Allelic distributions of CYP2D6 gene copy number variation in the Eastern Han Chinese population
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Allelic distributions of CYP2D6 gene copy number variation in the Eastern Han Chinese population1

Hai-hui Sheng2,6, Ai-ping Zeng3,6, Wen-xiang Zhu2, Ren-fang Zhu3, Hong-mei Li2, Zhi-dong Zhu2, Ying Qin2, Wei Jin4, Yan Liu4, Yun-lan Du5, Jian Sun5, Hua-sheng Xiao2,7

2National Engineering Center for Biochip at Shanghai, Shanghai 201203, China; 3Department of Clinical Laboratory, Wenling No 1 People’s Hospital, Wenzhou Medical College, Wenzhou 317500, China; 4Department of Cardiology, Ruijin Hospital, Medical College, Shanghai Jiaotong University, Shanghai 200025; 5Department of Neurology, Shanghai Gongli Hospital, Shanghai 200135, China

1Project supported by grants from the Shanghai Science and Technology Committee Fund (No 05dz52022 and No 05dz19318).

6These authors contributed equally to this work.

7Correspondence to Dr Hua-sheng XIAO.
Phn 86-21-5132-0288. Fax 86-21-5132-0266.
E-mail huasheng_xiao@shbiochip.com

Aim: The human cytochrome P450 2D6 (CYP2D6) gene copy number variation, involving CYP2D6 gene deletion (CYP2D6*5) and duplication or multiduplication (CYP2D6*×N), can result in reduced or increased metabolism of many clinically used drugs. The identification of CYP2D6*5 and CYP2D6*×N and the investigation of their allelic distributions in ethnic populations can be important in determining the right drug and dosage for each patient.

Methods: The CYP2D6*5 and CYP2D6 genes, and CYP2D6 gene duplication were identified by 2 modified long PCR, respectively. To determine duplicated alleles, a novel long PCR was developed to amplify the entire duplicated CYP2D6 gene which was used as template for subsequent PCR amplification. A total of 363 unrelated Eastern Han Chinese individuals were analyzed for CYP2D6 gene copy number variation.

Results: The frequency of CYP2D6*5 and CYP2D6*×N were 4.82% (n=35) and 0.69% (n=5) in the Eastern Han Chinese population, respectively. Of the 5 duplicated alleles, 3 were CYP2D6*1×N and 2 were CYP2D6*10×N. One individual was a carrier of both CYP2D6*5 and CYP2D6*1×N. Taken together, the CYP2D6 gene rearrangements were present in 10.74% of subjects.

Conclusion: Allelic distributions of the CYP2D6 gene copy number variation differ among Chinese from different regions, indicating ethnic variety in Chinese. Long PCR are convenient, cost effective, specific and semiquantitative for the detection of the CYP2D6 gene copy number variation, and amplification of the entire duplicated CYP2D6 gene is necessary for the accurate identification of duplicated alleles.

Keywords: CYP2D6; long PCR; deletion; duplication; copy number variation; rearrangement

Submitted Jun 21, 2006. Accepted for publication Aug 29, 2006.

doi: 10.1111/j.1745-7254.2007.00479.x

Introduction

Human cytochrome P450 (CYP) enzymes play important roles in the metabolism of a wide variety of exogenous and endogenous compounds. Approximately 57 CYP genes encoding cytochrome P450 proteins and 58 pseudogenes are present in the human genome and are classified into distinct families and subfamilies according to their sequence similarity[1]. The CYP2D subfamily comprises the CYP2D6 gene and 2 pseudogenes (CYP2D7 and CYP2D8), located in tandem on chromosome 22q13.1. The CYP2D6 gene is the only functional gene present in the human CYP2D gene locus. Although CYP2D6 is expressed at low levels in the liver, it plays crucial roles in the metabolism of over 65 commonly used drugs, including β-adrenergic blocking agents, antiarrhythmics, antipsychotics, antidepressants, and narcotic analgesics[2].

The CYP2D6 gene is highly polymorphic and has the most variations among the CYP450 gene superfamily, with more than 80 variations identified so far[3]. The variations can result in absent, decreased, normal, increased or qualitatively altered catalytic activity of CYP2D6, and consequently cause clinically relevant interindividual differences in therapeutic efficacy or adverse drug reactions. The CYP2D6 genotype importantly determines the metabolism of approximately 12% of all clinically used drugs[4].

The functionally deficient alleles are caused by detrimental mutations that range from single base pair changes to partial or whole gene deletion. The incidence of poor metabolizers (PMs), the homozygous or compound homozygous carriers of 2 functionally deficient alleles, is approximately 3%–10% in Caucasians, but only 1%–2% in Orientals[5]. In Caucasians, common deficient alleles include CYP2D6*3, *4, *5, and *6, accounting for about 98% of PMs[6]. However, the most common allele in Han Chinese is CYP2D6*10, which is associated with reduced activity[7–10]. The occurrence of deficient alleles of CYP2D6 is less frequent in Chinese, except for CYP2D6*5. CYP2D6*5, representing the deletion of the entire CYP2D6 gene, occurs in 2%–7% of Caucasians[9] and 3%–7% of Chinese[7,8,10,11]. The frequency of CYP2D6*5 is similar across populations, and therefore, CYP2D6*5 is not a major cause of the difference of PM prevalences between populations[5,8]. In contrast to PMs, ultrarapid metabolizers (UMs) usually carry a duplicated, or even multiduplicated (up to 13 copies of CYP2D6), active CYP2D6 allele (CYP2D6*×N). CYP2D6*2×N and CYP2D6*41×N are the most common alleles with CYP2D6 gene duplications[9]. UM may have therapeutic failure with drugs on account of increased enzymatic activity. The frequencies of CYP2D6*×N vary greatly between races[9], which is significantly different from the allelic distribution of CYP2D6*5. CYP2D6*×N is relatively rare in South-East Asians and Northern Europeans, but occurs at a frequency of 29% in an Ethiopian population owing to dietary selective pressure in the past that favored preservation of duplicated genes. The North-South gradient of the presence of the CYP2D6*×N in the European population is due to migrations of subjects from North-East Africa to the Mediterranean areas[9,12]. Both CYP2D6*5 and CYP2D6*×N result from CYP2D6 gene rearrangement[13] and comprise CYP2D6 gene copy number variation.

The methods for determining the CYP2D6 gene copy number variation can be divided into quantitative and qualitative analyses. Quantitative methods, including pyrosequ-encing, InvaderTM and real-time PCR, are based on the assessment of the relative CYP2D6 gene copy number by comparison between the amount of PCR product reflecting the number of CYP2D6 genes and the co-amplified region from an unrelated constitutive single-copy gene[12,14–17]. However, quantitative methods sometimes result in errors when determining the CYP2D6 gene copy number[12,14,17] and are unable to discriminate alleles with duplicated CYP2D6 genes. Qualitative methods often use long PCR spanning the repeated regions (CYP-REP) flanking the CYP2D6 gene to identify CYP2D6*5 and CYP2D6*×N[18–21]. Long PCR is one of the most commonly used methods for the detection of CYP2D6*5 and CYP2D6*×N, with its simplicity, convenience and cost effectiveness. Due to high polymorphism, it is important for the identification of the duplicated alleles to obtain the entire duplicated CYP2D6 gene. Two methods, the restriction digestion of genomic DNA[22] and PCR amplification, can obtain fragments containing the entire duplicated gene. The former is very time-consuming and not suitable for large scale and high-throughput detection and clinical practice. Although PCR is highly efficient and convenient, a new method which can specifically amplify the entire duplicated CYP2D6 gene is still needed.

The CYP2D6 gene copy number variation obviously affects the metabolic rates of drugs which are substrates of CYP2D6[23], and its alleles vary in frequency among populations. Although CYP2D6 gene rearrangements have been studied in the Central Han Chinese population, there are no data regarding the Eastern Han Chinese population. Therefore, the aims of this study were to develop assays for detecting CYP2D6 gene copy number variation and to assess the prevalence of the CYP2D6 gene copy number variation in the Eastern Han Chinese population.

Materials and methods

Subjects This study included 363 unrelated healthy individuals (51% women; mean±SD, 55±10.39 years) recruited through several hospitals in Shanghai and the Zhejiang province. All subjects were ethnically Eastern Han Chinese and were informed about the experimental procedure and the purpose of the study. Written consent was obtained from each participant. Genomic DNA was extracted from blood samples of the subjects using the Flexi Gene DNA Kit (Qiagen, Hilden, Germany) according to the manufacture’s protocol.

Detection for CYP2D6*5 To identify the CYP2D6*5 allele, the assay was carried out by a duplex long PCR method. A forward primer DuplF, binding in the 5'UTR of the CYP2D6 gene, was designed by using Primer Premier 5 (PREMIER Biosoft International, Palo Alto, CA, USA) and BLAST search (http://www.ncbi.nlm.nih.gov/BLAST/) (Table 1) and a reverse primer DPKlow is specific for the downstream of the CYP2D6 gene[24]. Primers cyp-13 and cyp-24 are specific for the downstream of the CYP2D7 and CYP2D6 genes, respectively. Duplex long PCR was carried out in a total volume of 50 µL containing 0.32 µmol/L of primers cyp-13 and cyp-24, 0.4 µmol/L of primers DuplF and DPKlow, 0.4 mmol/L of each deoxynucleoside triphosphate, 1× PCR reaction buffer, 2.85 mmol/L MgCl2, 2.5 U LA Taq (TaKaRa, Otsu, Shiga, Japan) and 200 ng of genomic DNA. Cycling conditions were as follows: 94 °C for 3 min, followed by 35 cycles of 94 °C for 35 s, 66 °C for 1 min and 72 °C for 5 min, followed by 72 °C for 6 min. Five µL of amplification products were then separated on 0.8% agarose gel electrophoresis and identified.

Detection of the duplicated CYP2D6 gene To identify CYP2D6 gene duplications, the assay was carried out by a modified long PCR method, as described by Lovlie et al[20]. The PCR was performed in a total volume of 25 µL containing 0.36 µmol/L of each primer, 0.4 mmol/L of each deoxy-nucleoside triphosphate, 1× PCR reaction buffer, 2.65 mmol/L MgCl2, 1.25 U LA Taq (TaKaRa, Otsu, Shiga, Japan) and 150 ng of genomic DNA. Cycling conditions were as follows: 94 °C for 3 min, followed by 35 cycles of 94 °C for 35 s, 64 °C for 1 min and 72 °C for 5.2 min, followed by 72 °C for 6 min. Five µL of PCR products were then separated on 0.8% agarose gel electrophoresis and identified.

Amplification of the entire duplicated CYP2D6 gene and sequencing To amplify the entire duplicated CYP2D6 gene, a long PCR was performed using specific primers DuplF and DuplR (Table 1). Long PCR reactions were carried out in a total volume of 50 µL containing 0.4 µmol/L of each primer, 0.4 mmol/L of each deoxynucleoside triphosphate, 1× PCR reaction buffer, 2.85 mmol/L MgCl2, 3 U LA Taq (TaKaRa, Otsu, Shiga, Japan) and 240 ng of genomic DNA. Cycling parameters were 2.5 min at 94 °C, followed by 35 cycles at 94 °C for 35 s, 66 °C for 1 min and 72 °C for 8.5 min, and then a final extended step of 72 ℃for 10 min. The PCR product was purified by QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) to avoid contamination of genomic DNA, and was then used as template for subsequent PCR amplification.

Table 1
Table 1 PCR primer sequences.
Full table

To amplify the coding region and part of the introns of the duplicated CYP2D6 genes, primers were designed using primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_slow.cgi); the amplicons were 494–752 bp. The sequences of the primers are listed in Table 1. Each fragment was amplified by touchdown[25]. Amplification reactions were carried out in a total volume of 30 µL containing 0.3 mmol/L of each deoxynucleoside triphosphate, 10 mmol/L Tris-HCl, 50 mmol/L KCl, 2 mmol/L MgCl2, 20% Q solution (Qiagen, Hilden, Germany), 0.16 µmol/L of each primer, 10 ng purified long PCR product, and 1.2 U Taq (TaKaRa, Otsu, Shiga, Japan). Cycling conditions were as follows: 94 °C for 3 min, followed by 10 cycles of 94 °C for 30 s, 66 °C for 30 s with a 0.5 °C decrement of the annealing temperature per cycle and 72 °C for 30 –45 s, followed by 30 cycles of 94 °C for 30 s, 61 °C for 30 s and 72 °C for 30–45 s, followed by 72 °C for 10 min. Amplified products were purified by the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) and sequenced with forward and reverse primer by ABI 3700 sequencer according to the Big-Dye chemistry reaction protocol (Applied Biosystems, Foster City, CA, USA). For definition of CYP2D6 alleles, see the CYP allele website[3].

Determination of genotype of carriers of CYP2D6*×N To determine the genotype of carriers of CYP2D6*×N, all exons and most parts of introns were amplified using genomic DNA as templates and sequenced. The primers used for amplification and sequencing and the reaction conditions were as described earlier.

Statistical analyses The frequencies were compared using the χ2 test and Fisher’s exact test. A value of P<0.05 was considered statistically significant. These analyses were carried out with SAS (SAS Institute Inc, Cary, NC, USA).

Results

With primer combination DuplF and DPKlow, a 4.8 kb fragment was amplified from the CYP2D6 gene locus, indicating the presence of the CYP2D6 gene (Figure 1). Because individuals homozygous for CYP2D6*5 are very rare, with a frequency of less than 0.5%[10,26], the 4.8 kb fragment also functions as a positive control for the amplification. In order to increase the efficiency of amplification, we used primer DuplF instead of DPKup, since the former does not form a hairpin structure and yields a shorter PCR product when combined with primer DPKlow[24]. A 3.5 kb fragment produced with primers cyp-13 and cyp-24 indicated the deletion of the CYP2D6 gene[19]. The 4.8 kb fragments were observed in all 363 samples, indicating a reliable and effective long PCR amplification in every sample. No subject with homozygous CYP2D6*5 was found. A long PCR analysis showed the existence of a 3.5 kb fragment in 35 of 363 subjects (Figure 2). CYP2D6*5 was found with a frequency of 4.82% in the study population (Table 2).

Figure 1 Schematic overview of the CYP2D genes and methods for the detection of CYP2D6*5 and CYP2D6 gene duplication. Large boxes represent the CYP2D genes. Small black and gray boxes represent 0.6 kb repeats and 2.8 kb CYP-REP units, respectively. The 2.8 kb CYP-REP units are almost completely identical the downstream of the CYP2D7 and CYP2D6 gene and duplicated CYP2D6 gene. The 0.6 kb repeats and 2.8 kb CYP-REP units the downstream of the CYP2D7 gene are separated by a 1.6 kb DNA fragment. The horizontal arrowheads show specific primer sets. (A) For individuals carrying single copy of CYP2D6 gene, 5.2 kb fragment and 4.8 kb fragment are amplified by primer sets, cyp-17 and cyp-32, and DuplF and DPKlow, respectively. The 15.7 kb fragment is too large to amplify with primers cyp-13 and cyp-24. (B) When the CYP2D6 gene is deleted (CYP2D6*5), a 3.5 kb fragment is amplified with primers cyp-13 and cyp-24. (C) When duplicated or multiduplicated CYP2D6 genes are present, a 3.6 kb and a 5.2 kb fragment are co-amplified with primers cyp-17 and cyp-32. The 4.8 kb fragment for the presence of the CYP2D6 gene may be potential mixture of PCR products of duplicated and single copy of the CYP2D6 gene. An 8.2 kb fragment yielded by primers DuplF and DuplR only contains the upstream and central CYP2D6 genes.
Figure 2 Long-PCR for the detection of CYP2D6*5. The 4.8 kb fragment for the presence of the CYP2D6 gene and the 3.2 kb for CYP2D6*5 were co-amplified in a single multiples reaction. Samples 19, 56, 84, and 110 are heterozygous for CYP2D6*5, whereas samples 70 and 200 have no CYP2D6 gene deletion.
Table 2
Table 2 Comparison of CYP2D6 gene rearrangement across populations.
Full table

CYP2D6*×N was detected by long PCR with primers cyp-17 and cyp-32 (Figure 1). The 5.2 kb fragment, an internal control for the reaction, was observed in all 363 samples. The 3.6 kb fragment representing the duplicated CYP2D6 gene was seen in 5 individuals (0.69%), whereas no amplification product was observed in the others. One of the 5 carriers of CYP2D6*×N is also a carrier of CYP2D6*5. Taken together, 39 individuals carried CYP2D6 gene rearrangements with an incidence of 10.74%. The CYP2D6 gene copy number variation showed no statistically significant difference between Eastern and Central Han Chinese (χ2=4.215, P=0.112), or between Eastern Han Chinese and Malaysian Chinese (χ2=4.323, P=0.115), but a statistically significant difference between Central Han Chinese and Malaysian Chinese (χ2=13.252, P=0.0008; Table 2).

To characterize the duplicated CYP2D6 gene, 2 specific primers DuplF and DuplR were used to amplify the entire duplicated CYP2D6 gene (Figure 1). Forward primer DuplF is specific for the 5'UTR of the CYP2D6 gene and reverse primer DuplR can bind in CYP2D6–CYP2D6 intergenic regions and the downstream of the CYP2D7 gene. The primer DuplR was modified from cyp-32 in order to increase amplification specificity and yield. This primer combination can amplify an 8.2 kb fragment spanning the 5'UTR of the CYP2D6 gene and CYP2D6–CYP2D6 intergenic regions, which only allows for the amplification of the upstream and central CYP2D6 genes, not the downstream or single-copy CYP2D6 gene. Therefore, the 8.2 kb fragment contains the entire duplicated CYP2D6 genes, not the single-copy CYP2D6 gene. The 8.2 kb fragment was observed in all 5 carriers of the duplicated CYP2D6 gene, whereas no amplification product was seen in any other sample, as expected (Figure 3). Direct sequencing showed that the 8.2 kb fragment contained all 9 exons and indicated that the entire duplicated CYP2D6 genes were specifically amplified by the long PCR. Among the 5 carriers of the duplicated CYP2D6 gene, the most frequent duplicated allele was CYP2D6*1 (60%), followed by CYP2D6*10 (40%; Table 2). Since we were not able to exclusively sequence the downstream CYP2D6 gene, the downstream alleles were indirectly determined by sequencing both genomic DNA and the entire duplicated CYP2D6 gene. The downstream CYP2D6 alleles in the 5 carriers were identical to the corresponding upstream and/or central CYP2D6 alleles. The genotypes of 2 of the carriers of CYP2D6*×N were CYP2D6*1×N/*1, while the others were CYP2D6*1×N/*5, CYP2D6*10×N/*1 and CYP2D6*10×N/*2, respectively.

Figure 3 Results of the PCR amplified fragments containing the entire duplicated CYP2D6 gene. The 8.2 kb fragment was obtained from samples 56, 107, 226, 239, and 311, whereas this fragment was not seen in sample 320 with a single copy of the CYP2D6 gene.

Discussion

Recently, several studies revealed that numerous copy number variations are in the human genome[27–31]. Many copy number variations contain entire genes and their numbers lead to differential levels of gene expression. Copy number variations account for many normal phenotypic variations and are involved in diseases[32], thus becoming a major focus of research in human genetics. In the present study, CYP2D6*5 and CYP2D6*×N were detected to characterize the CYP2D6 gene copy number variation in a large Eastern Han Chinese population. The frequency of CYP2D6*5 is lower (4.82%) than in previous findings of 7.17% in the Central Han Chinese population[10] and 5.7% in Chinese living in Sweden[11], and very close to the 4.6% found in a Hong Kong Chinese population[7]. The frequency of CYP2D6*×N was also lower (0.66%) in the study population than that in the Central Han Chinese (1.35%)[10]. In Malaysian Chinese, the frequency of CYP2D6*5 and CYP2D6*×N are both lower than those found on the Chinese mainland[8]. Our study showed that the CYP2D6 gene copy number variation was the frequent variant in the Eastern Han Chinese population. Furthermore, the most common duplicated allele was CYP2D6*1, followed by CYP2D6*10, which was inconsistent with the high prevalence of CYP2D6*10 in Chinese[7,8,10,11] and different from Central Han Chinese and other populations[9,10]. In the Chinese population, the frequency of CYP2D6*10 ranges from 51% to 70%[7,8,10,11]. The structure, CYP2D6*36+*10 tandem, was not observed in 5 carriers of CYP2D6*×N. However, our results need further verification in a larger population since only a few cases of CYP2D6 gene duplication were included in our study. Differences of allelic distributions of the CYP2D6 gene were observed between not just Eastern and Central Han Chinese, but also Taiwanese[33] and Hong Kong Chinese[7], implying that there is genetic diversity in Chinese from different regions.

The sequences of the CYP2D7 and CYP2D6 genes, as well as their downstream sequences, are almost completely identical, thus it is difficult to design PCR-based methods for detecting the CYP2D6 gene copy number variation. Long PCR is the original method for the determination of CYP2D6 gene rearrangement and has been widely used. In this study, we combined 2 long PCR for the detection of the CYP2D6*5 and CYP2D6 gene, and CYP2D6*×N, respectively, to investigate the CYP2D6 gene copy number variation. This approach offers a unique ability to distinguish among 0, 1, 2 or more CYP2D6 genes. It is a semiquantitative method for the detection of the CYP2D6 gene copy number. Unusual CYP2D6 gene rearrangement may confuse the determination of the CYP2D6 gene copy number, but it occurs very rarely[26,34,35]. Long PCR are specific, costless and convenient for detecting CYP2D6 gene rearrangements. Since TaqMan PCR, InvaderTM and pyrosequencing for the detection of the CYP2D6 gene copy number variation require pre-amplifying specific regions of the CYP2D6 gene, it seems unlikely that these methods are less time-consuming than long PCR. Furthermore, polymorphic sites and gene conversions in target sequences may affect the accuracy of these methods[12,14,17]. The AmpliChip CYP450 test can detect simultaneously CYP2D6 alleles and determine 7 duplicated alleles[36], but it remains relatively expensive.

The CYP2D genes are rich in specific DNA elements for recombination, and thus the CYP2D locus is a hot spot region for unequal crossover events[13]. The breakpoint is located downstream of both the CYP2D7 and CYP2D6 genes. According to the recombination pattern of the CYP2D locus, several methods for identification of the CYP2D6 gene deletion and duplication, respectively, were developed by amplification of fragments spanning the potential crossover sites[19,20]. In order to identify alleles with duplicated CYP2D6 genes, additional assays should be carried out. Routine methods are to amplify fragments spanning exon 9 in the upstream extra CYP2D6 gene to intron 2 in the downstream CYP2D6 gene used as template for restriction fragment length polymorphism (RFLP) assay[20,24] and allele-specific PCR[10]. However, gene duplications such as CYP2D6*36×N and the CYP2D6*36+*10 tandem can not amplify due to the gene conversion in exon 9. The new method described by Gaedigk et al can amplify all duplication arrangements with forward and reverse primers binding to intron 6 and intron 2, respectively[37]. Because tag single nucleotide polymorphisms (SNPs) and/or mutations are distributed widely across the entire CYP2D6 gene, duplicated alleles could be misclassified when analyzed on the basis of only part of the sequence of the duplicated CYP2D6 gene. Amplification of the fragment containing the entire duplicated CYP2D6 gene is necessary for accurate identification of the duplicated alleles. Although the method described by Johansson et al can amplify a 5.1 kb fragment containing the entire CYP2D6 gene by the DPKup/DPKlow primer pair[24], both single-copy and duplicated CYP2D6 genes were amplified. The method described by Gaedigk et al[37] suffers from the same drawback. Therefore, duplicated alleles can not be exactly identified as a result of the mixture of PCR products. To accurately determine variant alleles with duplication of the CYP2D6 gene, a novel long PCR was developed to specifically amplify the entire duplicated CYP2D6 gene and confirmed by sequencing. The long PCR can always amplify upstream and central CYP2D6 genes whenever sequential CYP2D6 genes are duplicated comprising same allele or CYP2D6*36 in tandem with CYP2D6*10. The 8.2 kb fragment, containing the entire duplicated CYP2D6 gene, can be used as template for the identification of alleles with duplication of the CYP2D6 gene. The long PCR is less time-consuming than both the restriction digestion of genomic DNA, which takes at least 2 days[22], and the previously described PCR-based methods[20,24,37], since our method yields a shorter PCR product. In addition, Lovlie et al showed that the amplification of larger genomic DNA fragments, in contrast to shorter fragments, is more prone to failure[20]. Therefore, the long PCR is more maneuverable than the aforementioned methods[20,24,37]. The amplified duplicated CYP2D6 gene is useful for further genotyping of duplicated genes to avoid misclassification of PM as UM due to the duplication of an inactive allele, or extensive metabolizer (EM) as UM due to the duplication of an allele associated with reduced activity. In addition, the long PCR can be readily adapted for other applications, such as the detection of CYP2D6 gene duplication.

In conclusion, we screened CYP2D6 gene rearrangements by long PCR in the Eastern Han Chinese population and developed a long PCR to amplify the entire duplicated CYP2D6 gene. The allelic distributions of the CYP2D6 gene copy number variation vary among Chinese from different regions, indicating ethnic variety in Chinese.

Acknowledgement

We thank Dr Yi QU for reviewing this manuscript.

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Cite this article as: Sheng Hh, Zeng Ap, Zhu Wx, Zhu Rf, Li Hm, Zhu Zd, Qin Y, Jin W, Liu Y, Du Yl, Sun J, Xiao Hs. Allelic distributions of CYP2D6 gene copy number variation in the Eastern Han Chinese population1. Gland Surg 2007;28(2):279-286. doi: 10.1111/j.1745-7254.2007.00479.x