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Clinical Chemistry 48: 983-988, 2002;
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(Clinical Chemistry. 2002;48:983-988.)
© 2002 American Association for Clinical Chemistry, Inc.

Single-Step Assays to Analyze CYP2D6 Gene Polymorphisms in Asians: Allele Frequencies and a Novel *14B Allele in Mainland Chinese

Ling Ji1,2, Shixiu Pan2, Jacqueline Marti-Jaun1, Edgar Hänseler1, Katharina Rentsch1 and Martin Hersberger1a

1 Institute of Clinical Chemistry, University Hospital Zurich, Raemistrasse 100, CH-8091 Zurich, Switzerland

2 Clinical Medical Laboratory, Union Hospital Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Republic of China

aAuthor for correspondence. Fax 41-1-255-4590; e-mail hmr{at}ikc.unizh.ch.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Cytochrome P450-dependent monooxygenase 2D6 (CYP2D6) activity can be estimated by investigating the metabolism of model drugs or by genotyping the most common CYP2D6 alleles. For Caucasians, the CYP2D6 allele frequencies are well investigated, and single-step assays are available for genotyping, whereas allele analysis in mainland Chinese is limited.

Methods: Two tetra-primer assays and one allele-specific amplification assay were developed to easily genotype the CYP2D6 alleles *8, *10, and *14 previously detected in Asians. Applying these assays in combination with established single-tube assays, we analyzed 223 DNA samples from Chinese volunteers for the CYP2D6 alleles *3, *4, *5, *6, *8, *10, and *14 and for duplication of CYP2D6.

Results: Six different alleles were detected in mainland Chinese. The most frequent mutant allele was the intermediate metabolizer allele, CYP2D6*10, with a prevalence of 51.3%, followed by the poor metabolizer alleles CYP2D6*5 (7.2%) and a novel variant of CYP2D6*14. This novel *14B allele (2.0%) differs from the *14 allele by the absence of the C188T substitution and by the additional G1749C substitution. Furthermore, six duplication alleles of CYP2D6 were detected, including one duplication of the *10 allele (*10X2).

Conclusions: The CYP2D6 allele frequencies in mainland Chinese shows some genetic diversity compared with Chinese from other regions: a novel *14B allele, a slightly higher frequency of the *5 allele, and a slightly lower frequency of the *10 allele than in most other Chinese populations.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The cytochrome P450 2D6 (CYP2D6)1 enzyme debrisoquine 4-hydroxylase metabolizes >40 widely prescribed medications and several illegal amphetamine derivatives (1)(2)(3). The activity of the CYP2D6 enzyme can be measured in vivo after oral intake of a single dose of a probe drug with subsequent determination of the ratio between the urinary recovery of the drug and the metabolite. According to the activity of the enzyme, patients can be classified into three different phenotypes: poor metabolizers (PMs), extensive metabolizers, and ultrarapid metabolizers (UMs). The different phenotypes have profound effects on the efficacy of a drug and on its adverse reactions (4).

There are significant interethnic differences in the prevalence of the different phenotypes. The prevalence of the PM phenotype ranges from 0–2% in Asians (5)(6)(7) to 5–10% in Caucasians (8), whereas the prevalence of UMs varies from 1–2% in Asians (9)(10) to 29% in some African populations (11)(12)(13). The combination of two inherited CYP2D6 alleles from at least 73 known CYP2D6 alleles (14) produces the three different phenotypes. Individuals homozygous for deficient CYP2D6 alleles metabolize drugs at a low rate (PMs), whereas individuals with duplication of the active CYP2D6 gene metabolize drugs at ultrarapid rates (UMs).

In Caucasians, the PM phenotype is caused mainly by the mutant alleles CYP2D6*3, *4, and *5 (15)(16). In contrast, CYP2D6*3 and *4 are basically absent in Asians, explaining the low frequency of PM phenotypes in these populations (10)(17)(18). Nevertheless, an Asian-specific PM allele (CYP2D6*14) was recently described, which was not detected in Caucasians (19).

Asians also have a high prevalence of the intermediate metabolizer (IM) allele, CYP2D6*10, which encodes an enzyme with reduced activity (20). Genotype-to-phenotype correlation studies showed that homozygotes for the CYP2D6*10 allele have a lower CYP2D6 metabolic activity (21)(22). Because of the lower CYP2D6 activity and the high prevalence of CYP2D6*10 in Asians compared with Caucasians (50–70% vs 5.1%), this observed lower mean activity in Asians has been attributed to the *10 allele (9)(23)(24). Four allelic variants of CYP2D6*10 are known, including *10A, *10B, *1C (renamed CYP2D6*36), and *10D (renamed CYP2D6*37), but for diagnostic analysis, combining the four under CYP2D6*10 seems reasonable.

The genetics for the UM phenotype are not yet entirely clear, with only some UMs explained by the duplication of the CYP2D6 gene (25)25. However, these CYP2D6*1X2 and CYP2D6*2X2 alleles, which encode two to several copies of CYP2D6, cause increased enzyme activity and increased metabolism (26)(27)(28).

Several smaller studies investigated a set of CYP2D6 polymorphisms in Chinese from different areas, but no study investigated the major PM, IM, and UM alleles. Hence, we developed single-step assays and genotyped genomic DNA from 223 Chinese volunteers from the Hubei province (central China) for CYP2D6*3, *4, *5, *6, *8, *10, and *14 and for the duplication of CYP2D6 by allele-specific amplification (ASA) PCR assays, by tetra-primer PCR assays, and by multiplex long PCR.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
A total of 223 unrelated mainland Chinese volunteers participated in the study after giving written informed consent. These Chinese were all of Han nationality, and 88% were born and raised in Hubei Province. They were all healthy volunteers who were recruited during their yearly routine examination at the Union Hospital in Wuhan, China.

EDTA-anticoagulated blood samples were extracted with the QIAamp DNA Blood Mini Kit (Qiagen AG). Oligonucleotides (Table 1 ) were purchased from Microsynth and were used as 10 µmol/L solutions in water. The AmpliTaq GoldTM System (Applied Biosystems/Perkin-Elmer Corp.) was used to amplify the tetra-primer and ASA PCR, and the ExpandTM Long Template PCR System (Roche Molecular Biochemicals) was used for long PCR. Alleles and genotypes were assigned according to the nomenclature for human CYP2D6 alleles, with the data in Table 2 used for discrimination (29).


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  		Table 1. Oligonucleotides used in this study.


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  		Table 2. Allele assignment.

For detection of A2637del (*3), G1934A (*4), CYP2D6del (*5), and T1795del (*6), we performed three tetra-primer PCR assays and one multiplex long PCR assay as described previously (30).

For detection of G1846T (CYP2D6*8) and G1846A (CYP2D6*14), we developed two 25-µL tetra-primer PCR reactions. In the first set of cycles, preamplification of the 750-bp CYP2D6 region with primers 1new and 2new (Fig. 1 , control) ensured the specificity of the subsequent ASA for G1846T (CYP2D6*8) or G1846A (CYP2D6*14). In the second set of cycles, ASA (primers L14wt and U8mut or U14mut) produced a 472-bp PCR product for the G1846 allele and a 305-bp PCR product for the T1846 (CYP2D6*8) and A1846 (CYP2D6*14) alleles. The reaction mixture contained 18.1 µL of water, 2.5µL of buffer 1 (1.5 mM MgCl2), 0.2 µL of Gold Taq (5 U/µL), 0.5 µL of deoxynucleotide triphosphate (dNTP) mixture (10 mM), 0.2 µL of primer 1new, 0.3 µL of primer 2new, 0.7 µL of primer L14wt (G), 0.5 µL of primer U8mut (T) or U14mut (A), and 2.0 µL of genomic DNA (~50 ng/µL). Cycling conditions were as follows: 10 min at 94 °C, followed by 20 cycles (first set) of 94 °C for 30 s, 63 °C for 30 s, and 72 °C for 60 s; 22 cycles (second set) of 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 60 s; and a final extension at 72 °C for 7 min.



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  		Figure 1. Analysis of the G1846T (CYP2D6*8) and G1846A (*14) mutations by tetra-primer PCR.

Preamplification of the CYP2D6 locus with gene-specific primers produces a 750-bp product (control). Reducing the annealing temperature then allows ASA on this newly amplified template in the same tube. ASA produces a 472-bp product for G1846 and a 305-bp product for T1846 (*8) or A1846 (*14).

The PCR products were separated by 2.0% agarose gel electrophoresis. The genotypes of eight genomic DNA samples were confirmed by sequence analysis: four DNA samples were heterozygous for G1846A (G/A), and four were wild type (G/G). These genomic DNAs were subsequently reanalyzed 11 times as controls for the G1846A (CYP2D6*14) analysis.

For detection of C188T (CYP2D6*10), a single-step ASA assay was developed in two tubes, one for detection of C188 and one for detection of T188 (Fig. 2 ). Preamplification of the 790-bp CYP2D6 region with primers U1595 and L2362 (Fig. 2 , control) ensured the specificity of the subsequent ASA for C188 or T188. The ASA (primers L10wt or L10mut) produced a 137-bp PCR product for the C188 allele or the T188 allele. The reaction mixture contained 18.3 µL of water, 2.5 µL of buffer 1 (1.5 mM MgCl2), 0.2 µL of Gold Taq (5 U/µL), 0.5 µL of dNTP mixture (10 mM), 0.5 µL of primer U1595, 0.5 µL of primer L2362, 0.5 µL of primer L10wt (C) or primer L10mut (T), and 2.0 µL of genomic DNA (~50 ng/µL). Cycling conditions were as follows: 10 min at 94 °C, followed by 15 cycles (first set) of 94 °C for 30 s, 61 °C for 30 s, and 72 °C for 60 s; 27 cycles (second set) of 94 °C for 30 s, 53 °C for 30 s, and 72 °C for 60 s; and a final extension at 72 °C for 7 min. PCR products were separated by 2.0% agarose gel electrophoresis. The genotype of 11 genomic DNA samples was confirmed by sequence analysis: 4 were heterozygous for C188T (C/T), 6 were homozygous for C188 (C/C), and 1 was homozygous for T188 (T/T). These genomic DNAs were subsequently reanalyzed four times as controls for the C188T analysis.



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  		Figure 2. Analysis of the C188T (CYP2D6*10) mutation by ASA.

Amplification is performed in two tubes. A 790-bp product amplifying the CYP2D6 locus serves as a control and as template for ASA in both tubes. Reducing the annealing temperature then allows ASA on this newly amplified template in the same tube. In the first tube, a 137-bp product is amplified in the presence of C188. In the second tube, a 137-bp product is amplified in the presence of the T188.

For detection of the CYP2D6X2 gene duplication, two 25-µL long-PCR reactions were performed as described previously (31).

For verification of the CYP2D6*10X2 allele, an ~10-kb fragment specific for the duplicated allele was first amplified in a 50-µL long-PCR reaction adapted from Johansson et al. (32). This ~10-kb fragment includes exons 9 and 1 of the duplicated CYP2D6 coding region. The reaction mixture contained 34.75 µL of water, 5 µL of buffer 3 (2.25 mM MgCl2), 0.75 µL of enzyme mixture (3.5 U/µL), 2.5 µL of dNTP mixture (10 mM), 1.5 µL of primer P2x2f, 1.5 µL of primer L2362, and 4.0 µL of genomic DNA (~50 ng/µL). The cycling conditions were as follows: 2 min at 94 °C, followed by 36 cycles of 94 °C for 15 s, 63 °C for 30 s, and 68 °C for 8 min; and a final extension at 72 °C for 7 min. The PCR products were then separated by 0.8% agarose gel electrophoresis. For alleles with the CYP2D6X2 gene duplication, an ~10-kb fragment was produced, which was subsequently used as template (diluted 1:100 000) for the ASA to detect the C188T mutation. C188T of the ~10-kb fragments was also confirmed by sequence analysis.

To confirm the new CYP2D6*14B allele, a 5.1-kb region that included the entire CYP2D6 coding region was amplified with primers DPKup and DPKlow (30) and was subcloned into vector pCR2.1, using the Original TA Cloning Kit (Invitrogen). The QIAprep Miniprep Purification Kit (Qiagen) was used for plasmid purification. Plasmids were analyzed for A1846 (CYP2D6*14) with the tetra-primer assay described and then sequenced on both strands. All CYP2D6 exons, including the intron-exon boundaries, were sequenced.

To distinguish between the CYP2D6*14B/*10 genotype and the *14/*1 genotype, a 750-bp fragment that included exon 3 of CYP2D6 was amplified with primers 1new and 2new. This PCR product was then sequenced to analyze G1749C. In contrast to CYP2D6*14, which contains G1749, the *14B allele contains C1749. Hence, the *14B/*10 genotype shows a homozygous C1749 signal, whereas the *14/*1 genotype shows a homozygous G1749 signal. The reaction mixture contained 37.6 µL of water, 5.0 µL of buffer 1 (1.5 mM MgCl2), 0.4 µL of Gold Taq (5 U/µL), 1.0 µL of dNTP mixture (10 mM), 1.0 µL of primer 1new, 1.0 µL of primer 2new, and 4.0 µL of genomic DNA (~50 ng/µL). Cycling conditions were as follows: 10 min at 94 °C, followed by 36 cycles of 94 °C for 30 s, 61 °C for 30 s, and 72 °C for 60 s; and a final extension of 7 min at 72 °C.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
This study investigated the allele frequencies of the CYP2D6 gene in 223 Chinese volunteers from the central part of China. To investigate most CYP2D6 key mutations prevalent in Asians, we developed and evaluated two tetra-primer PCR assays and one ASA PCR assay to distinguish among G1846T (*8), C188T (*10), and G1846A (*14) substitutions in CYP2D6 (Figs. 1Up and 2Up ). The combination of the previously developed assays to detect A2637del (CYP2D6*3), G1934A (*4), CYP2D6del (*5), and T1795del (*6) (30) and the assay to detect the gene duplication of CYP2D6 (31) detected six different alleles, which are listed in Table 3 . In this study, CYP2D6*10 was the most frequent mutant allele (51.3%), followed by two PM alleles, CYP2D6*5 (7.2%) and CYP2D6*14B (2.0%).


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  		Table 3. Frequencies of the CYP2D6 alleles identified.

In general, our genotype results are in agreement with the published CYP2D6 allele sequences (29), and assignment of the alleles was unambiguous (Table 2Up ). However, three DNA samples showed heterozygous results (G/A) in the G1846A assay and homozygous C/C results in the C188T assay. This contradicted the published sequence of the CYP2D6*14 allele, which contains the T188 mutation. To further investigate this potentially new allelic variant of *14, we amplified and subcloned the entire CYP2D6 coding region from such genomic DNAs. Three individual subclones were sequenced for all exons of CYP2D6, revealing the A1846, T2938, and C4268 mutations intrinsic to CYP2D6*14. In contrast, the novel allele lacks the T188 mutation but contains the C1749 mutation and the intron 1 gene conversion to CYP2D7 (nucleotides 302–333), which are both part of the CYP2D6*2 allele (33). Hence, the 5' region of the allele is identical to the *2 allele, whereas the 3' region is identical to the *14 allele, suggesting that the novel *14B allele originated from recombination between these two alleles.

One DNA sample showed a homozygous T/T result in the C188T assay and the presence of a CYP2D6 gene duplication, which suggests the presence of a duplication of the *10 allele, as described recently (17). To confirm the presence of the *10X2 allele, we amplified an ~10-kb region (32) specific for the CYP2D6 gene duplication and used it as template to detect C188T. ASA PCR and direct sequencing of this ~10-kb template confirmed T188, supporting the presence of the *10X2 gene duplication allele (data not shown). To investigate whether the *10X2 duplication was more prevalent, the above procedure was repeated with the three DNA samples that showed heterozygous results in the C188T assay and the presence of the CYP2D6 gene duplication. However, none of these gene duplications had T188, excluding that they were *10X2 duplication alleles. Furthermore, none of these CYP2D6X2 duplication alleles included the G1934A substitution part of the CYP2D6*4X2 allele (PM) or any other investigated mutation; they therefore possibly represent the UM alleles CYP2D6*1X2 and CYP2D6*2X2.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The CYP2D6 allele frequencies in this study are comparable to the previously determined frequencies in Chinese, with some slight alterations. The allele frequency of CYP2D6*10 in volunteers from this study was 51.3%, which is lower than in Chinese from other areas. In Singapore, Taiwan, and Hong Kong, the CYP2D6*10 allele frequencies ranged from 62% to 70% (19)(34)(35). In addition, the prevalence of CYP2D6*5 (7.2%) was slightly higher in this study than has been reported for Hong Kong Chinese (4.6%) (34) and Chinese living in Sweden (5.7%) (9). However, concordant with other studies of Chinese, the other PM alleles, CYP2D6*3, *4, *6, and *8, were not detected or were detected in only very low frequencies (34), and the frequency of the gene duplication (1.3%) was also similar to other investigations of Chinese (9)(17)(36). These minor differences in allele frequencies may reflect ethnic variety within the Chinese population. This is also supported by the presence of the CYP2D6*14B allele in mainland Chinese, which is present as an allelic variant (*14) in Taiwanese (19) and was not detected in Hong Kong Chinese (34).

It is not clear whether CYP2D6*14B is a PM allele or whether it encodes CYP2D6 with reduced activity (IM). Wang et al. (19) reported a patient with a CYP2D6*5/*14 genotype, which was phenotyped as PM. They showed, in vitro, that the G1846A substitution in *14 decreased CYP2D6 activity to 43% of the wild-type enzyme and argued that its activity may be further reduced in combination with the C2938T and G4268C substitutions (19). However, the novel allele CYP2D6*14B lacks the T188 mutation, which was also shown to decrease enzyme activity in CYP2D6*14 (20). Hence, to classify the *14B, it will be necessary to determine the enzyme activity encoded by CYP2D6*14B or to phenotype *14B/*5 compound heterozygotes.

Genotyping of CYP2D6 polymorphisms in Caucasians is facilitated by the knowledge of the majority of the polymorphisms, which were detected by screening the entire CYP2D6 coding sequence of 672 Caucasians (15). Such a screening approach has not been done in Asians. Nevertheless, several IM, PM, and UM alleles prevalent in Asians have been characterized that should be assayed in a genotyping approach. We therefore aimed to set up an assay strategy based on the current knowledge of alleles in Asians, which could allow determination of the major IM and PM alleles in these populations.

The developed assays do not rely on preamplification of the entire CYP2D6 coding region, but directly amplify the CYP2D6 region of interest in a first set of amplification cycles, before the ASA is performed on this newly produced template in the second set of cycles in the same tube (30)(37). Such a design has the advantage that no transfer of amplified DNA is necessary, but it has the disadvantage that pseudogenes could be coamplified if the amplification primers are not gene specific. We used specific CYP2D6 amplification primers for each assay with the exception of the primer pair U1595/L2362, which has only one mismatch in the 3' end region of the lower primer to the CYP2D7BP pseudogene sequence and hence could amplify the pseudogene. Because such coamplification of the CYP2D7BP would cause misclassification of homozygous C188 DNA as heterozygous (C/T), we reanalyzed 68 DNA samples, including 54 heterozygous DNA samples (C/T), by a modified two-step assay, as described by Sachse et al. (16). This assay preamplifies the entire CYP2D6 coding region in a first step and applies this product diluted in a second tube as template for ASA. All results for C188T assigned with our new single-step assay were confirmed by the two-step assay (data not shown), suggesting that misclassification through coamplification of CYP2D7BP is rare.

To investigate the accuracy of the assays, we amplified the specific region for CYP2D6 in 8–11 genomic DNA samples for each assay and determined the genotype by sequence analysis (see Materials and Methods). All sequences confirmed the results of the tetra-primer and ASA analysis. These genomic DNAs were then reanalyzed 4–11 times with identical results (data not shown), demonstrating that the presented assays are reproducible.

Recently, Garcia-Barcelo et al. (17) found discordant results among three long-PCR amplification assays for detecting the CYP2D6 gene duplication in DNA samples from Hong Kong Chinese. We applied two of the same assays in our study to detect the CYP2D6 gene duplication and obtained identical results for all gene duplications. This difference in results may be attributable to the low frequency of samples that did not amplify with these two assays in their study (17). The third assay used by Garcia-Barcelo et al. (17) produced the most discordant results, and this assay was not applied in our study. Hence, we cannot exclude that we missed certain CYP2D6 duplication alleles prevalent in Asians, but to answer this, a better understanding of the CYP2D6 gene structure in Asians is necessary. It is noteworthy that genotyping for UMs is limited not only in Asians, but also in Caucasians, in whom the CYP2D6 gene duplications account for only 10–30% of the UMs (15)(27). Recent investigations of the genetics of duplication-negative UMs showed association of promoter polymorphisms with the UM phenotype and with increased CYP2D6 enzyme activity, but direct evidence for other UM alleles has not been found (25)(38).

In conclusion, we describe a set of one-step assays to genotype the most prevalent IM and PM alleles of CYP2D6 in a Chinese population. Applying these assays, this study in mainland Chinese shows some genetic diversity compared with Chinese from other regions: We found a novel CYP2D6*14B allele, a slightly higher frequency of the *5 allele, and a slightly lower frequency of the *10 allele than in most other Chinese populations. These differences suggest ethnic diversity in Chinese.


   Footnotes
 
1 Nonstandard abbreviations: CYP, cytochrome P450-dependent monooxygenase; PM, poor metabolizer; UM, ultrarapid metabolizer; IM, intermediate metabolizer; ASA; allele-specific amplification; and dNTP, deoxynucleotide triphosphate.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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