The decreased in vivo clearance of CYP2D6 substrates by CYP2D6*10 might be caused not only by the low-expression but also by low affinity of CYP2D6

CYP2D6 exhibits genetic polymorphism with interindividual differences in metabolic activity. We have found a significant influence on the pharmacokinetics of venlafaxine by the CYP2D6*10 allele in a Japanese population. CYP2D6.10, which is translated from CYP2D6*10, has two amino acid substitutions: Pro34 --> Ser and Ser486 --> Thr. In this study, CYP2D6.10 was expressed in Saccharomyces cerevisiae and its catalytic activity for CYP2D6 substrates was investigated. The CYP2D6*10B- and *10C-associated cDNA were isolated from human lymphocyte genotyped as CYP2D6*10. In addition, three forms of CYP2D6, Pro34/Thr486 (PT), Ser34/Ser486 (SS), and Pro34/Ser486 (wild type, CYP2D6.1), were constructed by PCR-site mutagenesis to clarify the effects of the two amino-acid substitutions. The expression of CYP2D6 protein was confirmed by immunoblotting using CYP2D antibody. The absorbance at 450 nm was measured by CO-reduced difference spectra from five all microsome preparations. The CYP2D6 forms with Pro34 --> Ser amino acid substitution were at a lower expression than CYP2D6.1 from the findings of immunoblotting and spectral analysis. The apparent K(m) values of CYP2D6.1, CYP2D6.10A, and CYP2D6.10C were 1.7, 8.5, and 49.7 microM, respectively, for bufuralol 1'-hydroxylation, and 9.0, 51.9, and 117.4 microM, respectively, for venlafaxine O-demethylation, respectively. The V(max) values were not significantly different among the three variants. These findings suggest that the decreased in vivo clearance by CYP2D6*10 was caused not only by low expression of but also the increased K(m) value of CYP2D6.     

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.     

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.     

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.     

CYP1A1 and CYP2D6 polymorphism and risk of lung cancer in a North Indian population

This case–control study was conducted to examine the association between the CYP1A1 and CYP2D6 genotypes and lung cancer risk among North Indians. The estimated relative risk for lung cancer associated with the CYP1A1 Val/Val allele was 2.68, and was four-fold when cases with small cell lung cancer (SCLC) were considered alone. With regard to the metabolism of debrisoquine, no poor metabolizers were found amongst the subjects. The odds ratio of risk with the heterozygous extensive metabolizer (HEM) genotype was 1.5. However, in the presence of at least a single copy of the variant CYP1A1 MspI allele and the CYP2D6 HEM genotype, the risk was two-fold for squamous cell carcinoma (SQCC). When the CYP1A1 Val/Val and CYP2D6 HEM genotypes were taken together, the risk for SCLC was four-fold. Stratified analysis indicated an interaction between bidi smoking and variant CYP1A1 genotypes on the risk for SQCC and SCLC. Heavy smokers (Brinkman index>400) with Val/Val genotypes were at a very high risk of developing lung cancer (odds ratio 29.30, 95% confidence interval 2.42–355, p=0.008). Heavy smokers with CYP1A1 MspI (CYP1A1*1/2A or CYP1A1*2A/*2A) genotype had a seven-fold risk for SCLC compared with non-smokers. This study is the first to be carried out on a North Indian population, and, although small, suggests that CYP1A1 and CYP2D6 polymorphisms might have a role in determining the risk for lung cancer and should be investigated further.     

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.     

CYP1A1 and CYP2D6 polymorphism and risk of lung cancer in a North Indian population.

This case-control study was conducted to examine the association between the CYP1A1 and CYP2D6 genotypes and lung cancer risk among North Indians. The estimated relative risk for lung cancer associated with the CYP1A1 Val/Val allele was 2.68, and was four-fold when cases with small cell lung cancer (SCLC) were considered alone. With regard to the metabolism of debrisoquine, no poor metabolizers were found amongst the subjects. The odds ratio of risk with the heterozygous extensive metabolizer (HEM) genotype was 1.5. However, in the presence of at least a single copy of the variant CYP1A1 MspI allele and the CYP2D6 HEM genotype, the risk was two-fold for squamous cell carcinoma (SQCC). When the CYP1A1 Val/Val and CYP2D6 HEM genotypes were taken together, the risk for SCLC was four-fold. Stratified analysis indicated an interaction between bidi smoking and variant CYP1A1 genotypes on the risk for SQCC and SCLC. Heavy smokers (Brinkman index>400) with Val/Val genotypes were at a very high risk of developing lung cancer (odds ratio 29.30, 95% confidence interval 2.42-355, p=0.008). Heavy smokers with CYP1A1 MspI (CYP1A1*1/2A or CYP1A1*2A/*2A) genotype had a seven-fold risk for SCLC compared with non-smokers. This study is the first to be carried out on a North Indian population, and, although small, suggests that CYP1A1 and CYP2D6 polymorphisms might have a role in determining the risk for lung cancer and should be investigated further.     

Inhibitory Potency of 8-Methoxypsoralen on Cytochrome P450 2A6 (CYP2A6) Allelic Variants CYP2A6*15, CYP2A6*16, CYP2A6*21 and CYP2A6*22: Differential Susceptibility Due to Different Sequence Locations of the Mutations

Human cytochrome P450 2A6 (CYP2A6) is a highly polymorphic isoform of CYP2A subfamily. Our previous kinetic study on four CYP2A6 allelic variants (CYP2A6*15, CYP2A6*16, CYP2A6*21 and CYP2A6*22) have unveiled the functional significance of sequence mutations in these variants on coumarin 7-hydroxylation activity. In the present study, we further explored the ability of a typical CYP2A6 inhibitor, 8-methoxypsoralen (8-MOP), in inhibition of these alleles and we hypothesized that translational mutations in these variants are likely to give impact on 8-MOP inhibitory potency. The CYP2A6 variant and the wild type proteins were subjected to 8-MOP inhibition to yield IC₅₀ values. In general, a similar trend of change in the IC₅₀ and K_m values was noted among the four mutants towards coumarin oxidation. With the exception of CYP2A6*16, differences in IC₅₀ values were highly significant which implied compromised interaction of the mutants with 8-MOP. Molecular models of CYP2A6 were subsequently constructed and ligand-docking experiments were performed to rationalize experimental data. Our docking study has shown that mutations have induced enlargement of the active site volume in all mutants with the exception of CYP2A6*16. Furthermore, loss of hydrogen bond between 8-MOP and active site residue Asn297 was evidenced in all mutants. Our data indicate that the structural changes elicited by the sequence mutations could affect 8-MOP binding to yield differential enzymatic activities in the mutant CYP2A6 proteins.     

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.     

High frequency of CYP1A1*2C allele in Brazilian populations

The genetic variability of the CYP1A1 I462V polymorphism (CYP1A1*2C) was investigated in four Brazilian populations: three groups of African descent and one group of European descent. The CYP1A1 polymorphism was analyzed by two different procedures, first by the allele-specific polymerase chain reaction (PCR) method and then by the PCR-restricted fragment length polymorphism (PCR-RFLP) method before digestion with BsrDI. The frequency of CYP1A1 *2C was 11% in Brazilians of European descent, a frequency that is slightly higher but not statistically different from that observed in European populations. In Brazilians of African ancestry this value was very high (12% to 15%). This allele was not observed in the only two African populations investigated thus far. By themselves, the two factors of interethnic admixture (with populations of European descent and/or Amerindian populations) and genetic drift cannot explain the high values observed here. Our findings suggest that the CYP1A1 *2C allele may possibly be present in Africa, but restricted to some ethnic groups not yet investigated. Environmental factors in South America might also have acted as selective factors increasing the CYP1A1 *2C gene frequency. Our data also suggest that the CYP1A1 *2C allele might possibly have originated in Africa.     

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.     

Frequency of the ESD*5 allele in a Spanish population

The red blood cell esterase D (ESD) polymorphism was studied by means of IEF in a North-East Spanish population (Barcelona). Gene frequencies in 430 unrelated individuals were ESD*1: 0.888, ESD*2: 0.091 and ESD*5: 0.021. Our data confirm previous results showing that ESD*5 occurs in polymorphic frequency and has a Caucasian origin.     

CYP1A1*2 and CYP1A1*3 polymorphisms cosegregate in the Indian population

Several ethnic groups have been genotyped for polymorphisms at the CYP1A1 gene locus that encodes the enzyme that catalyzes the initial step in the metabolism of polycyclic aromatic hydrocarbons. Two of the CYP1A1 polymorphisms, namely, CYP1A1*2 and CYP1A1*3 are reported to cosegregate among the Japanese and to a lesser extent in Caucasians, but not in people of African descent. In the absence of such information in the Indian population, the frequency of the CYP1A1*2 polymorphism was determined in this study, using DNA samples from 649 ethnic Indians who had been earlier genotyped for the CYP1A1*3 polymorphism. Analysis of the combined genotype data revealed that the two polymorphisms cosegregate in the Indian population.     

An in vivo pilot study characterizing the new CYP2A6*7, *8, and *10 alleles.

We developed genotyping assays for CYP2A6*7 (Ile471Thr) and CYP2A6*8 (Arg485Leu). We found higher allelic frequencies in Japanese and Chinese versus Caucasians and identified an allele in which both substitutions occur together (CYP2A6*10). We created a homology model for predicting the impact of allelic variants on enzymatic activity and subsequently tested this in vivo in a pilot kinetic study. Consistent with our homology model predictions, we found (i) that CYP2A6*7 produces an enzyme that has decreased (not inactive) activity for metabolizing nicotine and coumarin; (ii) that CYP2A6*8 is unlikely to affect catalytic activity in vivo; and (iii) that having both substitutions together on an allele (CYP2A6*10) dramatically reduces function and may be fully inactive for some substrates. In conclusion, this study identifies, at relatively high frequency in Asians, an allele with decreased activity (may be substrate selective), a fully functional allele, and an allele containing both substitutions in which function is dramatically reduced.     

Allele and genotype frequencies of CYP2B6 in a Turkish population

Increasing interest in cytochrome P450 2B6 (CYP2B6) genetic polymorphism was stimulated by revelations of a specific CYP2B6 genotype significantly affecting the metabolism of various drugs in common clinical use in terms of increasing drug efficacy and avoiding adverse drug reactions. The present study aimed to determine the frequencies of CYP2B6*4 CYP2B6*5, CYP2B6*6, CYP2B6*7 and CYP2B6*9 alleles in healthy Turkish individuals (n = 172). Frequencies of three single nucleotide polymorphisms were 516G>T (28 %), 785A>G (33 %), and 1459C>T (12 %). The frequencies of CYP2B6*1, *4, *5, *6, *7, and *9 alleles were 54.3 (95 % CI 49.04–59.56), 6.4 % (95 % CI 3.81–8.99), 11 % (95 % CI 7.69–14.31), 25.3 % (95 % CI 20.71–29.89), 0.87 % (95 % CI ?0.11–1.85) and 2.0 % (95 % CI 0.52–3.48), respectively. Allele *6 was more frequent (25.3 %) than the other variant alleles in Turkish subjects. The frequencies of CYP2B6*4, *5, *6, *7, and *9 alleles were similar to European populations but significantly different from that reported for Asian populations. This is the first study to document the frequencies of the CYP2B6*4, *5, *6, *7, *9 alleles in the healthy Turkish individuals and our results could provide clinically useful information on drug metabolism by CYP2B6 in Turkish population.     

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.     

Apolipoprotein E*3-Leiden allele results from a partial gene duplication in exon 4

The apolipoprotein E3-Leiden variant has been shown to be associated with familial dysbetalipoproteinemia (FD) in a dominant manner (Havekes et al., Hum Genet 1986;73:157-163). Applying the polymerase chain reaction technique, we have cloned and sequenced relevant parts of both APOE alleles of the original proband. In exon 4 of the E*3-Leiden allele a partial gene duplication encompassing 21 nucleotides was found, leading to a tandem repeat of the codons 120-126 or 121-127. Using an E3-Leiden mutation specific oligonucleotide probe, the same mutation was found in two additional independently ascertained FD patients with an E3E3 phenotype based on isoelectric focusing. The E*3-Leiden mutation will be useful in the elucidation of the etiology of dominantly inherited forms of FD.     

Towards a polymeric binding mimic for cytochrome CYP2D6

A series of fluorescent molecularly imprinted polymers has been prepared with a view to generating material capable of mimicking the binding characteristics of the metabolically important cytochrome isoform CYP2D6. Such polymers would have the possibility to form the sensing element in a high-throughput assay for the prediction of CYP2D6 affinity. The imprinted polymers possessed binding-dependent fluorescence. They re-bound their templates and various cross-reactivities were encountered for test compound/drug recognition. One polymer in particular exhibited a rational discrimination amongst the related synthetic templates and was reasonably successful in recognising CYP2D6 substrates from a drug panel.     

High prevalence of CYP2C19*2 allele in Roma samples: study on Roma and Hungarian population samples with review of the literature

The purpose of our study was to characterise the CYP2C19*2 and CYP2C19*3 alleles in healthy Roma and Hungarian populations. DNA of 500 Roma and 370 Hungarian subjects were genotyped for CYP2C19*2 (G681A, rs4244285) and CYP2C19*3 (G636A, rs4986893) by PCR–RFLP assay and direct sequencing. Significant differences were found comparing the Roma and Hungarian populations in CYP2C19 681 GG (63.6 vs. 75.9 %), GA (31.8 vs. 23.0 %), AA (4.6 vs. 1.1 %), GA+AA (36.4 vs. 24.1 %) and A allele frequencies (0.205 vs. 0.125) (p < 0.004). Striking differences were found between Roma and Hungarian samples in CYP2C19*1 (79.5 vs. 87.4 %) and CYP2C19*2 (20.5 vs. 12.6 %) alleles, respectively (p < 0.001). None of the subjects was found to carry the CYP2C19*3 allele. Frequencies of the intermedier metabolizer phenotype defined by the *1/*2 genotype (0.318 vs. 0.230, p < 0.005) and poor metabolizer predicted by the *2/*2 genotype (0.046 vs. 0.011, p < 0.005) was significantly higher in Roma than in Hungarians, respectively. Genotype distribution of the Roma population was similar to those of the population of North India, however, a major difference was found in the frequency of the CYP2C19*2 allele, which is likely a result of admixture with European lineages. In conclusion, the frequencies of the CYP2C19 alleles, genotypes and corresponding extensive, intermediate and poor metabolizer phenotypes studied here in the Hungarian population are similar to those of other European Caucasian populations, but display clear differences when compared to the Roma population.