CYP2D6 Genotype Dependent Oxycodone Metabolism in Postoperative Patients

Background

The impact of polymorphic cytochrome P450 CYP2D6 enzyme on oxycodone''s metabolism and clinical efficacy is currently being discussed. However, there are only spare data from postoperative settings. The hypothesis of this study is that genotype dependent CYP2D6 activity influences plasma concentrations of oxycodone and its metabolites and impacts analgesic consumption.

Methods

Patients received oxycodone 0.05 mg/kg before emerging from anesthesia and patient-controlled analgesia (PCA) for the subsequent 48 postoperative hours. Blood samples were drawn at 30, 90 and 180 minutes after the initial oxycodone dose. Plasma concentrations of oxycodone and its metabolites oxymorphone, noroxycodone and noroxymorphone were analyzed by liquid chromatography-mass spectrometry with electrospray ionization. CYP2D6 genotyping was performed and 121 patients were allocated to the following genotype groups: PM (poor metabolizer: no functionally active CYP2D6 allele), HZ/IM (heterozygous subjects, intermediate metabolizers with decreased CYP2D6 activity), EM (extensive metabolizers, normal CYP2D6 activity) and UM (ultrarapid metabolizers, increased CYP2D6 activity). Primary endpoint was the genotype dependent metabolite ratio of plasma concentrations oxymorphone/oxycodone. Secondary endpoint was the genotype dependent analgesic consumption with calculation of equianalgesic doses compared to the standard non-CYP dependent opioid piritramide.

Results

Metabolism differed between CYP2D6 genotypes. Mean (95%-CI) oxymophone/oxycodone ratios were 0.10 (0.02/0.19), 0.13 (0.11/0.16), 0.18 (0.16/0.20) and 0.28 (0.07/0.49) in PM, HZ/IM, EM and UM, respectively (p = 0.005). Oxycodone consumption up to the 12^th hour was highest in PM (p = 0.005), resulting in lowest equianalgesic doses of piritramide versus oxycodone for PM (1.6 (1.4/1.8); EM and UM 2.2 (2.1/2.3); p<0.001). Pain scores did not differ between genotypes.

Conclusions

In this postoperative setting, the number of functionally active CYP2D6 alleles had an impact on oxycodone metabolism. The genotype also impacted analgesic consumption, thereby causing variation of equianalgesic doses piritramide : oxycodone. Different analgesic needs by genotypes were met by PCA technology in this postoperative cohort.     

The AmpliChip CYP450 genotyping test: Integrating a new clinical tool

The AmpliChip CYP450 Test, which analyzes patient genotypes for cytochrome P450 (CYP) genes CYP2D6 and CYP2C19, is a major step toward introducing personalized prescribing into the clinical environment. Interest in adverse drug reactions (ADRs), the genetic revolution, and pharmacogenetics have converged with the introduction of this tool, which is anticipated to be the first of a new wave of such tools to follow over the next 5-10 years. The AmpliChip CYP450 Test is based on microarray technology, which combines hybridization in precise locations on a glass microarray and a fluorescent labeling system. It classifies individuals into two CYP2C19 phenotypes (extensive metabolizers [EMs] and poor metabolizers [PMs]) by testing three alleles, and into four CYP2D6 phenotypes (ultrarapid metabolizers [UMs], EMs, intermediate metabolizers [IMs], and PMs) by testing 27 alleles, including seven duplications. CYP2D6 is a metabolic enzyme with four activity levels (or phenotypes): UMs with unusually high activity; normal subjects, known as EMs; IMs with low activity; and PMs with no CYP2D6 activity (7% of Caucasians and 1-3% in other ethnic groups). Levels of evidence for the association between CYP2D6 PMs and ADRs are relatively reasonable and include systematic reviews of case-control studies of some typical antipsychotics and tricyclic antidepressants (TCAs). Evidence for other phenotypes is considerably more limited. The CYP2D6 PM phenotype may be associated with risperidone ADRs and discontinuation due to ADRs. Venlafaxine, aripiprazole, duloxetine, and atomoxetine are newer drugs metabolized by CYP2D6 but studies of the clinical relevance of CYP2D6 genotypes are needed. Non-psychiatric drugs metabolized by CYP2D6 include metoprolol, tamoxifen, and codeine-like drugs. CYP2C19 PMs (3-4% of Caucasians and African Americans, and 14-21% of Asians) may require dose adjustment for some TCAs, moclobemide, and citalopram. Other drugs metabolized by CYP2C19 are diazepam and omeprazole. The future of pharmacogenetics depends on the ability to overcome serious obstacles, including the difficulties of conducting and publishing studies in light of resistance from grant agencies, pharmaceutical companies, and some scientific reviewers. Assuming more studies are published, pharmacogenetic clinical applications may be compromised by economic factors and the lack of physician education. The combination of a US FDA-approved test, such as the AmpliChip CYP450 Test, and an FDA definition of CYP2D6 as a 'valid biomarker' makes CYP2D6 genotyping a prime candidate to be the first successful pharmacogenetic test in the clinical environment. One can use microarray technology to test for hundreds of single nucleotide polymorphisms (SNPs) but, taking into account the difficulties for single gene approaches such as CYP2D6, it is unlikely that very complex pharmacogenetic approaches will reach the clinical market in the next 5-10 years.     

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.     

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.     

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.     

Characterization of variant alleles of cytochrome CYP2D6 in a Spanish population

The CYP2D6 gene codes for a P450 monooxygenase which is involved in the biotransformation of a large number of commonly prescribed drugs. Adverse drug effects and therapeutic failure can be related to abnormal CYP2D6 activity. We investigated the allele and genotype frequencies of cytochrome P4502D6 in a Spanish population to predict the prevalence of ultra-rapid and poor metabolizer phenotypes in our population and to design a feasible CYP2D6 genotyping protocol. The study included 105 healthy unrelated Spanish Caucasian volunteers. CYP2D6 genotyping was performed by a combination of long-PCR, direct sequencing and allele-specific real-time PCR. The frequency of the wild-type CYP2D6*1 allele was 31%. The alleles coding for slightly (CYP2D6*2) or moderately (*9 and *10) reduced activity showed frequencies of 40.47, 2.38 and 1.90%, respectively. Frequencies of defective alleles *3, *4, *5 and *6 were 0.95, 13.8, 3.33 and 0.95%, respectively. The defective CYP2D6 alleles *7, *8, *12, *14, *15 and *21 were not found. Duplicated CYP2D6 alleles were detected at a frequency of 4.27%. Our protocol allows the identification of the four inactive CYP2D6 alleles (*3, *4, *5 and *6) and the detection of alleles with CYP2D6 *1, CYP2D6 *2 and CYP2D6*4 gene duplications. Testing for this reduced CYP2D6 allele set would facilitate its use in clinical practice by assisting in the development of individualized pharmacotherapy.     

Genetic variations of S-mephenytoin 4'-hydroxylase (CYP2C19) in the Chinese population

Most of phenotyping studies have shown that Chinese populations have a higher incidence of poor metabolizers (PMs) of S-mephenytoin 4'-hydroxylation compared with populations of African and European descent. The present study was aimed at defining an exact population frequency of the genetic defect of S-mephenytoin 4'-hydroxylase (CYP2C19) in native and overseas Chinese healthy populations. All the related data were systematically summarized and re-analyzed using meta-analysis method, and consistency between phenotypic and genotypic frequencies of the PM was tested. A statistically significant homogeneity was across all 11 phenotyping studies (chi2 = 15.17, d.f. = 10; P > 0.05) and also across the remaining 4 genotyping studies (chi2 = 2.61, d.f. = 3; P > 0.05) except for a non-randomly selected population analysis. An approximate estimate of the PM phenotypic and genotypic frequencies was 13.6% (212 of 1555; 95% CI: 11.9%-15.3%) and 13.8% (79 of 573; 95%CI: 11.0%-16.6%), respectively. There was a good consistency between phenotyped and genotyped PM frequencies. The half of all genotyped EMs (50.3%, 276 of 549) were heterozygotes. The data estimate that 14% of Chinese would be homozygotes of CYP2C19 defective alleles, and that 176 million Chinese would be slow metabolizers of CYP2C19 substrates.     

Miotic action of tramadol is determined by CYP2D6 genotype

Polymorphic CYP2D6 is the enzyme that activates the opioid analgesic tramadol by O-demethylation to its active metabolite O-demethyltramadol (M1). Our objective was to determine the opioid effects measured by pupillary response to tramadol of CYP2D6 genotyped volunteers in relation to the disposition of tramadol and M1 in plasma. Tramadol displayed phenotypic pharmacokinetics and it was possible to identify poor metabolizers (PM) with >99% confidence from the metabolic ratio (MR) in a single blood sample taken between 2.5 and 24 h post-dose. Homozygous extensive metabolizers (EM) differed from PM subjects by an almost threefold greater (P=0.0014) maximal pupillary constriction (Emax). Significant correlations between the AUC and Cmax values of M1 versus pupillary constriction were found. The corresponding correlations of pharmacokinetic parameters for tramadol itself were weaker and negative. The strongest correlations were for the single-point metabolic ratios at all sampling intervals versus the effects, with rs ranging from 0.85 to 0.89 (p<0.01). It is concluded that the concept of dual opioid/non-opioid action of the drug, though considerably stronger in EMs, is valid for both EM and PM subjects. This is the theoretical basis for the frequent use and satisfactory efficacy of tramadol in clinical practice when given to genetically non-selected population.     

Polymorphic Cytochromes P450 and Drugs Used in Psychiatry

1. The cytochrome P450 monooxygenases, CYP2D6, CYP2C19, and CYP2C9, display polymorphism. CYP2D6 and CYP2C19 have been studied extensively, and despite their low abundance in the liver, they catalyze the metabolism of many drugs.2. CYP2D6 has numerous allelic variants, whereas CYP2C19 has only two. Most variants are translated into inactive, truncated protein or fail to express protein.3. CYP2C9 is expressed as the wild-type enzyme and has two variants, in each of which one amino acid residue has been replaced.4. The nucleotide base sequences of the cDNAs of the three polymorphic genes and their variants have been determined, and the proteins derived from these genes have been characterized.5. An absence of CYP2D6 and/or CYP2C19 in an individual produces a poor metabolizer (PM) of drugs that are substrates of these enzymes.6. When two drugs that are substrates for a polymorphic CYP enzyme are administered concomitantly, each will compete for that enzyme and competitively inhibit the metabolism of the other substrate. This can result in toxicity.7. Patients can be readily phenotyped or genotyped to determine their CYP2D6 or CYP2C19 enzymatic status. Poor metabolizers (PMs), extensive metabolizers (EMs), and ultrarapid metabolizers (URMs) can be identified.8. Numerous substrates and inhibitors of CYP2D6, CYP2C19, and CYP2C9 are identified.9. An individual's diet and age can influence CYP enzyme activity.10. CYP2D6 polymorphism has been associated with the risk of onset of various illnesses, including cancer, schizophrenia, Parkinson's disease, Alzheimer's disease, and epilepsy.     

Deletion of the entire cytochrome P450 CYP2D6 gene as a cause of impaired drug metabolism in poor metabolizers of the debrisoquine/sparteine polymorphism.

The debrisoquine/sparteine polymorphism is associated with a clinically important genetic deficiency of oxidative drug metabolism. From 5% to 10% of Caucasians designated as poor metabolizers (PMs) of the debrisoquine/sparteine polymorphism have a severely impaired capacity to metabolize more than 25 therapeutically used drugs. The impaired drug metabolism in PMs is due to the absence of cytochrome P450IID6 protein. The gene controlling the P450IID6 protein, CYP2D6, is located on the long arm of chromosome 22. A pseudogene CYP2D8P and a related gene CYP2D7 are located upstream from CYP2D6. This gene locus is highly polymorphic. After digestion of genomic DNA with XbaI endonuclease, restriction fragments of 11.5 kb and 44 kb represent mutant alleles of the cytochrome CYP2D6 gene locus associated with the PM phenotype. In order to elucidate the molecular mechanism of the mutant allele reflected by the XbaI 11.5-kb fragment, a genomic library was constructed from leukocyte DNA of one individual homozygous for this fragment and screened with the human IID6 cDNA. The CYP2D genes were isolated and characterized by restriction mapping and partial sequencing. We demonstrate that the mutant 11.5-kb allele results from a deletion involving the entire functional CYP2D6 gene. This result provides an explanation for the total absence of P450IID6 protein in the liver of these PMs.     

Pharmacogenetic basis for therapeutic optimization in Alzheimer's disease

Alzheimer's disease is a major health problem in developed countries. Approximately 10-15% of direct costs in dementia are attributed to pharmacological treatment, and only 10-20% of the patients are moderate responders to conventional antidementia drugs, with questionable cost effectiveness. The phenotypic expression of Alzheimer's disease is characterized by amyloid deposition in brain tissue and vessels (amyloid angiopathy), intracellular neurofibrillary tangle formation, synaptic and dendritic loss, and premature neuronal death. Primary pathogenic events underlying this neurodegenerative process include genetic factors involving more than 200 different genes distributed across the human genome, accompanied by progressive cerebrovascular dysfunction, and diverse environmental factors. Mutations in genes directly associated with the amyloid cascade (APP, PSEN1, PSEN2) are present in less than 5% of the Alzheimer's disease population; however, the presence of the epsilon4 allele of the apolipoprotein E gene (APOE) represents a major risk factor for more than 40% of patients with dementia. Genotype-phenotype correlation studies and functional genomics studies have revealed the association of specific mutations in primary loci and/or APOE-related polymorphic variants with the phenotypic expression of biological traits. It is estimated that genetics accounts for between 20% and 95% of the variability in drug disposition and pharmacodynamics. Recent studies indicate that the therapeutic response in Alzheimer's disease is genotype specific, depending on genes associated with Alzheimer's disease pathogenesis and/or genes responsible for drug metabolism (e.g. cytochrome P450 [CYP] genes). In monogenic studies, APOEepsilon4/epsilon4 genotype carriers are the worst responders to conventional treatments. Some cholinesterase inhibitors currently being use in the treatment of Alzheimer's disease are metabolized via CYP-related enzymes. These drugs can interact with many other drugs that are substrates, inhibitors or inducers of the CYP system, this interaction eliciting liver toxicity and other adverse drug reactions. CYP2D6 enzyme isoforms are involved in the metabolism of more than 20% of drugs used in CNS disorders. The distribution of the CYP2D6 genotypes in the European population of the Iberian peninsula differentiates four major categories of CYP2D6-related metabolizer types: (i) extensive metabolizers (EM) [51.61%]; (ii) intermediate metabolizers (IM) [32.26%]; (iii) poor metabolizers (PM) [9.03%]; and (iv) ultra-rapid metabolizers (UM) [7.10%]. PMs and UMs tend to show higher transaminase activity than EMs and IMs. EMs and IMs are the best responders, and PMs and UMs are the worst responders to pharmacologic treatments in Alzheimer's disease. At this early stage of the development of pharmacogenomic/pharmacogenetic procedures in Alzheimer's disease therapeutics, it seems very plausible that the pharmacogenetic response in Alzheimer's disease depends on the interaction of genes involved in drug metabolism and genes associated with Alzheimer's disease pathogenesis.     

Characterization of pharmacogenetically relevant CYP2D6 and ABCB1 gene polymorphisms in a Portuguese population sample

Differences in metabolism of drugs can lead to severe toxicity or therapeutic failure. In addition to cytochrome P450 2D6, which plays a critical role in drug metabolism, ABCB1 encoded P‐glycoprotein (PGP) is also an important determinant in drug bioavailability. The genes encoding these molecules are highly variable among populations and, given their clinical importance in drug therapy, determining CYP2D6 and ABCB1 allele frequencies in specific populations is very important for useful application in clinical settings. In this study the frequency of the pharmacologically relevant CYP2D6*3, *4, *5, *6 allelic variants and gene duplication, and ABCB1 C1236T and C3435T gene polymorphisms and their haplotypes was determined in a population sample of 100 Portuguese healthy subjects. CYP2D6 allele frequencies were 1.4% (*3), 13.3% (*4), 2.8% (*5), 1.8% (*6) and 6.1% (gene duplication), with 5% of the individuals classified as PM and 8.4% as UM. The frequencies obtained for the non‐functional alleles and for the CYP2D6 gene duplication are in agreement with other South European populations, and reinforce the previously suggested south/north gradient of CYP2D6 duplications. Allelic frequencies for the ABCB1 polymorphisms were 52% (3435C) and 54% (1236C) and the most common haplotype (1236C‐3435C) occurred with a frequency of 45.5%. Although allele and haplotype frequency data for ABCB1 in Southern Europe is limited, some discrepancies were found with other European populations, with possible therapeutic implications for PGP substrate drugs. Copyright © 2009 John Wiley & Sons, Ltd.     

Individualized therapy for gastroesophageal reflux disease: potential impact of pharmacogenetic testing based on CYP2C19

The main therapeutic agent for gastroesophageal reflux disease (GERD) is a proton pump inhibitor (PPI). Plasma levels and the acid inhibitory effect of PPIs depend on the activity of cytochrome P450 (CYP) 2C19, which is polymorphic. Genotypes of CYP2C19 are classified into three groups: rapid metabolizers (RMs: *1/*1), intermediate metabolizers (IMs: *1/*X), and poor metabolizers (PMs: *X/*X), where *1 and X represent the wild type and the mutant allele, respectively. RMs include ultra-rapid metabolizers, who possess the CYP2C19*17 allele. The pharmacokinetics and pharmacodynamics of PPIs differ among different CYP2C19 genotype groups. Plasma PPI levels and intragastric pH values during PPI treatment are lowest in the RM group, intermediate in the IM group, and highest in the PM group. These CYP2C19-genotype-dependent differences in the pharmacokinetics and pharmacodynamics of PPIs influence the healing and recurrence of GERD during PPI treatment, suggesting the need for CYP2C19 genotype-based tailored therapy for GERD. CYP2C19 pharmacogenetics should be taken into consideration for the personalization of PPI-based therapy. However, the clinical usefulness of CYP2C19 genotype testing in GERD therapy should be verified in clinical studies.     

Alternative Reliable Method for Cytochrome P450 2D6 Poor Metabolizers Genotyping

High-resolution melting curve analysis (HRM) of polymerase chain reaction (PCR) amplicons has been described as a fast, cheap, and reliable closed-tube method of genotyping with no need for labeled primers or labeled probes. We adapted this melting analysis assay for the detection of the most common nonfunctional alleles of cytochrome P-450 (CYP) 2D6 in the Caucasian population that affect the metabolism of many commonly used drugs. We used this method to genotype 91 patients under paroxetine therapy. The presence and the constitution of the most common single-nucleotide polymorphisms (1846G>A, 2988G>A, 100C>T, 2549delA, 2615_2617delAAG, and 1707delT) in poor and intermediate metabolizers from the Caucasian population were detected in short amplicons (??148 bp). After fluorescence normalization, the wild-type, homozygous, and heterozygous samples were easily distinguishable from each other by their specific melting curve shape. A total of 92.6% of the 1846G>A heterozygotes, 96% of the 100C>T heterozygotes, and 100% of the 2988G>A, 2549delA, 2615_2617delAAG, and 1707delT heterozygotes have been correctly distinguished from the wild types. One hundred percent of all the homozygotes in this group of patients have been detected without any error. HRM of short amplicons is a simple tool for effective, rapid, and reliable CYP2D6 genotyping that does not require real-time PCR, labeled probes, processing or any separations after PCR. The reaction is performed in a closed-tube system and is highly specific and sensitive. We proved that this technique is highly reliable for use in routine diagnostics.     

Identification and characterisation of novel polymorphisms in the CYP2A locus: implications for nicotine metabolism.

The polymorphic human cytochrome P450 2A6 (CYP2A6) metabolises a number of drugs, activates a variety of precarcinogens and constitutes the major nicotine C-oxidase. A relationship between CYP2A6 genotype and smoking habits, as well as incidence of lung cancer, has been proposed. Two defective alleles have hitherto been identified, one of which is very common in Asian populations. Among Caucasians, an additional defective and frequently distributed allele (CYP2A6*3) has been suggested to play a protective role against nicotine addiction and cigarette consumption. Here, we have re-evaluated the genotyping method used for the CYP2A6*3 allele and found that a gene conversion in the 3' flanking region of 30-40% of CYP2A6*1 alleles results in genotype misclassification. In fact, no true CYP2A6*3 alleles were found among 100 Spaniards and 96 Chinese subjects. In one Spanish poor metaboliser of the CYP2A6 probe drug coumarin, we found two novel defective alleles. One, CYP2A6*5, encoded an unstable enzyme having a G479L substitution and the other was found to carry a novel type of CYP2A6 gene deletion (CYP2A6*4D). The results imply the presence of numerous defective as well as active CYP2A6 alleles as a consequence of CYP2A6/CYP2A7 gene conversion events. We conclude that molecular epidemiological studies concerning CYP2A6 require validated genotyping methods for accurate detection of all known defective CYP2A6 alleles.     

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.     

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.     

Phenotype-genotype analysis of CYP2C19 in Colombian mestizo individuals

Background  

Omeprazole is metabolized by the hepatic cytochrome P450 (CYP) 2C19 enzyme to 5-hydroxyomeprazole. CYP2C19 exhibits genetic polymorphisms responsible for the presence of poor metabolizers (PMs), intermediate metabolizers (IMs) and extensive metabolizers (EMs). The defective mutations of the enzyme and their frequencies change between different ethnic groups; however, the polymorphism of the CYP2C19 gene has not been studied in Colombian mestizos. The aim of this study was to evaluate the genotype and phenotype status of CYP2C19 in Colombian mestizos, in order to contribute to the use of appropriate strategies of drug therapy for this population.