Direct assessment of cytochrome P450 2D6 genotypes by high-resolution melting analysis and DNA sequencing

We developed a CYP2D6 genotyping method that required only one polymerase chain reaction (PCR) followed by a high-resolution melting curve analysis (HRM) and DNA sequencing. DNA was extracted from peripheral blood samples obtained from 100 normal individuals. From the HRM analysis using three fragments of amplicons (exons 1, 6, and 9), we successfully identified four common CYP2D6 gene polymorphisms (100C>T, 2850C>T, 2988G>A, and 4180G>C). Exons 3 and 7 were also screened by HRM analysis. The heteroduplexes, wild-type homoduplexes, and homoduplexes of compound mutations showed distinct melting plots. The other four exons (exons 2, 4, 5, and 8) were directly analyzed by DNA sequencing. In conclusion, we developed an HRM and DNA sequencing based method to assess the CYP2D6 gene directly without the need for nested PCR. This method is quick and cost-effective; it reduces the chance of PCR contamination and is suitable for clinical application.     

CYP2D6 and CYP2C19 genotypes in an elderly Swedish population

Objective: Eighty-three healthy elderly Swedish subjects (age 87 ± 4 years, mean ± SD, range 80–98 years) were genotyped with respect to the two genetic polymorphisms of oxidative drug metabolism, CYP2D6 and CYP2C19, using allele-specific polymerase chain reaction (PCR). A control population consisted of 248 younger unrelated healthy volunteers (age 31 ± 9 years, range 19–63 years) for CYP2D6, and 162 (age 30 ± 8 years, range 19–55 years) for CYP2C19. Results: No significant differences were found between the control groups and the elderly subjects with respect to the frequencies of the defect alleles CYP2D6*3, CYP2D6*4, CYP2C19*2 and CYP2C19*3. Neither were there any differences in the genotype frequencies, or the predicted phenotype frequencies. The study indicates that the CYP2D6 and CYP2C19 genotypes play no major role in the probability of reaching high age. Conclusion: No genetically determined differences in the pharmacokinetics of drugs metabolized by these two polymorphic enzymes are to be expected in the oldest age groups compared with younger adults. Received: 10 March 1998 / Accepted: 1 May 1998     

Sequence-based analysis of the CYP2D6*36-CYP2D6*10 tandem-type arrangement, a major CYP2D6*10 haplotype in the Japanese population

The frequency of the CYP2D6*10 allele (100C>T) in the Japanese is relatively high (0.3-0.4), and the two *10-related genes, Ch1 (currently *10B) and Ch2 (*36), and their tandem arrangement Ch(2)-Ch(1) (*36-*10B) have been reported. Although the tandem form of *36-*10 is assumed to be a major form, no detailed information has been reported for its intervening and flanking regions. Thus in this study, the tandem-type *36-*10B and the single-type *10 were analyzed by long-range PCR and sequencing of the subsequent nested PCR products. The sequence of the entire *36-*10 region confirmed the recombination of CYP2D6*10 with CYP2D7P. Also, we found that most of the *10B-harboring haplotypes have the upstream *36 gene and that the majority of the remaining haplotypes are the single-type *10B. Haplotype frequencies of the single-type *10 and *36-*10B were 0.06 and 0.30, respectively, in the subjects analyzed. Additionally, several novel single nucleotide polymorphisms (SNPs) were found in the *36 region and several *36 haplotypes were identified. This sequence information is an important addition to the CYP2D6 sequence data that was obtained by the human genome project.     

Phenotypes and genotypes for CYP2D6 and CYP2C19 in a black Tanzanian population

AIMS: CYP2D6 and CYP2C19 are polymorphically expressed enzymes that show marked interindividual and interethnic variation. The aim of this study was to determine the frequency of the defective alleles in CYP2D6 and CYP2C19 in Africans and to test whether the genotype for CYP2C19 is better correlated with the proguanil/cylcoguanil ratio than the mephenytoin S/R ratio. METHODS: Two hundred and sixteen black Tanzanians were phenotyped for CYP2D6 with the use of sparteine, and for CYP2C19 with the use of mephenytoin and proguanil. Of these 196 subjects were also genotyped for CYP2D6 (including the CYP2D6*1, CYP2D6*3 and CYP2D6*4 alleles) and 195 were genotyped for CYP2C19 (including the CYP2C19*1, CYP2C19*2 and the CYP2C19*3 alleles). Furthermore 100 subjects were examined for the allele duplication in CYP2D6, leading to ultrarapid metabolism, with long PCR. RESULTS: The sparteine metabolic ratio (MR) was statistically significantly higher in the Tanzanian group of homozygous, extensive metabolizers compared to a historical control group of white Danish extensive metabolizers. Only one poor metabolizer for CYP2D6 (MR=124 and genotype CYP2D6*1/CYP2D6*4 ) was found. The gene frequencies were 0.96 for the CYP2D6*1 allele and 0.04 for the CYP2D6*4 allele. No CYP2D6*3 alleles were found. Nine subjects had an allele duplication in CYP2D6 (9%). For CYP2C19 there were seven subjects (3. 6%) who were phenotyped as poor metabolizers, but only three subjects (1.5%) had a genotype (CYP2C19*2/CYP2C19*2 ) indicative of poor metabolism. The gene frequencies were 0.90 for the CYP2C19*1 allele and 0.10 for the CYP2C19*2 allele. No CYP2C19*3 alleles were found. The mephenytoin S/R ratios were not bimodally distributed. CONCLUSIONS: Both the genotyping and phenotyping results show that there is a substantial difference between an African black population and a Caucasian population in the capacity to metabolize drugs via CYP2D6 and CYP2C19.     

Influence of donor and recipient genotypes on CYP2D6 phenotype after liver transplantation: a study of mutations CYP2D6*3 and CYP2D6*4

Objective: After liver transplantation (LT), genotypic differences between the recipient and the transplanted liver, medications and post-LT complications may all affect drug metabolism. We have studied the effect of two CYP2D6 mutations in the donor and the recipient on post-LT CYP2D6 phenotype. Method: The CYP2D6 phenotype was assessed in 48 patients before and after LT with debrisoquine or␣dextromethorphan. CYP2D6*3 (CYP2D6A) and CYP2D6*4 (CYP2D6B) mutations were detected in the donor and the recipient using polymerase chain reaction. Results: Before LT, 40 subjects were classified as extensive metabolisers (EM) and 8 as poor metabolisers (PM); after transplantation, 41 were EMs and 7 were PMs. Genotype and phenotype were in agreement in 100% of EMs and 40% of PMs. The low percentage of agreement in PMs could not be explained by severely altered liver function. The phenotype of 13 subjects was apparently changed by LT: 6 EMs became PMs and 7 PMs became EMs. All four subjects in whom genotype changed following LT had a corresponding change in phenotype: two EM subjects became PMs and two PM subjects became EMs. Conclusion: The low percentage of agreement in PMs may be partly explained by mutations other than CYP2D6*3 and CYP2D6*4. Nevertheless, our study shows that the CYP2D6 genotype of the donor controls the phenotype of the recipient of a liver transplantation. Received: 2 June 1997 / Accepted in revised form: 9 October 1997     

Drug extrapyramidal side effects. CYP2D6 genotypes and phenotypes

Objective: Among Caucasians, a lack of cytochrome P ₄₅₀ enzyme CYP2D6 is observed in 5–10% of individuals, named poor metabolizers (PMs). A consequence may be an impaired metabolism of many drugs such as most of the psychotropic drugs with an increased risk of drug side effects. This enzyme is also involved in the metabolism of endogenous compounds, including neurotransmitters such as dopamine and dopamine-related neurotransmitters which play a role in the mechanism of action of extrapyramidal drug side effects. The present study investigates whether patients who have developed and those who have not developed extrapyramidal drug side effects differ in their CYP2D6 genotypes and phenotypes. Methods: The CP2D6 genotype (method involving restriction length fragment polymorphism and polymerase chain reaction-single strand conformation polymorphism) was determined in 65 drug-treated in-patients, and the CYP2D6 phenotype (with dextromethorphan probe) in 62 of them. Two groups were constituted, one with 22 patients who had developed extrapyramidal drug side effects, and the second with 43 patients without such side effects. Results: In the whole population, there was an over-representation of PM phenotypes – more marked in the first group than the second (45% vs 14%). Concerning the genotypes, we observed that the percentage of functional alleles (with extensive metabolic capacity) was higher in group 2, whereas the percentage of non-functional alleles (without metabolic activity) was higher in group 1; this frequency difference was only marginally significant (χ² 5.95; P < 0.0509; degrees of freedom=2). Consequently, there was a higher percentage of genotypes with no (extensive) functional alleles in the group of patients suffering from extrapyramidal side effects than in the other group (P < 0.00001). Conclusion: CYP2D6-impaired metabolic capacity may be a contributory factor in extrapyramidal drug side effects. Received: 25 May 1999 / Accepted in revised form: 18 August 1999     

CYP2D6 and GSTM1 genotypes in a Polish population

Objective: The aim of the present study was to investigate the distribution of the CYP2D6 and GSTM1 genotypes in a Polish population. Methods: One hundred and forty-five unrelated healthy individuals from the western region of Poland were studied. The CYP2D6 genotype was analysed by means of polymerase chain reaction (PCR) amplification for the CYP2D6 ^* 3 and CYP2D6 ^* 4 alleles. The GSTM1 genotype was also analysed by means of a PCR assay to determine two genotypes: GSTM1-1 (positive) and GSTM1-0 (negative). Results: Fourteen subjects (9.6%) were classified as poor metabolisers. The frequency of CYP2D6 ^* 4 and CYP2D6 ^* 3 was 23.1% and 2.1%, respectively. The frequency of GSTM1 nulled genotype in a Polish population came to 49%. Conclusion: The frequencies of poor metabolisers for CYP2D6 and GSTM1 nulled genotype among a Polish population were similar to those observed in other Caucasian populations. Received: 29 October 1998 / Accepted in revised form: 15 March 1999     

The major fluvoxamine metabolite in urine is formed by CYP2D6

OBJECTIVE: Previous studies have shown that fluvoxamine is metabolized by CYP1A2 and CYP2D6, but there is no information on the impact the various CYP enzymes have on the different metabolic pathways of fluvoxamine biotransformation. The present study was designed to investigate this issue. METHODS: The major fluvoxamine metabolite, the 5-demethoxylated carboxylic acid metabolite, was analyzed in urine from 50 healthy volunteers after intake of a single oral dose of 50 mg fluvoxamine, and the formation clearance for the metabolite (CLm) was calculated. Of the subjects, 28 were non-smoking CYP2D6 and CYP2C19 extensive metabolizers (EMs), 12 were smokers and were thus considered to have an induced CYP1A2 activity, 5 were CYP2D6 poor metabolizers (PMs), and 5 were CYP2C19 PMs. In 11 of the non-smoking EMs, 200 mg caffeine was given at another occasion in order to calculate oral caffeine clearance as a measure of CYP1A2 activity. In addition, CLm was calculated in ten other subjects given increasing doses of fluvoxamine for 4 weeks. RESULTS: Oral clearance of fluvoxamine was significantly higher in smokers, and significantly lower in CYP2D6 PMs than in non-smoking EMs. CLm was 78% lower in CYP2D6 PMs than in the EMs. Smoking and being a CYP2C19 PM did not influence CLm. There was no significant correlation between oral caffeine clearance and CLm. CLm decreased with increasing fluvoxamine dosage, but the decrease in oral clearance was even higher. CONCLUSION: These results indicate that CYP2D6 catalyzes the major metabolic pathway of fluvoxamine, whereas CYP1A2 seems to catalyze other less important pathways. Both the CYP2D6 and the CYP1A2 pathways seem to be saturated in parallel with increasing fluvoxamine dosage.     

QTc interval, CYP2D6 and CYP2C9 genotypes and risperidone plasma concentrations

The role of certain drug metabolizing enzymes in cardiotoxicity, such as CYP2D6 for thioridazine, has been suggested. Risperidone has been shown to inhibit the delayed rectifier leading to lengthening of cardiac repolarization. The heart-rate corrected QT (QTc) interval lengthening has been reported in psychiatric patients receiving risperidone under steady-state conditions. CYP2D6 is involved in the metabolism of risperidone to 9-hydroxy (OH)-risperidone. CYP2C9 enzyme is also involved in the metabolism of several psychotropic drugs, although there are no data about its implication in risperidone metabolism. The present study aimed to evaluate the influence of CYP2D6 and CYP2C9 genotypes, and plasma concentrations of risperidone and 9-OH-risperidone on the QTc interval in patients under steady-state conditions. The relevance of CYP2D6 and CYP2C9 genotypes on risperidone metabolism was also analysed. Thirty-five White European psychiatric patients receiving risperidone monotherapy were studied. QTc interval was longer (p < 0.05) in subjects with one active CYP2D6 gene compared to those with two. The number of CYP2D6 active genes was related to the dose-corrected plasma concentration of risperidone (p < 0.05), the active moiety (risperidone plus 9-OH-risperidone) (p < 0.05) and the risperidone/9-OH-risperidone ratio (p < 0.05). CYP2C9 genotypes were not related to plasma concentrations of risperidone or 9-OH-risperidone, nor QTc interval. The results suggest that CYP2D6, but not CYP2C9, may be related to QTc lengthening during treatment with risperidone. The effect of the CYP2D6 genotype in risperidone metabolism is also shown.     

Effects of CYP2D6 genotypes on age-related change of flecainide metabolism: involvement of CYP1A2-mediated metabolism

AIMS

The aim of this study was to clarify the effects of CYP2D6 genotype on age-related change in flecainide metabolism in patients with supraventricular tachyarrhythmias. An in vitro study using microsomes was performed to identify other CYPs responsible for age-related change in flecainide metabolism.

METHODS

The study population comprised 111 genotyped patients: CYP2D6-homozygous extensive metabolizers (hom-EMs, n= 34), heterozygous EMs (het-EMs, n= 56), and intermediate and poor metabolizers (IMs/PMs, n= 21). Serum concentrations of flecainide and its metabolites [m-O-dealkylated flecainide (MODF) and m-O-dealkylated lactam of flecainide] were determined by use of a high-performance liquid chromatography. Metabolic ratio (MR) was expressed as serum concentrations of flecainide to its metabolites. In vitro formation of MODF was examined in human liver microsomes and cDNA-expressed CYP isoforms.

RESULTS

MR was higher in elderly patients (≥70 years) than in middle-aged patients (<70 years). The increase of MR in elderly patients differed among CYP2D6 genotypes: 1.6-fold in het-EMs [4.3, 95% confidence interval (CI) 2.8, 5.7 vs. 2.7, 95% CI 2.3, 3.1, P < 0.05], 1.5-fold in IMs/PMs (6.0, 95% CI 4.5, 7.6 vs. 4.1, 95% CI 2.9, 5.4, P < 0.05), and no change in hom-EMs. The in vitro study using microsomes revealed that both CYP2D6 and CYP1A2 were involved in the formation of MODF. MODF formation in CYP2D6 PM microsomes increased as CYP1A2 activity increased.

CONCLUSIONS

The results suggest that patients with poor CYP2D6-mediated metabolism (het-EMs and IMs/PMs) showed age-related reduction in flecainide metabolism because metabolism was taken over by CYP1A2, whose activity decreases with age.     

Frequencies of CYP2D6 mutant alleles in a normal Japanese population and metabolic activity of dextromethorphan O-demethylation in different CYP2D6 genotypes

AIMS: To determine the frequencies of 11 CYP2D6 mutant alleles (CYP2D6*2, *3, *4, *5, *8, *10, *11, *12, *14, *17 and *18), and their relation to the metabolic capacity of CYP2D6 in Japanese subjects. METHODS: One hundred and sixty-two unrelated healthy Japanese subjects were genotyped with the polymerase chain reaction amplification method and 35 subjects were phenotyped with dextromethorphan. RESULTS: The frequencies of CYP2D6*2,*5, *10 and *14 were 12.9, 6.2, 38.6 and 2.2% in our Japanese subjects, respectively. CYP2D6*3, *4, *8, *11, *12, *17 and *18 were not detected. The mean log metabolic ratio of dextromethorphan in subjects with genotypes predicting intermediate metabolizers was significantly greater than that of heterozygotes for functional and defective alleles. CONCLUSIONS: CYP2D6*5 and CYP2D6*14 are the major defective alleles found in Japanese subjects. In addition, CYP2D6*10 may play a more important role than previously thought for the treatment of Japanese patients with drugs metabolized by CYP2D6.     

The impact of the CYP2D6 and CYP2C19 genotypes on venlafaxine pharmacokinetics in a Japanese population

Objective: The cytochrome P ₄₅₀ isozymes CYP2D6 and CYP2C19 exhibit genetic polymorphism in human, including a marked interethnic difference. As the functional status of the isozymes CYP2D6 and CYP2C19 have an impact on the pharmacokinetics of some antidepressants, we investigated whether the disposition of venlafaxine was affected by the CYP2D6 and CYP2C19 genotypes. Methods: Twenty-eight adult Japanese men in good health participated in this study. Genomic DNA was isolated from peripheral lymphocytes, and the CYP2D6 genotype was determined using polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) analysis and XbaI-RFLP analysis. Subjects were categorized into the following four groups: group 1 CYP2D6*10/*10; group 2 CYP2D6*1/*10 and *2/*10; group 3 CYP2D6*1/*1, *1/*2 and *2/*2; and group 4 the other genotypes. Two defective CYP2C19 alleles (CYP2C19*2 and CYP2C19*3) were identified by means of PCR-RFLP analysis. Venlafaxine was administered orally following an overnight fast. Plasma concentrations of venlafaxine and O-desmethylvenlafaxine were monitored using high-performance liquid chromatography up to 24 h. Results: The peak plasma concentration and values of area under the concentration–time curve up to 24 h for venlafaxine were 298% and 453% higher for group 1 than group 3, and 91% and 120% higher for group 2 than for group 3, respectively. The homozygote for two defective alleles of CYP2C19 showed a higher concentration of venlafaxine within group 1 and group 2. Conclusion: The CYP2D6*10 allele and two CYP2C19 defective alleles, common in an Asian population, are the most likely genetic factors to use in determining interindividual differences in the pharmacokinetics of venlafaxine, although the results with respect to CYP2C19 are preliminary because of the few subjects used. Received: 6 July 1999 / Accepted in revised form 11 January 2000     

CYP2D6 and CYP2C19 genotypes of patients with terodiline cardiotoxicity identified through the yellow card system

AIMS: Terodiline has concentration dependent QT prolonging effects and thus the potential for cardiotoxicity. Pharmacogenetic variation in terodiline metabolism could be responsible for cardiotoxicity. We sought to determine whether CYP2D6 (debrisoquine hydroxylase) or CYP2C19 (S-mephenytoin hydroxylase) status is a risk factor for terodiline cardiotoxicity. METHODS: Using the UK Yellow Card scheme to identify patients, blood samples were obtained from eight patients who survived ventricular tachycardia or torsades de pointes suspected to be due to terodiline, for determination of CYP2D6 and CYP2C19 genotypes. Genotype prevalence was compared with that in published general population groups. RESULTS: One patient was a CYP2D6 poor metaboliser (CYP2D6*4 homozygous) and a second was heterozygous for CYP2D6*4, a slightly lower frequency for these genotypes compared with the general population (P = 0.31). In the case of CYP2C19, one patient was a poor metaboliser and four were heterozygous for the variant CYP2C19*2 allele, compared with general population frequencies of 2% and 23%, respectively (P = 0.035). CONCLUSIONS: These findings suggest that debrisoquine poor metaboliser status is not primarily responsible for terodiline cardiotoxicity. However, possession of the CYP2C19*2 allele appears to contribute to adverse cardiac reactions to terodiline. The present study demonstrates the feasibility of using spontaneous adverse drug reaction reporting schemes to determine the contribution of genotype for metabolizing enzymes to uncommon adverse drug reactions.     

The prevalence of CYP2D6 and CYP2C19 genotypes in a population of healthy Dutch volunteers

AIM: This study was performed in a sample of the Dutch population to estimate the prevalence of noncoding mutations of CYP2D6 and CYP2C19 as obtained by genotyping. In addition, the predictability of the genotyping strategy was assessed. METHODS: The CYP2D6 and CYP2C19 status of 765 unrelated healthy volunteers was evaluated. Dextromethorphan and mephenytoin were used for determining the phenotypes. Genotyping was performed by PCR on the most common null alleles for CYP2D6 (except for CYP2D6*5) and CYP2C19. RESULTS: For CYP2D6, the most frequently observed null allele was CYP2D6*4, which accounted for 89% of all null alleles. The prevalence of poor metabolizers (PMs) in healthy volunteers was 5.5%, which was lower than that found previously by phenotyping (8.0%; chi2 test P = 0.009). For CYP2C19*2 and CYP2C19*3, the frequencies were 13.3% and 0.2%, respectively. The S:R ratio was significantly higher in heterozygous subjects (S:R ratio 0.22) than in homozygous wild type subjects (S:R ratio 0.11). Comparison of all subjects below 45 years showed a significantly higher S:R ratio in the female ones compared to the male ones, especially in heterozygous subjects (S:R ratio 0.39 vs. 0. 19; P < 0.001). CONCLUSIONS: The frequencies of CYP2D6 and CYP2C19 allelic variants were in accordance with other European populations. Assessment of *3, *4, *6, *7, and *8 alleles for CYP2D6, and *2 and *3 for CYP2C19, predicted the phenotype with an accuracy of over 98.6%. A gene-dose effect was found for CYP2C19. CYP2C19 heterozygous female subjects had a decreased CYP2C19 activity that may be at least partly due to the use of oral contraceptives.     

The influence of CYP2B6, CYP2C9 and CYP2D6 genotypes on the formation of the potent antioestrogen Z-4-hydroxy-tamoxifen in human liver

AIMS: To investigate in a large panel of 50 human liver samples the contribution of CYP2C9, CYP2D6, and CYP3A4 to the overall formation of the potent antioestrogen Z-4-hydroxy-tamoxifen, and how various genotypes affect its formation from tamoxifen. METHODS: The formation of Z-4-hydroxy-tamoxifen from 10 microm tamoxifen was studied in human liver microsomes (n=50), characterized for CYP2B6, CYP2C9, CYP2D6 and CYP3A4 expression, and CYP2B6, CYP2C9 and CYP2D6 genotype. The effect of chemical and monoclonal antibody inhibitors, and the formation in supersomes expressing recombinant CYP isoforms was also investigated. Z-4-hydroxy-tamoxifen was quantified using LC-MS analysis. RESULTS: Z-4-hydroxy-tamoxifen was formed by supersomes expressing CYP2B6, CYP2C9, CYP2C19 and CYP2D6, but not CYP3A4. In agreement with these data, the mean formation of Z-4-hydroxy-tamoxifen was inhibited 49% by sulphaphenazole (P=0.001), 38% by quinidine (P<0.05) and 13% by monoclonal antibody against CYP2B6 (MAB-2B6, P<0.05). Furthermore, Z-4-hydroxy-tamoxifen formation significantly correlated with both CYP2C9 expression (r(s)=0.256, P<0.05) and CYP2D6 expression (r(s)=0.309, P<0.05). Genotypes of CYP2D6, CYP2B6 and CYP2C9 had an effect on metabolite formation in such a way that samples with two nonfunctional CYP2D6, or two variant CYP2C9 or CYP2B6 alleles, showed lower enzyme activity compared with those with two functional or wild-type alleles, (5.0 vs 9.9 pmol mg(-1) protein min(-1), P=0.046, 5.1 vs 9.9 pmol mg(-1) protein min(-1), P=0.053, and 6.8 vs 9.4 pmol mg(-1) protein min(-1), P=0.054, respectively). CYP2D6 and CYP2C9 contribute on average 45 and 46%, respectively, to the overall formation of Z-4-hydroxy-tamoxifen. CONCLUSIONS: CYP2B6, CYP2C9 and CYP2D6 genotypes all affected Z-4-hydroxy-tamoxifen formation and can predict individual ability to catalyse this reaction.