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Articles |
1
Institute of Clinical Chemistry, University Hospital Zurich, Raemistrasse 100, CH-8091 Zurich, Switzerland.
a Author for correspondence. Fax 41-1-255-4590; e-mail hmr{at}ikc.unizh.ch
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: Three tetra-primer PCR assays were developed to detect the mutations in the CYP2D6*3, *4, and *6 alleles. In these single-tube assays, the CYP2D6 locus is amplified directly, followed by the allele-specific amplification on this new template. In addition, a multiplex long PCR was developed to genotype the CYP2D6*5 allele. Two long PCR amplifications for detection of the deletion of CYP2D6 (*5) and for detection of the CYP2D6 gene region were combined in one tube.
Results: Analysis of 114 alleles showed no CYP2D6*3 allele, and allele frequencies of 28.1% for CYP2D6*4, 2.6% for CYP2D6*5, and 0.9% for CYP2D6*6. Re-analysis of the DNA samples by restriction fragment length polymorphism and sequencing analysis confirmed these results. Furthermore, re-analysis of sequenced genomic DNA by tetra-primer PCR analysis (7–11 times) always showed identical results.
Conclusions: Our set of single-tube assays allows rapid and reproducible genotyping of the majority of CYP2D6 poor metabolizers.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The individual CYP2D6 activity can be determined by phenotyping sparteine/debrisoquine metabolism, but these studies are hampered by complicated protocols (3). Recent comparative studies indicate that the CYP2D6 genotype predicts the sparteine/debrisoquine phenotype with high accuracy (4)(9), suggesting the use of CYP2D6 genotyping for classification into ultrarapid metabolizer, extensive metabolizer, or PM. Several PM alleles have been described that lead to inactive CYP2D6; however, evaluation of four PM alleles, CYP2D63, 4, 5, and 6 (10), can predict 93–97.5% of the PM phenotypes in the white Caucasian population (4)(9)(11).
Several methods based on PCR amplification of the CYP2D6 locus are used to genotype the PM alleles. The most widely used methods to detect PM alleles with small nucleotide mutations and deletions are restriction fragment length polymorphism (RFLP) analysis (12) and allele-specific amplification (ASA) (4)(13). To detect the deletion of the CYP2D6 gene, long PCR is used (14)(15). However, ASA and RFLP include several transfer steps that increase the risk of contamination and mix-up of samples, and long PCR has no internal control for PCR reliability. To facilitate genotyping of the CYP2D6 alleles that predict the majority of PMs, we developed three single-tube tetra-primer PCR assays to detect the CYP2D63, 4, and 6 alleles and a multiplex long PCR assay to detect the CYP2D65 allele.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For detection of CYP2D63, a 25-µL tetra-primer PCR reaction was performed. The reaction mixture contained 17.7 µL of water, 2.5 µL of buffer 1 (1.5 mmol/L MgCl2), 0.2 µL of Gold Taq (5 U/µL), 0.5 µL of dNTP mixture (10 mmol/L), 0.3 µL of primer 3, 0.75 µL of primer 6, 0.75 µL of primer Awt, 0.3 µL of primer 4new, and 2.0 µL of genomic DNA (~50 ng/µL), and 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; 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 of 7 min at 72 °C. The PCR products were then separated by 5% polyacrylamide gel electrophoresis for 2 h (17). DNA was detected by poststaining of the polyacrylamide gel with ethidium bromide (500 mg/L in water) and visualized under an ultraviolet transilluminator. A digital image was recorded with a charged-couple device camera and frame grabber (UV products). The genotypes of five genomic DNAs were confirmed by sequence analysis (18): three DNAs were heterozygous for the 3 allele (3/wt) and two were wild type (wt/wt). These genomic DNAs were subsequently re-analyzed seven times as controls for the CYP2D63 analysis of the 57 DNA samples.
For detection of CYP2D64, a 25-µL tetra-primer PCR reaction was performed. The reaction mixture contained 17.55 µL of water, 2.5 µL of buffer 1 (1.5 mmol/L MgCl2), 0.2 µL of Gold Taq (5 U/µL), 0.5 µL of dNTP mixture (10 mmol/L), 0.5 µL of primer 1new, 0.75 µL of primer Bmut, 0.5 µL of primer 7, 0.5 µL of primer 2new, and 2.0 µL of genomic DNA (~50 ng/µL), and the cycling conditions were as follows: 10 min at 94 °C; 15 cycles (first set) of 94 °C for 30 s, 63 °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 of 7 min at 72 °C. The PCR products were analyzed directly by 1.5% agarose gel electrophoresis for 1.25 h (17), followed by ethidium bromide staining and ultraviolet detection as described for the polyacrylamide gel. The genotypes of eight genomic DNAs were confirmed by sequence analysis: two DNAs were homozygous for the 4 allele (4/4), four were heterozygous (4/wt), and two were wild type (wt/wt). These genomic DNAs were subsequently re-analyzed 11 times as controls for the CYP2D64 analysis.
For detection of CYP2D66, a 25-µL tetra-primer PCR reaction was performed. The reaction mixture contained 17.3 µL of water, 2.5 µL of buffer 1 (1.5 mmol/L MgCl2), 0.2 µL of Gold Taq (5 U/µL), 0.5 µL of dNTP mixture (10 mmol/L), 0.5 µL of primer 1new, 0.75 µL of primer Tmut, 0.75 µL of primer 11, 0.5 µL of primer 2new, and 2.0 µL of genomic DNA (~50 ng/µL), and cycling conditions were as follows: 10 min at 94 °C; 15 cycles (first set) of 94 °C for 30 s, 63 °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 of 7 min at 72 °C. The PCR products were then analyzed directly by 2.5% agarose gel electrophoresis for 1.5 h. The genotype of six genomic DNAs was confirmed by sequence analysis: two DNAs were heterozygous for the 6 allele (6/wt) and four were wild type (wt/wt). These genomic DNAs were subsequently re-analyzed nine times as controls for the CYP2D66 analysis.
For detection of CYP2D65, a 50-µL long PCR reaction was performed. The reaction mixture contained 36.1 µL of water, 5.0 µL of buffer 3 (2.25 mmol/L MgCl2), 0.75 µL of Enzyme Mix (3.5 U/µL), 1.75 µL of dNTP mixture (10 mmol/L), 0.4 µL of primer Dup, 0.4 µL of primer Dlow, 0.8 µL of primer DPKup, 0.8 µL of primer DPKlow, and 4.0 µL of genomic DNA (~50 ng/µL), and the cycling conditions were as follows: 1 min at 94 °C, followed by 35 cycles of 94 °C for 60 s, 65 °C for 30 s, 68 °C for 5 min, and a final extension of 7 min at 68 °C. The PCR products were then analyzed directly by 0.8% agarose gel electrophoresis for 2 h.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) (19) was developed to analyze the
CYP2D63 allele. Two primers complementary to unique
intronic sequences of CYP2D6 (primers 3 and 4new) and two
primers designed for ASA of the wild-type (primer Awt) and the
3 allele (primer 6) were combined in one tetra-primer PCR
assay (13)(20)(21). During the first
set of cycles, a 1106-bp region of CYP2D6 amplified with
primers 3 and 4new (Table 1
) served as internal control for the quality of the PCR
amplification and as template for the subsequent ASA (Fig. 2
, control). During the second set of cycles, ASA (primers 6 and
Awt) produced the 580-bp PCR product specific for the 3
allele and the 553-bp PCR product specific for the wild-type allele.
Because the wild-type and 3 alleles were analyzed on the
coding and the noncoding DNA strands, respectively, both amplifications
could occur simultaneously in one tube (19). Separation by
5% polyacrylamide gel electrophoresis allowed the distinction of the
three PCR products and the interpretation of the CYP2D63
genotype (Fig. 2
). Analysis of 57 genomic DNA samples revealed no
CYP2D63 alleles (Table 2
). To confirm the accuracy of our new method, we re-analyzed the
DNAs for CYP2D63 by RFLP analysis (12) and
obtained identical results (data not shown).
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The CYP2D64 allele was detected by tetra-primer PCR.
Preamplification of the 750-bp CYP2D6 region (primers 1new
and 2new) ensured the specificity of the subsequent ASA for
CYP2D64 (Fig. 3
, control) (13)(20). ASA (primers Bmut
and 7) produced a 217-bp PCR product for the 4 allele and a
560-bp PCR product for the wild-type allele. The PCR products were
separated by 1.5% agarose gel electrophoresis. Thirty-two
CYP2D64 alleles were identified among 57 DNA samples
analyzed, including 3 individuals homozygous for CYP2D64
(Table 2
). Identical results were obtained by RFLP analysis
(12).
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The CYP2D66 allele was analyzed by tetra-primer PCR. The
same preamplification of the 750-bp CYP2D6 region was used
as for the 4 allele (primers 1new and 2new). The subsequent
ASA (primers Tmut and 11) specifically amplified a 421-bp PCR product
for the wild-type allele and a 356-bp PCR product for the
CYP2D66 allele (Fig. 4
). The PCR products were separated by 2.5% agarose gel
electrophoresis. One CYP2D66 allele (Table 2
) was detected
within the 57 DNAs analyzed and was confirmed by sequencing (data not
shown).
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The CYP2D65 allele was detected by multiplex long PCR. We
multiplexed two long PCR amplifications (14)(15)
to detect the deletion of CYP2D6 ((5)) and to
simultaneously control for the long PCR amplification (Fig. 5
). In this multiplexed long PCR, the 3.2-kb product indicated
the deletion of CYP2D6 (primers Dup and Dlow) and the 5.1-kb
product indicated the wild-type CYP2D6 allele (primers DPKup
and DPKlow) (15). Within the 57 genomic DNAs analyzed, we
detected 3 5 alleles. Identical results were obtained with
the original long PCR method in two tubes
(14)(15).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Three tetra-primer PCRs (Fig. 1
) (19) were developed and
evaluated to detect the single nucleotide polymorphisms of
CYP2D63, 4, and 6. For each assay,
four primers are combined in a single tube for the initial
amplification of the CYP2D6 locus and the subsequent ASA. In
the first set of cycles, preamplification of the CYP2D6 gene
region is ensured by the higher annealing temperature of the primers
used to amplify the CYP2D6 locus relative to the primers
used for the ASA (19). Decreasing the annealing temperature
in the second set of cycles then allows ASA on the newly synthesized
CYP2D6 gene region.
The tetra-primer PCRs described here are reliable and robust assays for
detecting the CYP2D63, 4, and 6
alleles. The analysis of 57 human DNA samples yielded frequencies for
these alleles (Table 2
) that were in agreement with previous studies in
Europeans (4)(9). Re-analysis of the DNAs for
CYP2D63 and 4 alleles by RFLP (12)
confirmed the results. Furthermore, we amplified the specific regions
for the 3, 4, and 6 alleles of five
to eight genomic DNAs each and determined the genotype by sequence
analysis (see Materials and Methods). All sequences
confirmed the results of the tetra-primer analysis. These genomic DNAs
were then re-analyzed 7–11 times by tetra-primer PCR with identical
results (data not shown), demonstrating that the tetra-primer PCR
assays for the 3, 4, and 6 allele
are reproducible. These findings seem to contrast the lack of
confidence in ASA that led to the use of RFLP to detect
CYP2D6 polymorphisms (4). It is known that ASA
assays can give false-positive results because of contamination and
false-negative results because of unsuccessful PCR (22).
However, the tetra-primer PCR assays presented here are less prone to
contamination because they are single-tube assays and do not require
transfer of amplified PCR products, and the risk for false-negative
results are omitted because the assays include an internal control for
PCR amplification. Furthermore, recent improvement in DNA extraction
and PCR amplification increased the reliability of PCR in general.
Several DNA extraction kits that yield high-quality genomic DNA from
blood and several PCR amplification kits that allow standardization
assure reliable and robust results.
To complete the set of single tube assays for genotyping the majority of the PMs, we developed a multiplex long PCR to simultaneously detect the deletion of CYP2D6 (5 allele) and the CYP2D6 gene. In the former long PCR assay for CYP2D65 (14)(15), misinterpretation could occur if the long PCR failed or if the genomic DNA was not added. This is omitted in the multiplex long PCR by inclusion of the simultaneous amplification of the CYP2D6 gene as an internal control for the reliability of the PCR. Detection of the CYP2D65 allele is not necessary unless the 5 allele is homozygous. Any PM allele that is transheterozygous with CYP2D65 would be genotyped as homozygous in the tetra-primer PCRs and would give the correct PM classification. However, in the absence of amplification in the tetra-primer PCRs, the multiplex long PCR is necessary to exclude handling errors or unsuccessful DNA extraction from misinterpretation as homozygous CYP2D65.
The four presented single-tube PCR assays allow genotyping of the majority of PMs in Caucasians. In addition to the wild-type allele, at least 52 different CYP2D6 alleles are known that are associated with deficient, decreased, normal, or increased enzyme activity (4)(9)(10). However, the four most common inactivating CYP2D6 alleles, 3, 4, 5, and 6, are associated with 93–97% of the PM phenotypes, whereas other inactivating mutations contribute <1% (4)(9). Therefore, testing of the CYP2D63, 4, 5, and 6 alleles seems reasonable and sufficient to routinely screen for inactivating mutations in CYP2D6.
The tetra-primer PCRs can be done during a typical work day in two thermal cyclers, whereas the multiplex long PCR to detect the 5 allele and the long PCR to detect ultrarapid metabolizers (23)(24) can be performed together overnight. This combination allows genotyping of CYP2D6 within 2 days.
In conclusion, we present three tetra-primer PCRs for genotyping the CYP2D63, 4, and 6 alleles, and a multiplex long PCR for genotyping the 5 allele. These single-tube PCR assays reduce the risk for handling errors and contamination, and facilitate genotyping of CYP2D6.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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