High throughput analysis of 10 microsatellite and 11 diallelic polymorphisms on the human Y-chromosome

We describe an integrated approach to the determination of complex Y chromosome haplotypes that is both fast and relatively inexpensive. The method employs GeneScan technology to enable a researcher to assay repeat number variation at ten microsatellite loci and determine the status of 11 diallelic polymorphisms. The method requires only four PCRs and four GeneScan runs per sample and is relatively insensitive to sample DNA concentration.     

Decreasing the CYP2D6 contribution to metabolism of a CK1ε inhibitor

Our internal casein kinase 1ε lead inhibitor, compound 1 was partially cleared by the polymorphic cytochrome P450 2D6. CYP2D6 involvement in metabolism implies more extensive clinical trials. We therefore wanted to reduce the contribution to clearance by this enzyme. We utilized metabolism reports for compound 1 performed in recombinant CYP2D6 together with structure-metabolism variation in structures of closely related analogs in order to see if we could incorporate similar substitution patterns in our lead compound. In addition, we utilized a previously established docking method using a modified CYP2D6 crystal structure to see if the metabolism patterns in CYP2D6 could be reproduced to afford the metabolites in the metabolism reports as well as those for the compounds used in the structure-metabolism relationship. All three of these steps, the metabolism report, the establishment of structure-metabolism relationships and the docking, lead to compound 10 where CYP2D6 was not involved in the clearance pathways.     

Determination of CYP2D6 gene copy number by multiplex polymerase chain reaction analysis

Cytochrome P450 2D6 (CYP2D6) copy number variation (CNV) influences the metabolism of 15-25% of clinical drugs. Here we describe a novel multiplex polymerase chain reaction (PCR) analysis method that accurately detects CYP2D6 CNV and CYP2D6*9 allele. It includes the amplification of 2 CYP2D6 and 7 control (AQP1, CYP3A4, MDR1, and SDHB) fluorescent PCR products that are separated on a capillary sequencer and normalized using reference samples. The technique was validated using 27 PCR-restriction fragment length polymorphism (RFLP) pregenotyped samples and further tested in 75 Caucasian samples. The method assigns the correct CYP2D6 copy number, independent of already characterized CYP2D6 single nucleotide polymorphisms (SNPs), and could easily be applied to clinical samples.     

Quantitation of cytochrome P450 mRNA levels in human skin

There is considerable interindividual variation in man's ability to metabolize drugs and foreign compounds. These differences can partly be attributed to genetic polymorphisms that result in the generation of multiple phenotypes with different drug-metabolizing capabilities. Genetically derived differences can easily be assessed by genotyping assays in cases where the polymorphism has been identified. However, many of the polymorphisms that result in these are not known, secondly not all the differences can be attributed to genetic polymorphisms, hence genotyping methods cannot be employed. We have therefore, developed real-time (Taqman) PCR assays to quantitate levels of P450 mRNAs in human tissues. These assays are highly sensitive, reproducible, and specific and will allow quantitation of P450 mRNA levels in various human tissues. We have applied these assays to quantitate cytochrome P450 mRNA levels in human skin samples from 27 healthy volunteers. The expression of 13 P450s was assessed. The major enzymes detected were CYP1B1, CYP2B6, CYP2D6, and CYP3A4 with mean values of 2.5, 2.6, 2.7, and 1.1 fg/18S rRNA in 50ng total RNA, respectively. Lower levels of CYP2C18, CYP2C19, and CYP3A5 were also detected while CYP1A2, 2A6, and 2C8 were below limits of detection. There was interindividual variation in the levels of mRNA among the 27 subjects studied although Poisson analysis showed data to be normally distributed, except for CYP2B6, as some individuals completely lacked CYP2B6 mRNA.     

CYP2D6 polymorphisms in patients with porphyrias

The cytochrome P-450 (CYP) isoenzymes, a superfamily of heme proteins which are the terminal oxidases of the mixed function oxidases system, metabolize more than 70% of all clinically approved drugs. The highly polymorphic CYP2D6 isoform metabolizes more than 25% of most common drugs, and the phenotypes of the 70-plus allelic variants range from compromised to excessive enzymatic activity. Porphyrias are a group of inherited or acquired metabolic disorders of heme biosynthesis, due to a specific decrease in the activity of one of the enzymes of the heme pathway. Clinical signs and symptoms of porphyrias are frequently associated with exposure to precipitating agents, including clinically approved drugs. CYP enzymes, including CYP2D6, participate in the metabolism of some porphyrinogenic drugs, leading to the deregulation of heme biosynthesis. Considering that some of the drugs not recommended for use in porphyric patients are metabolized by CYP2D6, the presence of CYP2D6 polymorphisms in porphyric patients would influence the triggering of the disease when these individuals receive a precipitating agent that is metabolized by CYP2D6. To investigate CYP2D6 polymorphisms in porphyric patients, healthy Argentinean volunteers, porphyric patients, and a group of individuals with high levels of iron were studied. Results indicated that the CYP2D6*3 and CYP2D6*4 alleles, in particular, would be linked to the onset of disease. Predictive genotyping for CYP2D6 in porphyric patients holds promise as a method to improve the clinical efficacy of drug therapy and to personalize drug administration for these patients.     

Polymorphisms and phenotypic analysis of cytochrome P450 2D6 in the Tibetan population

The cytochrome P450 2D6 enzyme (CYP2D6) metabolizes about 25% of prescribed drugs in the endoplasmic reticulum, and genetic polymorphisms in CYP2D6 can greatly affect its activity and lead to differences among individuals in drug efficacy and adverse drug reactions. To investigate genetic polymorphisms in CYP2D6 among Tibetan Chinese, we directly sequenced the whole gene in 96 unrelated, healthy Tibetans from The Tibet Autonomous Region of China and screened for genetic variants in the promoter, exons, introns, and 3′UTR. We detected fifty-one genetic polymorphisms in CYP2D6, and 16 of them are novel. The allele frequencies of CYP2D6*1, *2, *5, *10, *41, and *49 were 0.25, 0.43, 0.02, 0.29, 0.02, and 0.01, respectively. The frequency of CYP2D6*10, a putative poor-metabolizer allele, was lower in our sample population compared with that in the Han Chinese population (p < 0.001). In addition, haplotype analysis allowed 15 CYP2D6 haplotypes to be classified into three groups. In conclusion, our results provide basic information about CPY2D6 alleles in Tibetans and suggest that the enzymatic activities of CYP2D6 may differ among the diverse ethnic populations of China. Our results provide a basis for safer drug administration and better therapeutic treatment among Tibetans.     

High-throughput detection of multiple genetic polymorphisms influencing drug metabolism with mismatch primers in allele-specific polymerase chain reaction

Because of genetic polymorphisms of drug-metabolizing enzyme genes, the activities of the enzymes in humans vary widely and alter the metabolism of commonly used clinical agents. Severe adverse effects or resistance to therapy may result. We have developed a rapid and high-throughput genotyping method for detecting polymorphisms of the drug-metabolizing enzyme genes CYP2C9*3, CYP2C19*2, *3, CYP2D6*2, *4, *10, *14, *21, NAT2*5, *6, *7, and TPMT*3 using allele-specific polymerase chain reaction (PCR) with mismatch primers (ASPCR-MP) and CYP2D6*5, *36, and CYP2D6xN using stepdown PCR with detection by SYBR Green I. We analyzed genomic DNA from 139 Japanese volunteers. Identical genotyping results were obtained by using ASPCR-MP, stepdown PCR, and conventional PCR. We found that the methods clearly differentiate three specific profiles with no overlap in the signals. Moreover, both ASPCR-MP and stepdown PCR for genotyping took less than 3-4h. To our knowledge, this is the first report of successful simultaneous detection of multiple genetic polymorphisms with point mutations using ASPCR-MP or multiple genetic polymorphisms with large structural alterations using stepdown PCR. In conclusion, ASPCR-MP and stepdown PCR appear to be suitable for large clinical and epidemiological studies as methods that enable highly sensitive genotyping and yield a high-throughput.     

A Novel Simple Method for Determining CYP2D6 Gene Copy Number and Identifying Allele(s) with Duplication/Multiplication

Background

Cytochrome P450 2D6 (CYP2D6) gene duplication and multiplication can result in ultrarapid drug metabolism and therapeutic failure or excessive response in patients. Long range polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) and sequencing are usually used for genotyping CYP2D6 duplication/multiplications and identification, but are labor intensive, time consuming, and costly.

Methods

We developed a simple allele quantification-based Pyrosequencing genotyping method that facilitates CYP2D6 copy number variation (CNV) genotyping while also identifying allele-specific CYP2D6 CNV in heterozygous samples. Most routine assays do not identify the allele containing a CNV. A total of 237 clinical and Coriell DNA samples with different known CYP2D6 gene copy numbers were genotyped for CYP2D6 *2, *3, *4, *6, *10, *17, *41 polymorphisms and CNV determination.

Results

The CYP2D6 gene allele quantification/identification were determined simultaneously with CYP2D6*2, *3, *4, *6, *10, *17, *41 genotyping. We determined the exact CYP2D6 gene copy number, identified which allele had the duplication or multiplication, and assigned the correct phenotype and activity score for all samples.

Conclusions

Our method can efficiently identify the duplicated CYP2D6 allele in heterozygous samples, determine its copy number in a fraction of time compared to conventional methods and prevent incorrect ultrarapid phenotype calls. It also greatly reduces the cost, effort and time associated with CYP2D6 CNV genotyping.     

Characterization of cytochrome P450 2D6 alleles using the Invader system

The cytochrome p450 (CYP) superfamily comprises enzymes that play an essential role in the transformation of medically relevant compounds. Accurate genotyping of polymorphisms in members of this family is drawing increasing interest because certain allelic variants may result in either loss of efficacy or toxic accumulation of therapeutic agents. Debrisoquine 4-hydroxylase, or CYP2D6, is among the most widely studied of the CYPs. The complexity of the CYP2D6 genomic region, including pseudogenes, gene deletions, and gene duplications, has offered numerous challenges to developing a genotyping strategy. We describe a comprehensive CYP2D6 genotyping strategy that employs both a PCR/Invader genotyping assay system and an Invader genomic copy number assay The Invader system is a homogeneous, isothermal, highly specific, and robust signal amplification system. Resultsfrom II CYP2D6 assays in an alle frequency study compare well to published allele frequency values for Caucasians. Further, Invader assays provided unambiguous genotyping determinations for 100% of the 171 samples that yielded a visible PCR product on an agarose gel. A copy number assay yielded only one equivocal result in 205 samples. We identified 17 single-copy individuals and 17 three-copy (or more) individuals.     

Pharmacokinetic genes do not influence response or tolerance to citalopram in the STAR*D sample

Background

We sought to determine whether clinical response or tolerance to the Selective Serotonin Reuptake Inhibitor (SSRI) citalopram is associated with genetic polymorphisms in potentially relevant pharmacokinetic enzymes.

Methodology

We used a two-stage case-control study design in which we split the sample of 1,953 subjects from the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) trial into a discovery (n = 831) and validation set (n = 1,046). Fifteen polymorphisms from five (CYP2D6, ABCB1, CYP2C19, CYP3A4, and CYP3A5) pharmacokinetic genes were genotyped. We examined the associations between these polymorphisms and citalopram response and tolerance. Significant associations were validated in the second stage for those polymorphism found to be statistically significant in the first stage.

Conclusions

No genetic polymorphism in the pharmacokinetic genes examined was significantly associated with our response or tolerance phenotypes in both stages. For managing pharmacological treatment with citalopram, routine screening of the common pharmacokinetic DNA variants that we examined appears to be of limited clinical utility.     

Drug metabolizing enzyme activities versus genetic variances for drug of clinical pharmacogenomic relevance

Enzymes are critically important in the transportation, metabolism, and clearance of most therapeutic drugs used in clinical practice today. Many of these enzymes have significant genetic polymorphisms that affect the enzyme's rate kinetics. Regarding drug metabolism, specific polymorphisms to the cytochrome (CYP) P450 enzyme family are linked to phenotypes that describe reaction rates as "ultra", "intermediate", and "poor," as referenced to "extensive" metabolizers that are assigned to wildtype individuals. Activity scores is an alternate designation that provides more genotype-to-phenotype resolution. Understanding the relative change in enzyme activities or rate of clearance of specific drugs relative to an individual's genotypes is an important component in the interpretation of pharmacogenomic data for personalized medicine. Currently, the most relevant drug metabolizing enzymes are CYP 2D6, CYP 2C9, CYP 2C19, thiopurine methyltransferase (TPMT) and UDP-glucuronosyltransferase (UGT). Each of these enzymes is reactive to a host of different drug substrates. Pharmacogenomic tests that are in routine clinical practice include CYP 2C19 for clopidogrel, TPMT for thiopurine drugs, and UDP-1A1 for irinotecan. Other tests where there is considerable data but have not been widely implemented includes CYP 2C9 for warfarin, CYP 2D6 for tamoxifen and codeine, and CYP 2C19 for the proton pump inhibitors.     

人类细胞色素P450与药物氧化代谢遗传多态性分子机制的研究现状

近年,在表型及基因型上均发现存在药物氧化代谢多态性,特别是对于人类细胞色素Ｐ４５０氧化酶与药氧化代谢遗传多态性的关系进行了深入的研究。有关ＣＹＰ２Ｄ６、ＣＹＰ２Ｃ１９等的突变已大多被鉴定;ＣＹＰ１Ａ１、ＣＹＰ１Ａ２等在表型存在多态性而确切的遗传机制尚不清楚。     

Genetic variation at the mitochondrial DNA 9-bp repeat locus in the Sakha of Siberia

Genetic variation at the mitochondrial DNA 9-bp repeat locus was assayed in 779 Sakha from Siberia. Fourteen deletion (1.8%), nine triplication (1.2%), and two 4-repeat alleles (0.26%) were identified. Several of these alleles were also detected as heteroplasmies. Among the four heteroplasmic individuals identified (0.51%), three different combinations of repeat alleles were present: 1/2, 2/3, and 2/3/4 copies. Hypervariable region I (HVRI) sequencing revealed that three different sets of haplogroups were associated with the three most frequent 9-bp polymorphisms: (1) haplo-groups B, T, and W for deletions; (2) haplogroups C, D, and K for triplications; and (3) haplogroups C, D, and T for heteroplasmies. Both of the two 4-repeat alleles were associated with haplogroup D. We detected more types of 9-bp polymorphisms and more genetic variation within classes of polymorphism than previously reported for any single population. We also present the largest and most geographically diverse sampling of the Sakha population to date. No neighboring populations have been reported to carry a non-haplogroup B deletion, triplication, or heteroplasmy, suggesting that shared ancestry or admixture or both are unlikely explanations for the presence of these polymorphisms in the Sakha. The identification of high levels of variation may be a function of the large sample size and the in-depth analysis of all derived polymorphisms. Further study of the Sakha is warranted to determine whether the level of variation is unexpectedly high, especially in light of the presence of different heteroplasmies, which suggests multiple recent events.     

DNA haplotype-dependent differences in the amino acid sequence of debrisoquine 4-hydroxylase (CYP2D6): evidence for two major allozymes in extensive metabolisers

The molecular basis for DNA haplotype-dependent debrisoquine 4-hydroxylase (CYP2D6) expression was explored by sequencing all of the nine exons of the CYP2D6 gene. Two distinct exon sequence frameworks of the CYP2D6 gene were found, each associated with specific BamHI-defined DNA haplotypes of the CYP2D cluster. They corresponded to Arg²⁹⁶/Cys²⁹⁶ and Ser⁴⁸⁶/Thr⁴⁸⁶ amino acid polymorphisms in the CYP2D6 enzyme, and occurred in almost equal frequency among the Caucasians examined. These two major allozymes with amino acid differences in the presumed substrate recognition region and in the vicinity of the heme binding site could be the source of the observed DNA haplotype-dependent variation in phenotypic expression.     

CYP2D6 and CYP1A1 mutations in the Turkish population

Drugs and carcinogens are substrates of a group of metabolic enzymes including cytochrome p450 enzymes and gluthatione S-transferases. Many of the genes encoding these enzymes exhibit functional polymorphisms that contribute individual cancer susceptibility and drug response. Molecular studies based on these polymorphic enzymes also explain the aetiology of cancer and therapeutic management in clinics. We analysed the cytochrome p4501A1 (CYP1A1) and 2D6 (CYP2D6) variant genotype and allele frequencies by PCR-RFLP in Turkish individuals (n=140). The frequency of the CYP1A1*2A mutant allele was found to be 15.4%, and the CYP2D6*3 and *4 mutant allele (poor metabolizer) frequencies were 2.5% and 13.9%, respectively. This study presents the first results of CYP1A1 and CYP2D6 mutant allele distributions in the Turkish population and these data provide an understanding of epidemiological studies that correlate therapeutic approaches and aetiology of several types of malignancy in Turkish patients.     

Cloning and mapping of the porcine cytochrome-p450 2E1 gene and its association with skatole levels in the domestic pig

The porcine cytochrome-p450 2E1 (CYP2E1) gene was isolated by screening a pig BAC library and partially sequenced. This sequence information was used to identify six single nucleotide polymorphisms (SNPs) within the CYP2E1 gene and its promoter. In addition, a microsatellite marker tightly linked to the CYP2E1 gene was subcloned from the BAC. One of these markers was used to map the CYP2E1 gene distal of SWC27 on SSC14, well outside reported quantitative trait loci on SSC14 for skatole, indole and taste test measures of boar taint. However, in a population of commercial pigs scored for backfat skatole levels, there was evidence of association between a SNP in the CYP2E1 promoter and skatole deposition, although there was no significant association between this SNP and skatole levels in the experimental cross.     

Genetic variation at 9 autosomal microsatellite loci in Asian and Pacific populations.

Genetic variation at 9 autosomal microsatellite loci (CFS1R, TH01, PLA2A, F13A1, CYP19, LPL, D20S481, D20S473, and D20S604) has been characterized in 16 Asian and Oceanic populations, mostly from mainland and insular Southeast Asia. The neighbor-joining tree and the principal coordinates analysis of the genetic relationships of these populations show a clear separation of Papua New Guinea Highlanders and, to a lesser extent, Malayan aborigines (Orang Asli or Semai) from the rest of the populations. Although the number of markers used in this study appears to be inadequate for clarifying the patterns of genetic relationships among the studied populations, in the principal coordinates analysis a geographic trend is observed in the mainland and insular Southeast Asian populations. Furthermore, in an attempt to contrast the extent of variation between autosomal and Y-chromosome-specific microsatellite loci and to reveal potential differences in the patterns of male and female migrations, we have also compared genetic variation at these 9 autosomal loci with variation observed at 5 Y-chromosome-specific microsatellites in a common set of 14 Asian populations.     

Hereditary Nonpolyposis Colon Cancer: Analysis of Linkage to 2p15-16 Places the COCA1 Locus Telomeric to D2S123 and Reveals Genetic Heterogeneity in Seven Canadian Families

Hereditary nonpolyposis colon cancer (HNPCC) is an autosomal dominant trait responsible for approximately 6% of colorectal cancers. Linkage of the HNPCC trait to the D2S123 locus on 2p15-16 has previously been reported in two families. This HNPCC locus is now designated “COCA1.” We have tested seven Canadian HNPCC families, who have a variety of clinical presentations, for linkage to a panel of microsatellite polymorphisms in the vicinity of D2S123. One family was clearly linked to the COCA1 locus (LOD = 4.21), and a second family is likely to be linked (LOD = 0.92). In three families linkage was excluded. In the remaining two families the data were inconclusive. In the linked family, individuals with cancer of the endometrium or ureter share a common haplotype with 12 family members with colorectal cancer. This supports the suspected association between these extracolonic neoplasms and the HNPCC syndrome. In addition, five of the six individuals with adenomatous polyps (but no colorectal cancer) have the same haplotype as the affected individuals, while the sixth carries a recombination. One individual with colorectal cancer carries a recombination that places the COCA1 locus telomeric to D2S123. This study localizes the COCA1 gene to an 8-cM region that is consistent with the location of the hMSH2 gene. We also confirm that families presently classified as HNPCC are genetically heterogeneous.     

Data-mining methods as useful tools for predicting individual drug response: application to CYP2D6 data

OBJECTIVES: Selecting a maximally informative subset of polymorphisms to predict a clinical outcome, such as drug response, requires appropriate search methods due to the increased dimensionality associated with looking at multiple genotypes. In this study, we investigated the ability of several pattern recognition methods to identify the most informative markers in the CYP2D6 gene for the prediction of CYP2D6 metabolizer status. METHODS: Four data-mining tools were explored: decision trees, random forests, artificial neural networks, and the multifactor dimensionality reduction (MDR) method. Marker selection was performed separately in eight population samples of different ethnic origin to evaluate to what extent the most informative markers differ across ethnic groups. RESULTS: Our results show that the number of polymorphisms required to predict CYP2D6 metabolic phenotype with a high accuracy can be dramatically reduced owing to the strong haplotype block structure observed at CYP2D6. MDR and neural networks provided nearly identical results and performed the best. CONCLUSION: Data-mining methods, such as MDR and neural networks, appear as promising tools to improve the efficiency of genotyping tests in pharmacogenetics with the ultimate goal of pre-screening patients for individual therapy selection with minimum genotyping effort.