Pilot study for the characterization of pharmacogenetically relevant CYP2D6, CYP2C19 and ABCB1 gene polymorphisms in the Hungarian population

Polymorphisms of CYP450 metabolizer enzymes and transport proteins play crucial roles in the inter‐individual variability of drug efficiency. The aim of our study was to predict the frequency of functional variants of CYP2D6, CYP2C19 and ABCB1 genes in the Hungarian population. One hundred twelve unrelated healthy subjects donated DNA sample in the study. ABCB1 C3435T and G2677T/A single‐nucleotide polymorphisms (SNPs) were determined by LightCycler polymerase chain reaction. Because only limited amount of data is available on the rare allelic variants of CYP2D6 in the European populations, our study applied an expanded set of CYP2D6 and CYP2C19 alleles by using AmpliChip test. Our results show that the CYP2D6 phenotypes were 1.9% ultra‐rapid metabolizer, 6.5% intermediate metabolizer (IM), 8.3% poor metabolizer (PM) and 83.3% extensive metabolizer (EM), and the CYP2C19 phenotypes were 1.8% PM, 31.2% IM and 67% EM. The prevalence of the commonly observed CYP2D6 and CYP2C19 alleles in our study corresponds with that of other European populations. Nevertheless, our study confirms that extending the CYP2D6 allele set with loss‐of‐function variants such as CYP2D6*7, *9, *41 is worth considering. Frequency of the wild type ABCB1 3435C was 42.8% whereas the prevelance of 2677 G was 50.4%. Although frequency data of G2677T/A SNP in the European area are limited, some discrepancies with other studies were found. Copyright © 2011 John Wiley & Sons, Ltd.     

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.     

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.     

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.     

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.     

An inactive cytochrome P450 CYP2D6 allele containing a deletion and a base substitution

The cytochrome P450 CYP2D6 is a polymorphic enzyme, for which 5%–10% of Caucasians (poor metabolizers) lack activity. The majority of mutations giving rise to the deficiency have now been identified but some individuals show anomalous phenotype-genotype relationships when screened for the common mutant alleles. We have sequenced all nine exons and intron-exon boundaries in a subject who was phenotypically a poor metabolizer but genotypically heterozygous when screened for the common alleles. A single base-pair deletion (T₁₇₉₅) was detected in exon 3 and a base substitution (G₂₀₆₄A) resulting in an amino acid substitution (G₂₁₂E) in exon 4. The deletion results in premature termination of translation and a truncated protein. In a group of 50 white Americans, the allele frequency for the new mutant allele was 0.01. The new allele explains some cases of anomalous genotype/phenotype relationships for CYP2D6.     

Development of a PCR-based strategy for CYP2D6 genotyping including gene multiplication of worldwide potential use

There is growing consensus on the potential use of pharmacogenetics in clinical practice, and hopes have been expressed for application to the improvement of global health. However, two major challenges may lead to widening the "biotechnological gap" between the developing and the industrial world;first the unaffordability of some current technologies for poorer countries, and second the necessity of analyzing all described alleles for every clinical case due to the inability to predict the ethnic group of a given patient. Because of its role in the metabolism of a number of drugs, cytochrome P450 2D6 (CYP2D6) is an excellent candidate for use in the optimization of drug therapy. CYP2D6 is a highly polymorphic gene locus with more than 50 variant alleles, and subjects can be classified as poor metabolizers (PM), extensive metabolizers (EM), or ultrarapid metabolizers (UM) of a given CYP2D6 substrate. Several strategies and methods for CYP2D6 genotyping exist. Some, however, are expensive and laborious. The aim of this study was to design a PCR-based genotyping methodology to allow rapid, straightforward, and inexpensive identification of 90%-95% of CYP2D6 PM or UM genotypes for routine clinical use, independent of the individual's ethnic group. CYP2D6 is amplified in initial extra long PCRs (XL-PCRs), which subsequently undergo fragment-length polymorphism analysis for the determination of carriers of CYP2D6 allelic variants. The same XL-PCRs are also used for the determination of CYP2D6 multiplication and 2D6*5 allele (abolished activity). The application of this new strategy for the detection of CYP2D6 mutated alleles and multiplications to routine clinical analysis will enable the PM and UM phenotypes to be predicted and identified at a reasonable cost in a large number of individuals at most locations.     

Identification of four novel mutations in the CYP21 gene in congenital adrenal hyperplasia in the Chinese

Congenital adrenal hyperplasia (CAH) is a common autosomal recessive disorder mainly caused by defects in the steroid 21-hydroxylase (CYP21) gene. We have analyzed CYP21 gene sequences in 65 CAH families in Taiwan. All ten exons of the CYP21 gene were analyzed by differential polymerase chain reaction followed by single-strand conformation polymorphism electrophoresis and the amplification-created restriction site method. About 95% (123 chromosomes) contain mutations due to conversion of DNA sequences into its neighboring homologous pseudogene, CYP21P. Four novel mutations representing 5% of the total chromosomes have also been identified. The mutations were confirmed by sequencing an aberrant DNA fragment. These four mutations included a base change of the splicing donor site at intron 2 from GT to AT, a base substitution of C to T at codon 316, deletion of ten bases (TCCAGCTCCC) at codons 330–333 of exon 8, and duplication of 16 bases (CCTGGATGACACGGTC) at codons 393–397 of exon 9. The loss of the splicing donor site at intron 2 and the premature stop at codon 316 may result in aberrant splicing to reduce enzyme activity and a truncated protein with no enzyme activity, respectively. Likewise, both the duplication and the deletion forms create a frameshift and premature stop during translation. The resulting proteins lack the heme-binding domain and hence are expected to lose enzymatic activity. Since these mutations are not found in the neighboring CYP21P pseudogene, gene conversion should not be the cause of these novel mutations. Received: 20 April 1998 / Accepted: 30 May 1998     

Study of transcription of the human cytochrome P450IID6 gene by polymerase chain reaction]

Polymerase chain reaction was used to study the expression of the drug metabolism gene. Primers complementary to the 2070-2090 and 2912-2930 sites within exons 4 and 6 of the gene CYP2D6 were synthesized. The amplification of the cDNA from total human liver mRNA was achieved. The length of the fragment obtained (238 bp) was in accordance with the distance between the primers binding sites in cDNA. The amplification of the DNA from the same source led to the longer fragment due to the presence of introns. The total RNA from the blood cells of the extensive metabolizers was shown to contain the mRNA transcribed from the CYP2D6 gene. The Taq polymerase reaction in the presence of cDNA derived from a poor metabolizer did not lead to the synthesis of the 238 bp fragment.     

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.     

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.     

Molecular cloning and nucleotide sequence of a new P450 gene, CYP319A1, from the cattle tick, Boophilus microplus.

We have isolated and sequenced a novel P450 gene (CYP319A1) from the cattle tick, Boophilus microplus. The CYP319A1 cDNA encodes a protein of 531 amino acids with an estimated molecular weight of 60.9k. It contains all highly conserved motifs characteristic of P450 enzymes. Comparison of deduced amino acid sequence with other CYP members shows that the CYP319A1 is more closely related to CYP4 family, but its overall identity to the CYP4 family is less than 40%. Therefore, it was assigned to a new P450 family by the P450 nomenclature committee. A pseudogene which shares high homology with the CYP319A1 was identified. Analysis of genomic sequence of the pseudogene indicated that the pseudogene contains two additional DNA inserts in the coding region, which disrupt the open reading frame. RT-PCR analysis showed that CYP319A1 is expressed in both susceptible and acaricide-resistant ticks.     

Evolution of the CYP2D gene cluster in humans and four non-human primates

The human cytochrome P450 2D6 (CYP2D6) is a primary enzyme involved in the metabolism of about 25% of commonly used therapeutic drugs. CYP2D6 belongs to the CYP2D subfamily, a gene cluster located on chromosome 22, which comprises the CYP2D6 gene and pseudogenes CYP2D7P and CYP2D8P. Although the chemical and physiological properties of CYP2D6 have been extensively studied, there has been no study to date on molecular evolution of the CYP2D subfamily in the human genome. Such knowledge could greatly contribute to the understanding of drug metabolism in humans because it makes us to know when and how the current metabolic system has been constructed. The knowledge moreover can be useful to find differences in exogenous substrates in a particular metabolism between human and other animals such as experimental animals. Here, we conducted a preliminary study to investigate the evolution and gene organization of the CYP2D subfamily, focused on humans and four non-human primates (chimpanzees, orangutans, rhesus monkeys, and common marmosets). Our results indicate that CYP2D7P has been duplicated from CYP2D6 before the divergence between humans and great apes, whereas CYP2D6 and CYP2D8P have been already present in the stem lineages of New World monkeys and Catarrhini. Furthermore, the origin of the CYP2D subfamily in the human genome can be traced back to before the divergence between amniotes and amphibians. Our analyses also show that reported chimeric sequences of the CYP2D6 and CYP2D7 genes in the chimpanzee genome appear to be exchanged in its genome database.     

Expression and regulation of CYP6D3 in the house fly, Musca domestica (L.).

Recently, a new cytochrome P450 gene, CYP6D3, was identified from house fly. CYP6D3 was found upstream of a related gene (CYP6D1) on autosome 1. CYP6D3 cDNA sequences were obtained and compared from insecticide resistant (LPR) and susceptible (CS and Edinburgh) strains. Although each strain had a different CYP6D3 allele, the deduced amino acid sequences revealed no consistent differences between the susceptible and resistant strains. There was approximately 12-fold more CYP6D3 mRNA detected in adult LPR flies compared to CS, and the elevated level of expression in LPR was not due to gene amplification. Northern blots indicate expression of CYP6D3 mRNA is developmentally regulated with no expression in eggs, yet it is readily detectable in larvae as well as male and female adults. Phenobarbital is a well studied inducer of P450s in insects and it induced expression of CYP6D3 mRNA in both the CS (16-fold) and LPR (1.6 fold) strains. The CYP6D3 5' flanking regions were sequenced from the resistant and susceptible strains. Possible regulatory sequences within this region are discussed.