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Br J Clin Pharmacol. Jul 2000; 50(1): 31–34.
PMCID: PMC2014971

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

Abstract

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.

Keywords: CYP2D6*10, CYP2D6*14, dextromethorphan, Japanese

Introduction

CYP2D6 is an isoform of cytochrome P450 catalysing more than 50 clinically important drugs. Various CYP2D6 alleles carrying a point mutation or a combination of mutations as well as rearrangements of genes and pseudogenes of the CYP2D6 gene cluster on the chromosome have been reported. They are referred to by the unified nomenclature developed by Daly et al.[1]. Although the frequencies of CYP2D6 mutant alleles have been studied extensively in Caucasian populations, limited information is available for those of the Japanese population [2, 3].

In the present study, we examined the frequencies of 11 CYP2D6 mutant alleles, including CYP2D6*2,*3,*4,*5,*8,*10,*11,*12,*14,*17 and *18 in 162 Japanese subjects. We also studied the metabolic capacity for dextromethorphan O-demethylation in 35 subjects to assess its relationship to the genotypes of CYP2D6 present in these subjects.

Methods

One hundred and sixty-two unrelated, healthy Japanese volunteers (95 males and 67 females), aged 19–61 years, were recruited for the genotyping study. They were informed about the experimental procedure and the purpose of the study, and written consent was obtained from each subject. Approval for the study was obtained from the local Institutional Review Board.

Venous blood (7 ml) was obtained from each of the 162 subjects, and deoxyribonucleic acid (DNA) was isolated from peripheral leucocytes using an extraction kit (Genomix, Talent, Trieste, Italy). The CYP2D6 wild-type gene and the 11 mutated alleles were identified by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), by mismatch-or by long-PCR analysis directed against one or more relevant mutated sites using allele-specific primers [411].

Assays for the mutations of G→T1846 (CYP2D6*8) and G→A1846 (CYP2D6*14), and that of G→C4268 (CYP2D6*2, *4, *8, *10, *11, *12, *14, and *17) were performed by mismatch-PCR and PCR-RFLP methods, respectively, developed in our laboratory. Briefly, the allele-specific reverse primers to detect G→Τ1846 and G→Α1846 were R1846G (5′-CCTTCTGCCCATCACCCACC-3′) for the wild-type sequence, R1846T (5′-CCTTCTGCCCATCACCCACA-3′) for the altered sequence in CYP2D6*8, and R1846A (5′-CCTTCTGCCCATCACCCACT-3′) for the altered sequence in CYP2D6*14. Primer S1846 (5′-GACTGCGGGAGACCAGGG-3′) was used as the universal forward primer. To detect G→Τ1846 and G→Α1846, genomic DNA (100 ng) was amplified with first-PCR primer sets (S1846 and AS1846; 5′-TTTGTCCAAGAGACCGTTGG-3′) using 0.6–1.0 mm MgCl2. Amplification was performed for 35 cycles consisting of denaturation at 94 °C for 1 min, annealing at 61 °C for 1 min, and extension at 72 °C for 1.5 min. The first-PCR products (1.0 µl) were then amplified with second-PCR primer sets (S1846 and the allele-specific reverse primers mentioned above; R1846G, R1846T or R1846A). Amplification was performed for 20 cycles consisting of denaturation at 94 °C for 1 min, annealing at 64 °C for 30 s, and extension at 72 °C for 30 s. The size of amplified first-and second-PCR products was 374 and 270 base pair, respectively.

For the identification of the G→C4268 mutation, PCR-RFLP using allele-specific primers was performed as previously described [6], with the following modifications. The forward and reverse primers used were S4268 (5′-AGCTCTTCCTCTTCTTCACC-3′) and AS4268 (5′-TCAGCCTCAACGTACCCCT-3′), respectively. Amplification was performed for 35 cycles consisting of denaturation at 94 °C for 1 min, annealing at 63 °C for 1 min, and extension at 72 °C for 2 min. Restriction enzyme cleavage was conducted at 60 °C for 2 h after the addition of Bst EII. The G4268 allele was not digested, and a sample containing the C4268 allele produced fragments of 236 and 98 base pairs in size. The PCR products mentioned above were analysed on 3% agarose gels and stained with ethidium bromide.

Thirty-five of the 162 subjects genotyped were phenotyped by measuring the metabolic ratio (MR) of dextromethorphan/dextrorphan using a urine sample collected for 8 h after an oral dose of 30 mg dextromethorphan (Medicon, Shionogi Co., Ltd, Osaka, Japan). Urine concentrations of dextromethorphan and dextrorphan were measured by high-performance liquid chromatography as described previously [12]. The interassay (n = 5) and intra-assay (n = 5) coefficients of variations at 0.1–125 µm were less than 4.0 and 3.5% for determining dextromethorphan and dextrorphan, respectively.

Statistical analysis was performed by Student's unpaired t-test. A P value less than 0.05 was considered statistically significant.

Results

Four mutated alleles, CYP2D6*2,*5,*10 and *14, were detected, while CYP2D6*3,*4,*8,*11,*12,*17 and *18 were not detected in 162 Japanese subjects. The most frequent mutant allele found in the present study was CYP2D6*10, followed by *2, *5 and *14, and their frequencies were 38.6%, 12.9%, 6.2% and 2.2%, respectively.

The 11 different genotypes present in our subjects are listed together with their respective frequencies in Table 1. The most frequent genotypes were CYP2D6*1/*10,*10/*10,*1/*1 and*1/*2, which were present in 32.7%, 13.6%, 13.0% and 13.0% of our Japanese subjects, respectively. There was no subject whose genotype predicted the poor metabolizer (PM) phenotype (e.g. CYP2D6*5/*5,*14/*14 and *5/*14). However, one-fifth of our subjects showed genotypes predicting intermediate metabolizers such as CYP2D6*10/*10, *5/*10 and *10/*14. The frequencies of all the genotypes observed in the present study were within the 95% confidence interval estimated by the Hardy–Weinberg equation (Table 1).

Table 1
Distribution of CYP2D6 genotypes in 162 Japanese subjects.

Thirty-five of the 162 subjects genotyped were also phenotyped by measuring the MR of dextromethorphan/dextrorphan using 8 h urine. Judging from the antimode (MR = 0.3) reported previously for Caucasian population [12], all the subjects could be classified as extensive metabolizers. Their genotypes and mean (± s.d.) log MR are listed in Table 2. There were eight different genotypes of CYP2D6 found in the subjects studied. The mean log MR value of the subjects with CYP2D6*10/*10 was significantly (P < 0.05) higher than those with CYP2D6*1/*1,*1/*2,*1/*10 and *2/*10. In addition, when the mean log MR value of subjects with genotypes predicting intermediate metabolizers (e.g. CYP2D6*10/*10 and*14/*10) was compared with that of herozygotes for functional and inactivating alleles (e.g. CYP2D6*1/*5 and*2/*14), the value of former group was significantly (P < 0.05) greater than that of the latter group (−1.57 ± 0.48, n = 7 vs−2.19 ± 0.12, n = 3).

Table 2
The metabolic ratios (MR) of dextromethorphan in 35 Japanese subjects.

Discussion

The present study showed that CYP2D6*5 was the most frequently occurring defective allele in our Japanese subjects. The frequency observed is two-to three-times lower than that found in previous studies performed on small numbers of Japanese subjects [2]; however, it is similar to that of a Japanese population reported recently [3] and those of Korean [2] and German [14] populations, suggesting that CYP2D6*5 is a relatively common mutant allele in Oriental and Caucasian populations. On the other hand, we found that CYP2D6*14 was the second most frequently occurring defective allele in our Japanese subjects, although it was not detected among 1178 alleles present in Caucasian subjects [15]. The frequency of this mutant allele in our Japanese subjects is relatively low; however, CYP2D6*14 may be the major defective allele responsible for the presence of PMs in the Japanese population, since the frequency of PM in the Japanese population is very low. In fact, when the frequency of CYP2D6*14 is combined with that of CYP2D6*5, they could account for 83% of Japanese PMs, while CYP2D6*5 alone accounts for only 45% of PMs, when the incidence of PM in the Japanese population is estimated as 0.84% [4]. Therefore, CYP2D6*14 in addition to CYP2D6*5 is considered to be an important mutant allele contributing to the incidence of the PM phenotype in Japanese subjects.

The results of the present study showed that CYP2D6*10 is the most frequent mutant allele of CYP2D6 found in a Japanese population. The observed frequency of CYP2D6*10 is similar to those of earlier studies [2, 3], while it is much higher than that in Caucasians [14, 15]. In addition, the result of the present study showed that mean log MR of subjects with genotypes predicting intermediate metabolizers who are mainly homozygotes for CYP2D6*10 was significantly greater than that of heterozygotes for functional and inactivating alleles. Similarly, Tseng et al.[16] reported that the partial metabolic clearance of codeine to morphine in homozygotes for CYP2D6*10 is more than four times lower than that for the wild-type. The findings suggest that the polymorphism produced by CYP2D6*10 play a more important role than previously thought in the treatment of Japanese patients with drugs metabolized by CYP2D6.

In conclusion, the present study showed that CYP2D6*5 and *14 are the most frequently occurring defective alleles of CYP2D6 responsible for 83% of the PM phenotype in Japanese subjects. In addition, CYP2D6*10, which is mainly responsible for the intermediate metabolizers, appears to play more important role for the treatment of Japanese patients.

References

1. Daly AK, Brockmöller J, Broly F, et al. Nomenclature for human CYP2D6 alleles. Pharmacogenetics. 1996;6:193–201. [PubMed]
2. Dahl M-L, Yue Q-Y, Roh H-K, Johansson I, Säwe J, Sjöqvist F, Bertilsson L. Genetic analysis of the CYP2D locus in relation to debrisoquine hydroxylation capacity in Korean, Japanese and Chinese subjects. Pharmacogenetics. 1995;5:159–164. [PubMed]
3. Tateishi T, Chida M, Ariyoshi N, Mizorogi Y, Kamataki T, Kobayashi S. Analysis of the CYP2D6 gene in relation to dextromethorphan O-demethylation capacity in a Japanese population. Clin Pharmacol Ther. 1999;65:570–575. [PubMed]
4. Yokoi T, Kosaka Y, Chida M, et al. A new CYP2D6 allele with a nine base insertion in exon 9 in a Japanese population associated with poor metabolizer phenotype. Pharmacogenetics. 1996;6:395–401. [PubMed]
5. Tsuneoka Y, Matsuo Y, Iwahashi K, Takeuchi H, Ichikawa Y. A novel cytochrome P-450IID6 mutant gene associated with Parkinson's disease. J Biochem. 1993;114:263–266. [PubMed]
6. Wang S-L, Huang J-D, Lai M-D, Liu B-H, Lai M-L. Molecular basis of genetic variation in debrisoquin hydroxylation in Chinese subjects: Polymorphism in RFLP and DNA sequence of CYP2D6. Clin Pharmacol Ther. 1993;53:410–418. [PubMed]
7. Marez D, Sabbagh N, Legrand M, Lo-Guidice JM, Boone P, Broly F. A novel CYP2D6 allele with an abolished splice recognition site associated with the poor metabolizer phenotype. Pharmacogenetics. 1995;5:305–311. [PubMed]
8. Steen VM, Andreassen OA, Daly AK, Tefre T, Børresen A-L, Idle JR, Gulbrandsen A-K. Detection of the poor metabolizer-associated CYP2D6 (D) gene deletion allele by long-PCR technology. Pharmacogenetics. 1995;5:215–223. [PubMed]
9. Masimirembwa C, Persson I, Bertilsson L, Hasler J, Ingelman-Sundberg M. A novel mutant variant of the CYP2D6 gene (CYP2D6*17) common in a black African population: association with diminished debrisoquine hydroxylase activity. Br J Clin Pharmacol. 1996;42:713–719. [PMC free article] [PubMed]
10. Marez D, Sabbagh N, Legrand M, Lo-Guidice JM, Boone P, Broly F. A novel CYP2D6 allele with an abolished splice recognition site associated with the poor metaboliser phenotype. Pharmacogenetics. 1995;5:305-311. [PubMed]
11. Marez D, Legrand M, Sabbagh N, Lo-Guidice JM, Boone P, Broly F. An additional allelic variant of the CYP2D6 gene causing impaired metabolism of sparteine. Hum Genet. 1996;97:668–670. [PubMed]
12. Marinac JS, Foxworth JW, Willsie SK. Dextromethorphan polymorphic hepatic oxidation (CYP2D6) in healthy Black American adult subjects. Ther Drug Monit. 1995;17:120–124. [PubMed]
13. Schmid B, Bircher J, Preisig R, Küpfer A. Polymorphic dextromethorphan metabolism: Co-segregation of oxidative O-demethylation with debrisoquin hydroxylation. Clin Pharmacol Ther. 1985;38:618–624. [PubMed]
14. Griese E-U, Zanger UM, Brudermanns U, et al. Assessment of the predictive power of genotypes for the in-vivo catalytic function of CYP2D6 in a German population. Pharmacogenetics. 1998;8:15–26. [PubMed]
15. Sachse C, Brockmöller J, Bauer S, Roots I. Cytochrome P450 2D6 variants in a Caucasian population: allele frequencies and phenotypic consequences. Am J Hum Genet. 1997;60:284–295. [PMC free article] [PubMed]
16. Tseng C-Y, Wang S-L, Lai M-D, Lai M-L, Huang J-D. Formation of morphine from codeine in Chinese subjects of different CYP2D6 genotypes. Clin Pharmacol Ther. 1996;60:177–182. [PubMed]

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