have evaluated a modification of the previously reported Invader SNP-typing chemistry for use in a genotyping laboratory and tested its automation. The Invader technology uses a Flap Endonuclease for allele discrimination and a universal fluorescence resonance energy transfer (FRET) reporter system. Three hundred and eighty-four individuals were genotyped across a panel of 36 SNPs and one insertion/deletion polymorphism with Invader assays using PCR product as template, a total of 14,208 genotypes. An average failure rate of 2.3% was recorded, mostly associated with PCR failure, and the typing was 99.2% accurate when compared with genotypes generated with established techniques. An average signal-to-noise ratio (9:1) was obtained. The high degree of discrimination for single base changes, coupled with homogeneous format, has allowed us to deploy liquid handling robots in a 384-well microtitre plate format and an automated end-point capture of fluorescent signal. Simple semiautomated data interpretation allows the generation of approximately 25,000 genotypes per person per week, which is 10-fold greater than gel-based SNP typing and microsatellite typing in our laboratory. Savings on labor costs are considerable. We conclude that Invader chemistry using PCR products as template represents a useful technology for typing large numbers of SNPs rapidly and efficiently.     

Influence of CYP3A5 and ABCB1 gene polymorphisms and other factors on tacrolimus dosing in Caucasian liver and kidney transplant patients

Tacrolimus is a substrate of cytochrome P4503A (CYP3A) enzymes as well as of the drug transporter ABCB1. We have investigated the possible influence of CYP3A5 and ABCB1 single nucleotide polymorphisms (SNPs) and other factors (e.g. albumin, hematocrit and steroids) on tacrolimus blood levels achieved in a population of Caucasian liver (n=51) and kidney (n=50) transplant recipients. At 1, 3 and 6 months after transplantation, tacrolimus doses (mg/kg/day) and trough blood levels (C0) were recorded and the weight-adjusted tacrolimus dosage (mg/kg/day) was calculated. Polymerase chain reaction followed by restriction fragment length polymorphism analysis was used for genotyping CYP3A5*1 and *3 [6986A>G] as well as ABCB1 at exons 21 [2677G>T/A] and 26 [3435C>T] in both liver transplant donors and recipients and in kidney transplant recipients. Of the 152 subjects studied, 84.9% showed a CYP3A5*3/*3 genotype. The total frequency of the allelic variant *3 was 93%. For the G2677T/A and C3435T polymorphisms the total frequencies of the allelic variants T/A and T were 44.7 and 46.7%, respectively. At 1, 3 and 6 months after transplantation the dose-adjusted C0 levels were significantly lower in patients with one copy of the *1 allele compared to those homozygous for the *3 allele. In the case of liver transplant patients the tacrolimus dose requirements were dominantly influenced by the polymorphisms of the CYP3A5 gene in the donors. With regard to the ABCB1 SNPs, in general they did not show any appreciable influence on tacrolimus dosing requirements; however, kidney transplant recipients carrying the 2677T/A allele required significantly higher daily tacrolimus doses than subjects homozygous for the wild-type allele. Identification of CYP3A5 single nucleotide polymorphisms prior to transplantation could contribute to evaluate the appropriate initial dosage of tacrolimus in the patients.     

Can antiretroviral therapy be tailored to each human immunodeficiency virus-infected individual? Role of pharmacogenomics

Pharmacogenetics refers to the effect of single nucleotide polymorphisms(SNPs) within human genes on drug therapy outcome. Its study might help clinicians to increase the efficacy of antiretroviral drugs by improving their pharmacokinetics and pharmacodynamics and by decreasing their side effects. HLAB*5701 genotyping to avoid the abacavir-associated hypersensitivity reaction(HSR) is a cost-effective diagnostic tool, with a 100% of negative predictive value, and, therefore, it has been included in the guidelines for treatment of human immunodeficiency virus(HIV) infection. HALDRB*0101 associates with nevirapine-induced HSR. CYP2B6 SNPs modify efavirenz plasma levels and their genotyping help decreasing its central nervous system, hepatic and HSR toxicities. Cytokines SNPs might influence the development of drug-associated lipodystrophy. APOA5, APOB, APOC3 and APOE SNPs modify lipids plasma levels and might influence the coronary artery disease risk of HIV-infected individuals receiving antiretroviral therapy. UGT1A1*28 and ABCB1(MDR1) 3435 C T SNPs modify atazanavir plasma levels and enhance hyperbilirubinemia. Much more effort needs to be still devoted to complete large prospective studies with multiple SNPs genotyping in order to reveal more clues about the role played by host genetics in antiretroviral drug efficacy and toxicity.     

湖北地区房颤患者VKORC1、CYP2C9基因多态性及其对华法林用量的影响

目的:检测房颤患者维生素K环氧化物还原酶复合物1基因(VKORC1)和细胞色素P450酶2C9基因(CYP2C9)多态性及其对临床使用华法林用量的影响。方法:选择口服华法林抗凝治疗的房颤患者94例,采用PCR扩增和DNA直接测序技术检测患者VKORC1和CYP2C9基因型,同时记录不同基因型患者华法林日用量,比较不同基因型房颤患者华法林日用量的统计学差异。结果:94例房颤患者检出VKORC1 AA型84例,VKORC1AG型10例,CYP2C9*1/*1型88例,CYP2C9*1/*3型6例。不同VKORC1、CYP2C9基因型房颤患者华法林用量不同,VKORC1AA型并CYP2C9*1/*1型和VKORC1AG型并CYP2C9*1/*1型患者华法林日用量均高于VKORC1AA型并CYP2C9*1/*3型患者,VKORC1AA型并CYP2C9*1/*1型患者华法林用量又高于VKORC1AG型并CYP2C9*1/*1型患者。结论:湖北地区房颤患者基因型主要为VKORC1AA型与CYP2C9*1/*1型,少量VKORC1AG型与CYP2C9*1/*3型。VKORC1、CYP2C9基因型是华法林用量的重要影响因素。     

CYP3A5基因多态性与双环醇保肝降酶效应的关系

目的 研究CYP3A5酶基因多态性与双环醇保肝降酶作用临床效应的关系.方法 34例慢性乙型肝炎患者在常规治疗的基础上予双环醇片治疗24周;治疗前后检测肝功能指标(ALT、AST);采用PCR-RFLP法对患者CYP3A5酶基因型进行检测.结果 将检测结果基因型为CYP3A5*l(共16例,包括CYP3A5*1/*1型2例和CYP3A5*1/*3型14例)作为观察组,基因型为CYP3A5*3组(即CYP3A5*3/*3型,18例)作为对照组.治疗后,CYP3A5*1组和CYP3A5*3组患者ALT、AST水平均有显著下降(P＜0.05);CYP3A5*3组患者的ALT和AST下降幅度分别为79.73%和74.76%,显著大于CYP3A5*1组的65.90%和49.63%(P＜0.05);治疗后,两组之间的ALT及AST相比差异有统计学意义(P＜0.05),CYP3A5*3组ALT复常率和AST复常率均高于CYP3A5*1组,差异有统计学意义(P＜0.05).结论 CYP3A5酶基因型对双环醇治疗慢性乙肝的临床疗效抗炎保肝降酶效应有明显影响,基因型为CYP3A5*3的慢性乙肝患者双环醇疗效反应较强,该酶基因型可能成为双环醇疗效(尤其是降酶效应)的预测参照因子.     

Rapid detection of CYP2C9*3 alleles by real-time fluorescence PCR based on SYBR Green

CYP2C9 catalyzes the metabolism of important drugs such as phenytoin, S-warfarin, tolbutamide, losartan, and nonsteroidal anti-inflammatory drugs. A functional polymorphism of the CYP2C9 gene has been described. The single-base mutation of A1061C (Ile359Leu) in the CYP2C9 gene termed CYP2C9*3 was found at a frequency of about 2.1% in Japanese. We developed a rapid mutation analysis method for detecting the CYP2C9*1 genotype. This method is a marriage of two emerging technologies: allele-specific amplification primers for target DNA and a new double-stranded DNA-selective fluorescent dye, SYBR Green. 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 of healthy Japanese volunteers. The CYP2C9 wild-type CYP2C9*1/CYP2C9*1 and heterozygous CYP2C9*1/CYP2C9*3 genotypes of the CYP2C9 alleles detected by the assay were consistent with the results obtained from restriction enzyme cleavage. No genotype of CYP2C9*3/CYP2C9*3 was found in these samples. Using plasmid DNA containing a point mutation of CYP2C9*3 as template, the assay separated the three genotypes. We conclude that this simple, rapid, and inexpensive procedure is applicable to routine high-throughput assays.     

Combination Analysis in Genetic Polymorphisms of Drug-Metabolizing Enzymes CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A5 in the Japanese Population

The Cytochrome P450 is the major enzyme involved in drug metabolism. CYP enzymes are responsible for the metabolism of most clinically used drugs. Individual variability in CYP activity is one important factor that contributes to drug therapy failure. We have developed a new straightforward TaqMan PCR genotyping assay to investigate the prevalence of the most common allelic variants of polymorphic CYP enzymes CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A5 in the Japanese population. Moreover, we focused on the combination of each genotype for clinical treatment. The genotype analysis identified a total of 139 out of 483 genotype combinations of five genes in the 1,003 Japanese subjects. According to our results, most of subjects seemed to require dose modification during clinical treatment. In the near future, modifications should be considered based on the individual patient genotype of each treatment.     

Functional variants at CYP2A6: new genotyping methods, population genetics, and relevance to studies of tobacco dependence

Cytochrome P450CYP2A6 (CYP2A6) is the predominant enzyme responsible for the metabolism of nicotine to cotinine. Two variants have been identified that encode products presumed to have little or no activity. A previous study suggested that carriers of at least one copy of either null variant may be protected against tobacco dependence, while tobacco-dependent carriers smoke fewer cigarettes. However, different laboratories have reported widely disparate CYP2A6 allele frequencies across European populations. These differences prompted us to reexamine the genotyping methods for CYP2A6. We developed an improved genotyping strategy using CYP2A6-specific nested PCR, and differential restriction enzyme digestion to identify variant nucleotides in exon 3. We used sequencing to verify genotype results and to assess the sequence of exon 4, which previous work predicted should correspond to "wild-type" CYP2A6 sequence. In addition, we developed a new nomenclature in which CYP2A6*1 is designated CYP2A6*A1-*B1, CYP2A6*2 is CYP2A6*A2, and CYP2A6*3 is CYP2A6*B2. The frequencies of CYP2A6*A2 and CYP2A6*B2 were then estimated in samples from six populations. Sequencing confirmed CYP2A6*A2 genotypes in all cases. Unexpectedly, sequencing demonstrated exon 4 sequence corresponding to CYP2A7 in samples genotyped as CYP2A6*B2. In the population study, we found consistently low allele frequencies (相似文献   

CYP3A5基因的多态性及其临床意义的研究

目的 了解中国人群中细胞色素P450 3A5基因CYP3A5的多态性及其基因型与临床功能之间的关联。方法 应用变性高效液相色谱技术对180份样本进行基因变异的检测，并经测序验证；以免疫荧光偏振技术监测12例造血干细胞移植患者环孢素（cyclospofin A，CsA）血浓度，数据经统计学软件分析和处理。结果 180份样本中，共检测出1种等位基因即CYP3A5＊3，其频率为76．1％（274／360）；3种基因型，即纯合子CYP3A5＊1／＊1 10份，占总数的5．6％；杂合子CYP3A5＊1／＊3 66份，占总数的36．7％；纯合子CYP3A5＊3／＊3 104份，占总数的57．8％。12例造血干细胞移植患者中检测出两种基因型，即CYP3A5＊1／＊1 2例和CYP3A5＊l／＊3 10例。CYP3A5＊1／＊ l患者外周血均一化后的CsA浓度与CYP3A5＊1／＊3相比较，前者谷浓度C0较后者低，相差2．3～6．6倍，差异有统计学意义（P〈0．01）；服药后2 h峰值C2亦较后者低，相差1．4～3．8倍，同样差异有统计学意义（P〈0．01）。结论 CYP3A5＊3是中国人群中最主要的多态性，CYP3A5基因型与造血干细胞移植患者血中CsA浓度紧密关联，CYP3A5＊1／＊1比CYP3A5＊1／＊3需要更大剂量的CsA以维持相同的血浓度。应用变性高效液相色谱对CYP3A5进行基因型分型，可以预测患者CYP3A5的功能，进而预测其CsA的需求量，使临床应用CsA更加安全有效和个体化。     

High-Throughput Genotyping with Single Nucleotide Polymorphisms

To make large-scale association studies a reality, automated high-throughput methods for genotyping with single-nucleotide polymorphisms (SNPs) are needed. We describe PCR conditions that permit the use of the TaqMan or 5' nuclease allelic discrimination assay for typing large numbers of individuals with any SNP and computational methods that allow genotypes to be assigned automatically. To demonstrate the utility of these methods, we typed >1600 individuals for a G-to-T transversion that results in a glutamate-to-aspartate substitution at position 298 in the endothelial nitric oxide synthase gene, and a G/C polymorphism (newly identified in our laboratory) in intron 8 of the 11-beta hydroxylase gene. The genotyping method is accurate-we estimate an error rate of fewer than 1 in 2000 genotypes, rapid-with five 96-well PCR machines, one fluorescent reader, and no automated pipetting, over one thousand genotypes can be generated by one person in one day, and flexible-a new SNP can be tested for association in less than one week. Indeed, large-scale genotyping has been accomplished for 23 other SNPs in 13 different genes using this method. In addition, we identified three "pseudo-SNPs" (WIAF1161, WIAF2566, and WIAF335) that are probably a result of duplication.     

Lack of association of ACP1 gene with inflammatory bowel disease: a case-control study

The red cell acid phosphatease (ACP1) gene, which encodes a low molecular weight phosphotyrosine phosphatase (LMW-PTP), has been suggested as a common genetic factor of autoimmunity. In the present study, we aimed to investigate the possible influence of ACP1 polymorphisms in the susceptibility of inflammatory bowel disease (IBD). A total of 1271 IBD Spanish patients [720 Crohn's disease (CD) and 551 ulcerative colitis (UC)] and 1877 healthy subjects were included. Four single-nucleotide polymorphisms (SNPs), rs10167992, rs11553742, rs7576247 and rs3828329, were genotyped using TaqMan SNP genotyping assays. Common ACP1 alleles (i.e. ACP1*A, ACP1*B and ACP1*C) were determined by two of these SNPs. After the analysis, no evidence of association of the ACP1 genetic variants was found with CD or UC. Therefore, our results suggest that the ACP1 gene may not play a relevant role in the development of IBD.     

A novel intronic mutation that may affect genotyping result of <Emphasis Type="Italic">CYP2C8</Emphasis> by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) is strongly associated with <Emphasis Type="Italic">CYP2C8*3</Emphasis> in a South American population

During the investigation of a CYP2C8*3 genetic polymorphism in a South American population, we obtained a discrepant result using two different polymerase chain reaction (PCR) protocols. A single nucleotide polymorphism (SNP) (IVS3+43 G>C) was identified in the intron 3 region, which was used as a primer-annealing site of one of the two PCR protocols. A genotyping method was developed to enable discrimination of the CYP2C8*1A, CYP2C8*3 (416 G>A), and CYP2C8*3 (416G>A; IVS3+43 G>C) alleles. In a screen of a South American population, we found that individuals carrying the CYP2C8*3 (416 G>A) polymorphism also carried the CYP2C8*3 (416G>A; IVS3+43 G>C). However, we did not find any carriers of CYP2C8*3 (416G>A; IVS3+43 G>C) in a Japanese population.     

Effect of CYP2C9 gene polymorphisms on response to treatment with sulfonylureas in a cohort of Egyptian type 2 diabetes mellitus patients

Oral hypoglycemics are a widely prescribed group of drugs for the management of type 2 diabetes mellitus. Sulfonylureas (SUs) are the cornerstone of type 2 diabetes pharmacotherapy. The enzyme cytochrome P450 2C9 (CYP2C9) is the main enzyme that catalyzes the biotransformation of SUs. It is encoded by the polymorphic gene CYP2C9 with two allelic variants namely CYP2C9*2 and CYP2C9*3 coding for variant allozymes with reportedly decreased metabolic capacity resulting in decreased SUs clearance and consequently prolonged and exacerbated action. The aim of this study was to investigate the influence of genetic polymorphisms of CYP2C9 on the response to glibenclamide, a second-generation sulfonylurea. Hundred type 2 diabetic patients were enrolled in the study. Genotyping was done on the LightCycler 2 by real-time polymerase chain reaction (PCR) hybridization probe assay. The results are the following: 53 patients were carriers of the wild genotype (CYP2C9*1/*1), 20 were heterozygous for the variant CYP2C9*2 allele (CYP2C9*1/*2), 18 were heterozygous for the variant CYP2C9*3 allele (CYP2C9*1/*3), and 9 were double heterozygous for both mutant alleles (CYP2C9*2/*3); of those double mutant genotype patients, two were also homozygous for the mutant CYP2C9*2 allele (CYP2C9*2/*2) and one patient was homozygous for the mutant CYP2C9*3 allele (CYP2C9*3/*3). Although there was no significant difference in drug dosage between the four groups, there was however a significant association of the CYP2C9*2/*3 genotype with better glycemic control. As conclusion, the better glycemic control observed can probably be attributed to slower metabolism of SUs by the carriers of the CYP2C9*2/*3 genotype and consequently longer half-life or exacerbated effect of the SUs administered.     

CYP2C9 and VKORC1 gene polymorphism and acenocoumarol anticoagulant activity in Russian patients at high risk of thromboembolic complications

The study included 25 patients at high risk of thromboembolic complications. All of them were treated with acenocoumarol for 6 months under control of the frequency of hemorrhage and episodes of severe hypocoagulation (a more than 3-fold rise in INR). All the patients underwent CYP2C9 and VKORC1 genotyping. It was shown that the presence of CYP2C9*2 and CYP2C9*3 alleles in the CYP2C9 locus and the AA genotype of the polymorphous G-1639(3673)A marker of the VKORC1 gene was not associated with the development of severe hypocoagulation episodes (p = 0.261--for CYP2C9, p = 0.616 and 0.361 for VKORC1 in the total group and a subgroup of patients having the CYP2C9*1/*1 genotype respectively and treated with acenocoumarol. The search for other genetic markers of efficacy and safety of this drug should be continued.     

Elucidation of CYP2D6 Genetic Diversity in a Unique African Population: Implications for the Future Application of Pharmacogenetics in the Xhosa Population

Genetic variation of the CYP2D6 gene has been associated with altered drug metabolism; however, limited studies have investigated CYP2D6 sequence diversity in African populations. We devised a CYP2D6 genotyping strategy to analyse the South African Xhosa population and genotype a Xhosa schizophrenia cohort, as CYP2D6 metabolises many antipsychotics and antidepressants. The entire CYP2D6 gene locus was sequenced in 15 Xhosa control individuals and the data generated were used to design a comprehensive genotyping strategy. Over 25 CYP2D6 alleles were genotyped in Xhosa controls and Xhosa schizophrenia patients using long‐range PCR, DNA sequencing and single nucleotide primer extension analysis. Bioinformatic algorithms were used to predict the functional consequences of relevant mutations and samples were assigned CYP2D6 activity scores. A unique allele distribution was revealed and two rare novel alleles, CYP2D6*73 and CYP2D6*74, were identified. No significant differences in allele frequencies were detected between Xhosa controls and schizophrenia patients. This study provides i) comprehensive data on a poorly characterised population, ii) a valuable CYP2D6 genotyping strategy and iii) due to their unique genetic profile, provides the basis for pharmacogenetic intervention for Xhosa individuals.     

Detection and identification of cytochrome P-450 2C9 alleles *1, *2, and *3 by high-resolution melting curve analysis of PCR amplicons

High-resolution melting curve analysis using a fluorescent DNA binding dye can detect sequence variations in a closed-tube system without labeled primers or probes. We developed and verified a melting analysis assay for common single nucleotide polymorphisms of cytochrome P-450 (CYP) 2C9 that affect warfarin metabolism. We used this method to genotype 84 patients receiving warfarin. For wild-type, *1/*1, 50% fluorescence corresponded to a mean+/-SD of 87.17+/-0.05 degrees C, whereas *2/*2 was 0.4 degrees C lower. The *1/*2 melting curve was easily distinguished from *1/*1 and *2/*2 based on transition temperature and shape. Exon 7 showed a more complex melting curve; however, genotypes *1/*1, *1/*3, and *3/*3 were easily distinguishable. Melting curves were highly reproducible (SD of temperature for multiple fluorescence values 0.04 degrees C-0.11 degrees C; mean, 0.06 degrees C). Heterozygotes (*1/*2 or *1/*3) required significantly lower mean maintenance warfarin doses compared with wild-type (30.67 and 29.56 vs 42.81 mg/wk; P<.05). High-resolution melting analysis provides a simple and accurate method for genotyping of CYP2C9.     

CYP2C9 and VKORC1 genetic polymorphism analysis might be necessary in patients with Factor V Leiden and prothrombin gene G2021A mutation(s).

The annual incidence of venous thromboembolism is approximately 117 per 100,000 persons or about 1 per 1000 person-years, with the majority of the disease occurring in the older age groups. Factor V Leiden gene (most common) and the prothrombin G20210A gene mutation are inherited mild to moderate risk factors for hypercoagulability. The anticoagulant warfarin requires close monitoring of the patient's prothrombin time, normalized as the international normalization ratio. Patients with either Cytochrome P-450 CYP2C9*2, CYP2C9*3, or VKORC1*2 genotype (c.-1639G>A) require significantly reduced doses, and are at a higher risk of serious bleeding. Thirty-five samples in total, 15 with Factor V Leiden, 18 with prothrombin G2021A mutation, and 2 with both were analyzed for 2C9*2, 2C9*3, and VKORC1 (-1639) allele variants by using the Invader CYP2C9 and VKORC1 polymorphism analysis kit. Eight with CYP2C9*2 C/T, 2 with CYP2C9*3 A/C, 5 with VKORC1 (-1639) A/A, and 22 with VKORC1 (-1639) G/A genotypes or 29 out of 35 (83%) samples analyzed were found with CYP2C9*2 C/T, CYP2C9*3 A/C, VKORC1 (-1639) G/A, or/and VKORC1 (-1639) A/A genotypes. CYP2C9*2 C/T, CYP2C9*3 A/C, VKORC1 (-1639) G/A genotyping might be necessary for patients with Factor V Leiden and/or prothrombin G2021A mutation before warfarin anticoagulant therapy.     

Single nucleotide polymorphism detection: allelic discrimination using TaqMan

Candidate gene studies are one of the most widely used approaches in the dissection of the genetic basis of disease. High-throughput methods for genotyping single nucleotide polymorphisms (SNPs) are necessary to perform large-scale association studies. We describe the use of the TaqMan or 5' nuclease allelic discrimination assay for genotyping polymorphisms of the collagen I alpha 1 (COLIA1) and vitamin D receptor (VDR) genes. The basis for the assay is an allele specific oligonucleotide probe, labelled with a fluorescent reporter dye and a quencher dye, which is cleaved during the amplification process generating an increase in the intensity of fluorescence related to the accumulation of PCR product which is measured directly in the reaction well. Suitable for the discrimination of alleles differing by a single base change, this technique is robust, accurate, cost effective, and sufficiently high-throughput for a medium sized laboratory performing association analyses.