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.     

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.     

3'-UTR polymorphism in the human CYP2A6 gene affects mRNA stability and enzyme expression

Cytochrome P450 2A6 (CYP2A6) is the major nicotine C-oxidase in human and participates in the metabolism of drugs and precarcinogens. The CYP2A6 gene is highly polymorphic and more than 22 different alleles have been described. We here focused on the polymorphism in the 3'-UTR region, in particular the common CYP2A6*1B allele, carrying an unequal crossover element from the pseudogene CYP2A7. Analysis of CYP2A6 expression in a human liver bank (n=46) revealed that the protein level and catalytic activity using coumarin as a substrate were all higher, following a linear gene-dose relationship, in livers carrying one or two copies of CYP2A6*1B, as compared to other CYP2A6 allelic variants. Different variants of the CYP2A6 3'-UTR were cloned into a modified pGL3 plasmid downstream of the luciferase reporter gene. The plasmids, having the proximal promoter of CYP2A6 gene, were transfected into HeLa cells or injected into the tail veins of male CD1 mice. In both systems, the 3'-UTR CYP2A6*1B constructs caused higher reporter gene activity and the CYP2A7 3'-UTR construct lower activity, compared to the CYP2A6*1 3'-UTR constructs. Two SNPs differentiating the 3'-UTR between CYP2A7 and CYP2A6*1B were found to be of importance for the expression in both systems. Analysis of reporter enzyme degradation in HeLa cells showed that luciferase-3'-UTR-CYP2A6*1A had a half-life of approximately 4.9h as compared to 6.3h for luciferase-3'-UTR-CYP2A6*1B. In conclusion, we identified polymorphic motifs in the CYP2A6 3'-UTR of importance for CYP2A6 mRNA stabilization and enzyme expression. Such polymorphism has been described to influence the in vivo rate of nicotine elimination and possibly the cigarette consumption and risk of smoking induced lung cancer.     

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.     

A novel single nucleotide polymorphism altering stability and activity of CYP2a6

CYP2A6 is known as a major cytochrome P450 (CYP) responsible for the oxidation of nicotine and coumarin in humans. In this study, we explored genetic polymorphisms, which reduce CYP2A6 activity in Japanese. Two novel mutations in exon 9 of the CYP2A6 gene were found. A single nucleotide polymorphism of T1412C and G1454T resulted in Ile471Thr and Arg485Leu substitution, respectively. The frequency of the former variant allele was considerably high (15.7%), while the latter variant appeared to be a rare polymorphism. Heterologous expression of CYP2A6 using a cDNA possessing C instead of T-base at codon 471 in Escherichia coli caused remarkable reduction of the stability of holoenzyme at 37 degrees C. Furthermore, this variant enzyme almost lacked nicotine C-oxidase activity, although coumarin 7-hydroxylase activity was still observed. These data suggest that individuals homozygous for the T1412C variant allele or heterozygous for this and a defect allele such as the CYP2A6*4 may be poor metabolizer of nicotine, but not coumarin.     

CYP1A1, CYP2D6, CYP2E1, NAT2, GSTM1 and GSTT1 polymorphisms or their combinations are associated with the increased risk of the laryngeal squamous cell carcinoma

Polymorphisms in the selected genes controlling carcinogen metabolism (CYP1A1, CYP2D6, CYP2E1, NAT2, GSTM1, GSTT1) considered separately or in different combinations, were investigated for an association with tobacco smoke-associated squamous cell carcinoma (SCC) of the larynx. The case-control study was performed in 289 patients with laryngeal SCC and in 316 cancer-free controls; all were Caucasian males from the same region of Poland and current tobacco smokers. The DNA samples were genotyped using PCR-RFLP and multiplex PCR. The variants' frequencies in both groups were compared; odds ratios and their 95% confidence intervals were calculated by logistic regression analyses. The CYP1A1*1/*4, CYP2D6*4/*4, NAT2*4/*6A genotypes, as well as the CYP1A1*4, CYP2D6*4 and NAT2*4 alleles, were found at significantly higher frequencies in cases than in controls indicating their role as "risk-elevating" factors in laryngeal SCC. Combined genotypes, characterized by the presence of the "risk-elevating" variants at more than one locus, often occurred together with the null variant of the GSTM1 gene and homozygous XPD A/A (Lys751Gln, A35931C) genotype. Furthermore, we identified some "protective" variants, found more frequently in controls than in cases, i.e. the NAT2*6A/*6A and NAT2*5B/*6A genotypes. A distribution of "risk" or "protection" genotypes/alleles seems to be connected with age as an occurrence or risk genes was more frequent in the group of "young" cases (< or = 49 years). Accumulation of certain alleles or genotypes of the CYP1A1, NAT2, GSTM1 and XPD seems to be associated with either increased or decreased risk to develop laryngeal SCC. Therefore, polymorphisms in these genes may play a role in the laryngeal cancer etiology.     

CYP2A6 polymorphism reveals differences in Japan and the existence of a specific variant in Ovambo and Turk populations

CYP2A6 is a polymorphic enzyme, and CYP2A6 genotype has been shown to be associated with smoking habits and lung cancer. We investigated CYP2A6 polymorphism in Japanese from four different geographic areas of Japan and in the Ovambo and Turk populations. Using two polymerase chain reaction restriction fragment length polymorphisms (PCR-RFLPs), we identified the functionally important variants of CYP2A6: *1A, *1B, *1F, *1G, *4A, and *4D. In the Japanese population the highest frequencies of the CYP2A6*1A allele were observed in subjects from the Fukuoka (Kyushu Island) and Ehime (Shikoku Island) prefectures, whereas subjects in Shimane and Tottori (both located on the Japan Sea side of Honshu Island) showed the highest frequencies of the CYP2A6*1B allele. In the Tottori and Shimane groups no subject was homozygous for the CYP2A6*4A allele, a whole gene deletion type that is prevalent among Asians. In the Ovambo and Turk populations the CYP2A6*1A allele was predominant. Furthermore, two alleles undetected in the Japanese were observed in these latter two ethnic groups: CYP2A6*1G was found solely in the Ovambos, and CYP2A6*1F was found solely in the Turks. The present study is the first to show interprefecture differences in CYP2A6 polymorphism in Japanese who live in relatively close but distinct geographic areas; this is also the first study to evaluate CYP2A6 variations among these Japanese and the Ovambo and Turk populations. The distribution results of these alleles could help to define the true significance of CYP2A6 polymorphism as a genetic susceptibility marker in worldwide populations.     

Allele and genotype frequencies of the polymorphic cytochrome P450 genes (CYP1A1, CYP3A4, CYP3A5, CYP2C9 and CYP2C19) in the Jordanian population

Drug metabolizing enzymes participate in the neutralizing of xenobiotics and biotransformation of drugs. Human cytochrome P450, particularly CYP1A1, CYP2C9, CYP2C19, CYP3A4 and CYP3A5, play an important role in drug metabolism. The genes encoding the CYP enzymes are polymorphic, and extensive data have shown that certain alleles confer reduced enzymatic function. The goal of this study was to determine the frequencies of important allelic variants of CYP1A1, CYP2C9, CYP2C19, CYP3A4 and CYP3A5 in the Jordanian population and compare them with the frequency in other ethnic groups. Genotyping of CYP1A1(m1 and m2), CYP2C9 (*2 and *3), CYP2C19 (*2 and *3), CYP3A4*5, CYP3A5 (*3 and *6), was carried out on Jordanian subjects. Different variants allele were determined using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). CYP1A1 allele frequencies in 290 subjects were 0.764 for CYP1A1*1, 0.165 for CYP1A1*2A and 0.071 for CYP1A1*2C. CYP2C9 allele frequencies in 263 subjects were 0.797 for CYP2C9*1, 0.135 for CYP2C9*2 and 0.068 for CYP2C9*3. For CYP2C19, the frequencies of the wild type (CYP2C19*1) and the nonfunctional (*2 and *3) alleles were 0.877, 0.123 and 0, respectively. Five subjects (3.16?%) were homozygous for *2/*2. Regarding CYP3A4*1B, only 12 subjects out of 173 subjects (6.9?%) were heterozygote with none were mutant for this polymorphism. With respect to CYP3A5, 229 were analyzed, frequencies of CYP3A5*1,*3 and *6 were 0.071, 0.925 and 0.0022, respectively. Comparing our data with that obtained in several Caucasian, African-American and Asian populations, Jordanians are most similar to Caucasians with regard to allelic frequencies of the tested variants of CYP1A1, CYP2C9, CYP2C19, CYP3A4 and CYP3A5.     

Identification of novel CYP2A6 inhibitors by virtual screening

The human CYP2A6 enzyme metabolises several xenobiotics including nicotine, the addictive component in tobacco. Reduced activity of CYP2A6, either for genetic reasons or by administering inhibitors of CYP2A6, reduces tobacco smoking. The aim was to design novel inhibitors of CYP2A6 using 3D-QSAR analysis combined with virtual screening. A 3D-QSAR model was utilised to identify the most important features of the inhibitors, and this knowledge was used to design inhibitors for CYP2A6. Chemical database screening yielded several potent inhibitor candidates such as alkylamine derivatives (compound no. 5, IC(50)=0.1 μM) and 1-benzothiophene-3-carbaldehyde that can be used as lead compounds in the development of drugs for smoking reduction therapy.     

Influence of the genetic polymorphism in the 5'-noncoding region of the CYP1A2 gene on CYP1A2 phenotype and urinary mutagenicity in smokers

The functional significance of genetic polymorphisms on tobacco smoke-induced CYP1A2 activity was examined. The influence of three polymorphisms of the cytochrome P450 1A2 gene (CYP1A2) (-3860 G-->A (allele *1C), -2467 T-->delT (allele *1D), -163C-->A (allele *1F)), located in the 5'-noncoding promoter region of the gene, on CYP1A2 activity (measured as caffeine metabolic ratio, CMR), was studied in Caucasian current smokers (n=95). Tobacco smoke intake was calculated from the number of cigarettes/day. Also, studied was the influence of these CYP1A2 genotypes on smoking-associated urinary mutagenicity, detected in Salmonella typhimurium strain YG1024 with S9 mix, considering the urinary excretion of nicotine plus its metabolites as an internal indicator of tobacco smoke exposure. Smokers with at least one of the variant alleles CYP1A2 -3860A and -2467 delT showed a significantly increased CYP1A2 CMR (-3860 G/A versus G/G, p<0.05; -2467 delT/delT versus T/delT and T/T, p<0.01). Multiple regression analysis showed that the increase in CYP1A2 CMR (ln values) was again significantly related to the presence of CYP1A2 variants -2467delT and also to variant -163A (p<0.05), but moderately to -3860A (p=0.084). No influence of the number of cigarettes smoked per day by each subject was found. Heavy smokers (n=48, with urinary nicotine plus its metabolites>or=0.69 mg/mmol creatinine) with variant allele -2467delT or -163A had significantly increased urinary mutagenicity (p<0.01 and <0.05). CYP1A2 genetic polymorphisms are shown to influence the CYP1A2 phenotype in smokers, -2467 T-->delT having the main effect. This information is of interest for future studies assessing the possible role of tobacco smoke-inducible CYP1A2 genotypes as individual susceptibility factors in exposure to carcinogens.     

Homologous unequal cross-over within the human CYP2A gene cluster as a mechanism for the deletion of the entire CYP2A6 gene associated with the poor metabolizer phenotype.

To clarify the molecular mechanisms involved in the generation of the CYP2A6 gene deletion (E-type variant), we analyzed the CYP2A7 gene, which is located in the 5'-flanking region of the CYP2A6 gene, from individuals with the E-type variant and compared it with the sequences of wild type CYP2A7 and CYP2A6 genes. The 3'-downstream sequence (up to 324 bp from the SacI site in exon 9) of the CYP2A7 gene of the E-type variant is identical to that of the wild CYP2A7 gene. However, the 3'-downstream sequence (starting from 325 bp from the SacI site in exon 9) of the CYP2A7 gene of the E-type variant is identical to that of the wild CYP2A6 gene, indicating that the 3'-downstream region of CYP2A7 and the 3'-downstream region of CYP2A6 linked directly eliminating the whole CYP2A6 gene. PCR analysis using primers specific to the CYP2A7 gene and the CYP2A6 and CYP2A7 genes confirmed that all DNA samples obtained from 7 individuals carrying the E-type variant possessed the same break points. These results indicate that the breakpoint of the CYP2A6 gene deletion lies in the 3'-downstream region of the CYP2A7 and CYP2A6 genes.     

Genetic polymorphism of CYP2A6 in relation to cancer.

To clarify the roles of human cytochrome P450 (P450 or CYP) 2A6 and 2E1 on the metabolic activation of N-nitrosamines, we established genetically engineered Salmonella typhimurium strains harboring human CYP2A6 or CYP2E1 together with NADPH-P450 reductase (OR). The 5'-terminus of CYP cDNA was modified to achieve a high-level expression in S. typhimurium. Modified CYP2A6 or CYP2E1 cDNA and native OR cDNA were introduced into a pCW vector. S. typhimurium YG7108 cells were transformed with this vector. The mutagen producing ability of these enzymes for some N-nitrosamines were evaluated using the established S. typhimurium cells. We found that the substrate specificity of CYP2A6 and CYP2E1 was different among mutagens. CYP2A6 was responsible for the metabolic activation of N-nitrosamines possessing relatively long alkyl chains, whereas CYP2E1 was responsible for the metabolic activation of N-nitrosamines with relatively short alkyl chains. It is likely that CYP2A6 gene polymorphism is responsible for the interindividual variability on the cancer susceptibility. We found the whole deletion of CYP2A6 gene as a type of genetic polymorphism in Japanese. Thus, we developed a gene diagnosis method to detect the variant. We evaluated the relationship between the CYP2A6 gene whole deletion and the susceptibility to the lung cancer. The frequency of CYP2A6 gene whole deletion was significantly lower in the lung cancer patients than that of healthy volunteers.     

Identification of new alleles and the determination of alleles and genotypes frequencies at the CYP2D6 gene in Emiratis

CYP2D6 belongs to the cytochrome P450 superfamily of enzymes and plays an important role in the metabolism of 20-25% of clinically used drugs including antidepressants. It displays inter-individual and inter-ethnic variability in activity ranging from complete absence to excessive activity which causes adverse drug reactions and toxicity or therapy failure even at normal drug doses. This variability is due to genetic polymorphisms which form poor, intermediate, extensive or ultrarapid metaboliser phenotypes. This study aimed to determine CYP2D6 alleles and their frequencies in the United Arab Emirates (UAE) local population. CYP2D6 alleles and genotypes were determined by direct DNA sequencing in 151 Emiratis with the majority being psychiatric patients on antidepressants. Several new alleles have been identified and in total we identified seventeen alleles and 49 genotypes. CYP2D6*1 (wild type) and CYP2D6*2 alleles (extensive metaboliser phenotype) were found with frequencies of 39.1% and 12.2%, respectively. CYP2D6*41 (intermediate metaboliser) occurred in 15.2%. Homozygous CYP2D6*4 allele (poor metaboliser) was found with a frequency of 2% while homozygous and heterozygous CYP2D6*4 occurred with a frequency of 9%. CYP2D6*2xn, caused by gene duplication (ultrarapid metaboliser) had a frequency of 4.3%. CYP2D6 gene duplication/multiduplication occurred in 16% but only 11.2% who carried more than 2 active functional alleles were considered ultrarapid metabolisers. CYP2D6 gene deletion in one copy occurred in 7.5% of the study group. In conclusion, CYP2D6 gene locus is heterogeneous in the UAE national population and no significant differences have been identified between the psychiatric patients and controls.     

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.     

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.     

Bisoprolol responses (PK/PD) in hypertensive patients: A cytochrome P450 (CYP) 2D6 targeted polymorphism study

BackgroundBisoprolol is an effective β1-adrenergic blocker, an inter-individual genetic variability was recorded in its response. This study aimed at investigating the association of CYP2D6*2A (rs1080985) and CYP2D6*10 (rs1065852) single-nucleotide polymorphism (SNP) with Bisoprolol response in cardiac patients attending King Abdulaziz University Hospital, Jeddah, Kingdom of Saudi Arabia.Patients and methodsIn the study, 107 patients were enrolled. Five mL of venous blood was collected from each patient and genotyping for CYP2D6*2A and CYP2D6*10 using Vivid® CYP2D6 Green Screening Kit (Life Technologies, USA). Response to Bisoprolol was evaluated through assessment of diastolic and systolic blood pressure and by measuring Bisoprolol plasma level using triple quad mass spectrometer (TQ-MS).ResultsAll patients were found to carry homozygous wild type CYP2D6*10 (GG) and none were carrying heterozygous (GA) or mutant homozygous (AA) genotype. CYP2D6*2A allele was detected in the homozygous wild type (GG) in 70 out of 107 patients, the heterozygous (GC) in 19 patients, and the homozygous mutant (CC) in 18 patients with minor allele frequency (MAF) of 25.7%. The plasma concentrations of Bisoprolol in CC carriers were significantly lower than those in GG & CC carriers by 25%, and 51%; respectively. Higher systolic and diastolic blood pressures were also observed in CC carriers than GG and CC carriers.ConclusionThere is a possible association of CYP2D6*2A genotype with plasma concentration of bisoprolol. This could provide a helpful tool to choose the optimum dose for bisoprolol, depending on the patient’s genotyping, in order to increase effectiveness and ameliorate its toxicity.     

Characterisation and PCR-based detection of a CYP2A6 gene deletion found at a high frequency in a Chinese population

Cytochrome P450 2A6 is an important human hepatic P450 which activates pre-carcinogens, oxidises some drugs and constitutes the major nicotine C-oxidase. In fact, results have been presented in the literature which suggested a relationship between the distribution of defective CYP2A6 alleles and smoking behaviour as well as cigarette consumption. In the present report, we describe the structure of a novel CYP2A locus where the whole CYP2A6 gene has been deleted, resulting in an abolished cytochrome P450 2A6-dependent metabolism. The origin of this locus is apparently due to an unequal crossover event between the 3'-flanking region of the CYP2A6 and CYP2A7 genes. A rapid PCR-based method for the detection of the CYP2A6del allele was developed and the allele frequency was 15.1% among 96 Chinese subjects, but only 1.0% in Finns (n=100) and 0.5% in Spaniards (n=100). In the Chinese population, we did not detect any CYP2A6*2 alleles using an improved genotyping procedure, in contrast to the 11-20% previously reported. It is concluded that genotyping for the CYP2A6del allele is of great importance in studies correlating, for example, smoking behaviour, pre-carcinogen activation or drug metabolism to the CYP2A6 genotype, in particular when oriental populations are investigated.     

Genetic polymorphisms of CYP2C8, CYP2C9 and CYP2C19 in Ecuadorian Mestizo and Spaniard populations: a comparative study

This study was designed to investigate the potential differences between Spaniards and Ecuadorian Mestizo people regarding CYP2C8, CYP2C9, and CYP2C19 genetic polymorphisms. DNA from 282 Spaniard and 297 Ecuadorian subjects were analyzed by either a previously reported pyrosequencing method (CY2C8*3, CYP2C9*2, CYP2C9*3, CYP2C19*2 and CYP2C19*3) or a nested PCR technique (CYP2C19*17). Whereas CYP2C19*17 allele distribution was higher in Ecuadorians than in Spaniards (P < 0.001) and the frequency of CYP2C19*3 was similar in these two populations (P > 0.05), the other allelic variants were detected at significantly lower frequencies in Ecuadorians than in Spaniards (P < 0.05). According to the diplotype distributions, the prevalence of the presumed CYP2C9 and CYP2C8 extensive metabolizers was higher in Ecuadorians than in Spaniards (P < 0.05). Individuals genotyped CYP2C19*1/*17 and *17/*17 who were considered as ultrarapid metabolizers were overrepresented in Ecuadorians in relation to Spaniards (P < 0.001). By contrast, among Ecuadorians no poor metabolizers (PMs) of either CYP2C8 or CYP2C9 were found and only two individuals were CYP2C19 PMs. These data are compatible with a higher CYP2C8, CYP2C9, and CYP2C19 activity in Mestizo Ecuadorians as opposed to Spaniards, which could imply differences in dosage requirements for drugs metabolized by these cytochromes and should also be considered in allele-disease association studies.     

Characterization of CYP1A2, CYP2C19, CYP3A4 and CYP3A5 polymorphisms in South Brazilians

Potential causes of variability in drug response include intrinsic factors such as ethnicity and genetic differences in the expression of enzymes that metabolize drugs, such as those from Cytochrome P450 (CYPs) superfamily. Pharmacogenetic studies search for genetic differences between populations since relevant alleles occur with varying frequencies among different ethnic populations. The Brazilian population is one of the most heterogeneous in the world, resulting from multiethnic admixture of Amerindians, Europeans, and Africans across centuries. Since the knowledge of CYP allele frequency distributions is relevant to pharmacogenetic strategies and these data are scarce in the Brazilian population, this study aimed to describe genotype and allele distributions of 15 single nucleotide polymorphisms (SNPs) at CYP 1A2, 2C19, 3A4, and 3A5 genes in African and European descents from South Brazil. A sample of 179 healthy individuals of European and African ancestry was genotyped by the MassARRAY SNP genotyping system. CYP3A5*3, CYP1A2*1F, CYP3A4*1B, and CYP2C19*2 were the most frequent alleles found in our sample. Significant differences in genotype and allelic distribution between African and European descents were observed for CYP3A4 and CYP3A5 genes. CYP3A4*1B was observed in higher frequency in African descents (0.379) than in European descents (0.098), and European descents showed higher frequency of CYP3A5*3 (0.810) than African descents (0.523). Our results indicate that only a few polymorphisms would have impact in pharmacogenetic testing in South Brazilians. Further studies with larger sample sizes are required also among other Brazilian regions.     

Linkage between the CYP2C8 and CYP2C9 genetic polymorphisms

Cytochrome P450 (CYP) 2C8 and 2C9 are polymorphic enzymes. The CYP2C8*3 and CYP2C9*2 are the major variant alleles in Caucasian populations. The enzymes encoded by these variant alleles have impaired function for the metabolism of several drug substrates. In the present study 1468 subjects that were used as population-based controls in the Stockholm Heart Epidemiology Program (SHEEP) were genotyped by allelic discrimination using a 5'-nuclease assay for CYP2C8*1, 2C8*3, 2C9*1, 2C9*2, and 2C9*3 variant alleles in which the frequencies appeared to be 0.91, 0.095, 0.83, 0.11, and 0.066, respectively. Approximately, 96% of the subjects with CYP2C8*3 allele also carried a CYP2C9*2 and 85% of the subjects that had CYP2C9*2 variant also carried a CYP2C8*3. The number of subjects carrying both of the CYP2C8*1*3 and CYP2C9*1*2 was 4.5-fold higher than expected. This strong association may be of importance especially for the metabolism of common substrates of CYP2C8 and CYP2C9 like arachidonic acid that produces physiologically active metabolites.