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Technical Briefs |
Individuals homozygous or heterozygous for deficient CYP2D6 alleles metabolize drugs at a low rate, increasing the risks of side effects and drug toxicity. On the other hand, individuals with duplication/multiplication of the active CYP2D6 gene metabolize drugs at ultrarapid rate, hence requiring more than average doses of drugs to reach therapeutic plasma concentrations (5). The mechanism behind the duplication involves mainly the active allelic variant CYP2D62 (6). The occurrence of gene duplication varies widely, from 29% in Ethiopians (7), to 20% in Saudi Arabians (8), to 1–7% in Caucasians (9)(10), to 1–2% in Orientals (11)(12)(13), although not many data are available for the latter. Establishing the prevalence of duplicated CYP2D6 active alleles may help prevent therapeutic failure in UM individuals treated with CYP2D6 enzyme-substrates at standard doses.
We investigated the prevalence of the duplication of the CYP2D6 gene in a Hong Kong Chinese population. This is the first time that a Hong Kong Chinese population has been screened for duplication of CYP2D6. We believe that our data will assist the identification and quantification of metabolic explanations for treatment failure, thus facilitating rational therapeutics in our population.
A total of 114 unrelated Hong Kong Chinese volunteers participated in the genotyping tests after giving their informed consent. This research project was approved by the local ethics committee.
Blood samples (12 mL) were collected in Vacutte® (Greiner) tubes containing EDTA as anticoagulant. DNA was extracted using QIAamp Blood kit (Qiagen). DNA samples were initially screened for the functional CYP2D62 allele and the PM-associated alleles 4D, 5, 8/14, 10A, 10B, 15, 16 (3), and J9 (14) as we described previously (15).
Three different PCR tests were applied for the detection of duplicated CYP2D6 genes, two described by Løvlie et al. (16) and the third described by Lundqvist et al. (17). For test 1, primers cyp-17 and cyp-32 were used (16). This test yields a 5.2-kb and a 3.6-kb band in samples from subjects harboring duplicate alleles. The 5.2-kb band corresponds to the CYP2D7-CYP2D6 intergenic region, hence it is to be obtained from each sample and serves as internal control. The 3.6-kb band confirms the presence of the CYP2D6-CYP2D6 intergenic region formed during the duplication event (16). For test 2, amplification was carried out using the combination of primers cyp-207 and cyp-32, which yields a 3.2-kb product only from subjects carrying two copies of the CYP2D6 gene (16). For the third PCR assay, the primer combination used was 2D6dup-f and 2D6dup-r, which produce only a 3.5-kb amplification product from samples harboring the duplicated gene (17). For each test, 500 ng of genomic DNA was used as template and the Expand Long Template PCR SystemTM (Boehringer) was applied. Amplification reactions of 25-µL were performed on a PTC-200 DNA thermocycler (MJ Research). The reaction mixtures consisted of buffer 3 (supplied with the kit); 2.5 mmol/L MgCl2; each deoxynucleotide triphosphate at 500 µmol/L; 2 U of the enzyme mixture (Taq and Pwo polymerases), and each of the primers at 0.3 µmol/L. For PCR assays 1 and 2, the cycle profile was as follows: predenaturation at 93 °C, 1 min; 37 cycles of 93 °C for 1 min; 63 °C for 30 s, and 68 °C for 6 min. The final elongation step was 10 min at 72 °C. For PCR assay 3, cycling conditions were as described previously (17). PCR products were detected in 1% agarose gels.
Thirteen of 114 samples were positive for tests 1, 2, and 3. Six
samples were positive for tests 1 and 2 but negative for test 3, and
two samples were positive for test 1 but negative for tests 2 and 3
(Table 1
A). Interestingly, none of the samples positive for tests 1 and
2 harbored allele 2, whereas the samples that were positive
only for test 3 both presented allele 2. Considering only
the samples that scored positive for all three tests, the frequency of
duplicated CYP2D6 alleles was 5.7% (11.4% of the Hong Kong
Chinese population studied). However, only 7 of the 13 individuals had
genotypes involving duplication of functional gene copies. The
remaining samples positive for duplication presented a
10/10 genotype. To our knowledge, this is the
first time that duplication of allele 10 has been
described. Allele 10, which is the most frequently mutated
allele in Chinese populations, produces a CYP2D6 enzyme with
diminished metabolic activity (4). The consequences that
the duplication of an allele of such characteristics (neither fully
active nor inactive) may have on an individual phenotype are under
study. Genotypes in which a functional gene was duplicated included
1x2/10 (two subjects),
1x2/2 or 2x2/1 (one
subject), 2x2/5 (one subject),
1x2/5 (one subject),
2x2/10 (one subject), and
1x2/1 (one subject). On the basis of these
data, the frequency of duplication of functional CYP2D6
alleles was 3% (6% of Hong Kong Chinese subjects). Allele
2 was duplicated in only two to three samples
(1x2/2 or 2x2/1,
2x2/5 and
2x2/10). The frequency of
CYP2D62x2 was either 0.9% or 1.3% (based on two or three
2x2 alleles, respectively) and was comparable to that
found in other Chinese populations (Table 1
B). Differences in metabolic
capacity between constellations 1x2/2 and
2x2/1 should be very slight. These
constellations were not further investigated.
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To our knowledge, only two studies on duplicated CYP2D6
genes have been conducted on Chinese (Table 1
B). Dahl et al.
(12) reported a frequency of 2% for CYP2D62x2
in a Chinese population consisting of 21 subjects. In another study
conducted on 113 Chinese subjects (11), the frequency
reported for CYP2D62x2 was 0.9%.
This study has raised our concern about the methodology applied to screen for UM individuals in Chinese populations. In our experience, and as noted above, some PCR results are in disagreement. We wonder whether this could be attributable to differences in the structure of the CYP2D6 locus between races.
Duplication of the CYP2D6 gene has also been detected by the presence of a XbaI 42-kb fragment after restriction length polymorphism analysis (RFLP) (6). XbaI-RFLP analysis has identified several haplotypes of the gene cluster CYP2D6 (18)(19). The three most frequent XbaI fragments are 29 kb (active gene and two pseudogenes), 44 kb (in Caucasians, CYP2D64 and three pseudogenes), and 11.5 kb (deletion of the active gene) (17). The XbaI 42-kb fragment usually is indicative of a duplication of the functional CYP2D62 allele (6). However, the 42-kb haplotype may be heterogeneous and associated with both the PM and UM phenotypes. In a Zimbabwean population, the 42-kb XbaI fragment has been associated with the CYP2D64 mutation indicative of a duplication of a nonfunctional gene (20). Similarly, the 44-kb XbaI fragment, which is associated with 30% of mutant alleles in Caucasian PMs, is functional in Chinese (11)(21). The 44-kb XbaI fragments in Caucasians and Chinese differ in the composition of the genes of the CYP2D6 locus. Given the disagreement observed in PCR results and that XbaI-RFLP analysis could also be misleading in Chinese, we can only conclude that the composition of the 42-kb fragment in Chinese needs further study. Currently, our methodology does not allow us to proceed with further investigation. Pronounced differences between Caucasians and Orientals have been found in the CYP2D6 locus; consequently, primers used for PCR tests (designed from the CYP2D6 locus sequence in Caucasians) may not be specific enough.
PCR and RFLP tests detect the presence of duplicated genes but give no information about their activity or the metabolic capacity of an individual. Hence, an individual’s CYP2D6 complete genotype should be known, especially when interpreting the results from assays designed to detect duplicated CYP2D6 genes in "non-Caucasian" populations.
References
The following articles in journals at HighWire Press have cited this article:
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  N. Hosono, M. Kato, K. Kiyotani, T. Mushiroda, S. Takata, H. Sato, H. Amitani, Y. Tsuchiya, K. Yamazaki, T. Tsunoda, et al. CYP2D6 Genotyping for Functional-Gene Dosage Analysis by Allele Copy Number Detection Clin. Chem., August 1, 2009; 55(8): 1546 - 1554. [Abstract] [Full Text] [PDF]
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 S.-J. Lee, S. S. Lee, H.-J. Jung, H.-S. Kim, S.-J. Park, C.-W. Yeo, and J.-G. Shin Discovery of Novel Functional Variants and Extensive Evaluation of CYP2D6 Genetic Polymorphisms in Koreans Drug Metab. Dispos., July 1, 2009; 37(7): 1464 - 1470. [Abstract] [Full Text] [PDF]
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L. Ji, S. Pan, J. Marti-Jaun, E. Hanseler, K. Rentsch, and M. Hersberger Single-Step Assays to Analyze CYP2D6 Gene Polymorphisms in Asians: Allele Frequencies and a Novel *14B Allele in Mainland Chinese Clin. Chem., July 1, 2002; 48(7): 983 - 988. [Abstract] [Full Text] [PDF]
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