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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 °C 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.
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
frag-ment 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).
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.
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
misclassi-fied 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
sequenc-ing. 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
mis-classification 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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