| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Technical Briefs |
1 Department of Forensic Medicine, University of Helsinki, Helsinki, Finland;2 Department of Biology, University of Ferrara, Ferrara, Italy;
aaddress correspondence to this author at: Department of Forensic Medicine, PO Box 40, University of Helsinki, 00014 Helsinki, Finland; fax 358-9-19127518, e-mail johanna.sistonen{at}helsinki.fi
Great interindividual variability in drug response leads to variation in drug safety and efficacy. Although this can be the result of environmental and physiologic factors as well as drug–drug interactions, in many cases the response is inherited, arising from a polymorphism in genes encoding drug transporters, drug receptors, and especially, drug-metabolizing enzymes. The polymorphic cytochrome P450 2D6 (CYP2D6; OMIM 124030) is one of the most widely studied drug-metabolizing enzymes, being responsible for the metabolism of many commonly used drugs belonging to classes such as antidepressants, neuroleptics, beta-blockers, and antiarrhythmics (1). The CYP2D6 gene spans a 4.2-kb region located on chromosome 22q13.1 and is part of the CYP2D cluster together with CYP2D8P and CYP2D7 pseudogenes (2). At present, more than 50 different major polymorphic CYP2D6 alleles are known (3). These include variants produced by major rearrangements (whole-gene deletion or duplication), point mutations, and single or multiple base deletions or insertions. The phenotypic consequences of this variation are considerable: the CYP2D6 enzyme activity ranges from complete deficiency, possibly giving rise to profound toxicity of medication in the case of poor-metabolizing individuals, to ultrarapid metabolism, which can lead to therapeutic failure at recommended drug dosages. Individuals with a normal or slightly below normal rate of metabolism related to the CYP2D6 enzyme are usually defined as extensive or intermediate metabolizers, respectively.
CYP2D6 genotyping to predict metabolic status is considered a valid alternative to traditional phenotyping methods (4). Assessing the CYP2D6 genotype also offers several distinct advantages over the experimental determination of a CYP2D6 phenotype (5). The characteristics of the gene do not change during the lifetime, and genetic status is uninfluenced by environmental or physiologic factors. Genotyping requires only 1 sample and can be done before a drug is given to a patient. It therefore may facilitate improved drug efficacy and diminished risk for adverse drug reactions (6). At present, there are specific guidelines available on how genetic information could be taken into account in clinical dose adjustments for specific antidepressants and antipsychotics (7). Furthermore, in forensic medicine, CYP2D6 genotyping may provide important information on whether a polymorphism may have contributed to a drug fatality.
Because CYP2D6 genotyping has a wide range of applications in both routine and research investigations, methods for rapid and cost-effective genotyping are necessary. Different genotyping methods for the CYP2D6 gene have been developed, including multiplex allele-specific PCR (8), PCR with restriction fragment length polymorphism (PCR-RFLP) analysis (9), real-time PCR (10), pyrosequencing (11), and oligonucleotide microarray technology (12). Despite allele-specific PCR and RFLP analysis being laborious and low throughput, these methods are still widely used (13)(14) because the newer, high-throughput methods often require special laboratory facilities (15). Our aim was to develop a rapid and technically feasible genotyping method that is suitable for moderate-throughput laboratories and includes all clinically important CYP2D6 mutations that are known to alter the enzyme activity.
In this study, we describe a CYP2D6 genotyping protocol based on a combination of PCR and multiplex extension of unlabeled oligonucleotide primers with fluorescently labeled dideoxynucleotide triphosphates (SNaPshotTM; Applied Biosystems). The method has been designed to screen whole-gene deletion and duplication and 11 of the most relevant polymorphic positions of the gene (Table 1
) as well as the allelic composition of the gene duplication. It allows the identification of CYP2D6 alleles highly represented in different human populations (i.e., *2, *4, *10, *17, *29, *39, and *41) or alleles, even if rare, known to be responsible for low or null metabolic activity (i.e., *3, *6, and *9) (16). Alleles *10 and *17, in particular, have been included to extend the usability of the method because these are considered to be the major decreased-function variants in Asian and African populations, respectively. Alleles not carrying detected mutations were classified as *1.
|
The entire CYP2D6 gene (5.1 kb) was amplified in long-PCR reaction using primers CYP2D6-F (5'-CCAGAAGGCTTTGCAGGCTTCA-3') (17) and CYP2D6-R (5'-ACTGAGCCCTGGGAGGTAGGTA-3') (17) to separate the gene from the flanking highly homologous CYP2D8P and CYP2D7 pseudogenes. The 50-µL reaction mixture contained 1.5 U of GeneAmp® rTth DNA Polymerase XL (Applied Biosystems), 1x XL Buffer II, 1.25 mM Mg(OAc)2, 0.2 mM each deoxynucleotide triphosphate, 0.4 µM each primer, and 20–100 ng of genomic DNA. The PCR was conducted as follows: denaturation at 94 °C for 1 min; 10 cycles of 94 °C for 30 s and 68 °C for 10 min; 25 cycles of 94 °C for 30 s and 68 °C for 10 min and 15 s, plus 15 s per cycle; and a final extension at 72 °C for 30 min. Two additional long-PCR reactions were used to analyze the major rearrangements, i.e., duplication or deletion, of the entire CYP2D6 gene. We amplified a duplication-specific 3.2-kb fragment, using primers CYP-207-F (5'-CCCTCAGCCTCGTCACCTCAC-3') (18) and CYP-32-R (5'-CACGTGCAGGGCACCTAGAT-3') (18). To rule out false-negative results, we added primer CYP-13-F (5'-ACCGGGCACCTGTACTCCTCA-3') (19) to the reaction mixture, which yielded an internal control fragment of 3.8 kb. To detect the gene deletion, we again used a set of 3 primers; we amplified a deletion-specific 3.5-kb fragment and a 3.0-kb control fragment, using primers CYP-13-F, CYP-24-R (5'-GCATGAGCTAAGGCACCCAGAC-3') (19), and CYP-207-F. The 50-µL reaction contained 1.0 U of DyNAzymeTM EXT DNA Polymerase (Finnzymes), 1x magnesium-free EXT buffer, 1.0 mM MgCl2, and 0.2 mM each deoxynucleotide triphosphate. The primer concentrations were as follows: for the duplication-specific reaction, we used 0.6 µM CYP-207-F, 0.4 µM CYP-32-R, and 0.2 µM CYP-13-F; and for the deletion-specific reaction, 0.6 µM CYP-13-F, 0.4 µM CYP-24-R, and 0.2 µM CYP-207-F. The temperature cycling profile was the same as described above. The long-PCR products were analyzed on 1% agarose gels (Fig. 1A
), and the 5.1-kb fragments were purified by use of 1 µL of ExoSAP-IT® (USB Corporation) per 5 µL of PCR product, with incubation at 37 °C for 15 min and at 80 °C for 15 min to successively deactivate the enzymes.
|
The purified 5.1-kb product was used as a template to detect 11 polymorphic positions of the CYP2D6 gene (Fig. 1B
and Table 1
). In the following single-base extension reaction, the detection primers annealed adjacent to the single-nucleotide polymorphism (SNP) position. A difference of 5 nucleotides between primer lengths was chosen to avoid overlapping between the fluorescent signals of the final products in the capillary electrophoresis. To achieve the final length, 5' poly(dT) tails were added to the primers. The sequences of the detection primers and their final concentrations in the reaction are listed in Table 1
. The SNaPshot reaction contained 2.0 µL of purified PCR product, 2.0 µL of pooled detection primers, and 2.5 µL of SNaPshot ready reaction mixture in a final volume of 10 µL. The temperature cycling profile was 25 cycles of 96 °C for 10 s, 55 °C for 10 s, and 60 °C for 30 s. After the reaction, 5'-phosphoryl groups of unincorporated dideoxynucleotide triphosphates were removed by addition of 1.0 U of calf intestinal phosphatase (Finnzymes) and incubation of the samples at 37 °C for 1 h and at 75 °C for 15 min to successively deactivate the enzyme. Capillary electrophoresis of samples was performed on the ABI PRISM® 3100 genetic analyzer, and the results were analyzed with GeneMapper ID, Ver. 3.1 (Applied Biosystems; Fig. 1C
). Electropherograms presenting different CYP2D6 alleles are shown in Fig. 1 in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol51/issue7/.
We have used the method to genotype the HGDP-CEPH Human Genome Diversity Cell Line Panel, which includes 1064 individuals [Ref. (20); our unpublished data]; 247 samples extracted from fresh blood (21), and >200 postmortem cases. Altogether, these samples represent a worldwide distribution of populations; thus each detected mutation was found in at least 8 individuals. For method validation, we selected a subset of 50 individuals from the above-mentioned samples, representing all different detected alleles, and genotyped them by PCR-RFLP analysis (9)(22). The minimum allelic frequency in the subset was 3% (at least 3 chromosomes bearing the mutation). Polymorphic positions 2988G>A and 3183G>A, which were not included in our PCR-RFLP protocol, were verified by sequencing 5 carriers and 2 noncarriers according to the SNaPshot profile. In addition, we performed a SNaPshot genotyping to reanalyze 33 postmortem cases typed previously by PCR-RFLP analysis (22). Concordance was 100% between the new method and the conventional methods.
A new and interesting feature of our genotyping protocol is the possibility of determining the phase of gene duplication. We observed that the SNaPshot reaction described above presents as a byproduct a quantitative aspect, and we believe that it provides adequate information to determine which of the 2 alleles is actually duplicated (Fig. 1C
; also see Fig. 2 in the online Data Supplement). To confirm the previous result, we selected 11 samples from the CEPH dataset that were found duplication-positive (2 *1xN/*2, 2 *1/*2xN, 1 *1xN/*4, 2 *1/*4xN, 2 *2xN/*4, and 2 *2/*4xN). We amplified a 9.3-kb fragment (Fig. 1A
), using primers Lx2F (5'-GCCACCATGGTGTCTTTGCTTTC-3') (23) and Lx2R (5'-ACCGGATTCCAGCTGGGAAATG-3') (23). The reaction conditions and the temperature cycling profile were the same as described for the 5.1-kb fragment. The amplified region starts from exon 9 and ends at intron 2 of the 2 subsequent CYP2D6 genes. Three polymorphic positions, namely 4180 of the first gene and 100 and 1023 of the second gene, were examined by nested PCR-RFLP analysis. The duplicated genotype identifications obtained through the original 11-plex SNaPshot reaction were confirmed in all cases.
From our experience, the SNaPshot method is very robust, accurate, and easy to perform. Advantages of the SNaPshot technique include the detection of high numbers of mutations in a single reaction and the possibility to increase the number of detected SNPs without substantially increasing the costs of analysis. With our method the cost per genotype is 
5 
(from long PCR to the capillary electrophoresis), which is less than one-half the cost of the corresponding PCR-RFLP analysis. Samples can be processed in 96-well plates, and after the overnight PCR, the final genotypes can be obtained in 5 h.
In summary, we designed a cost-effective and technically feasible CYP2D6 genotyping technique through a 2-step assay that provides a straightforward interpretation of results. It allows detection of 11 of the most relevant polymorphic positions, the assessment of whole-gene deletion and duplication, and unlike most available typing methods, the allele composition of the gene duplication. The latter feature may be useful in the prediction of phenotype; a clear example is the difference between the genotype CYP2D6*1/*4xN, which produces a single full-function allele, and genotype CYP2D6*1xN/*4, which will produce at least twice the amount of enzyme.
Methods using the same principle have recently been published (24)(25), but to our knowledge this is the most exhaustive CYP2D6 SNaPshot assay in terms of number and type of polymorphic positions and validation. Covering the most frequent and clinically relevant CYP2D6 mutations, this method can be used in routine and research investigations worldwide. This feature is particularly important considering the mixed genetic background of modern societies (26). In addition, the results obtained by the proposed combination of PCR and SNaPshot were confirmed, with no exceptions, by conventional methods. This was true for both the definition of the 11 SNP haplotypes and the phase of gene duplication. The method is suitable for moderate-throughput laboratories and is easily extended to alternative or additional SNPs in the targeted CYP2D6 gene region.
References
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  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]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
indicate exons. (C), electropherograms of the SNaPshot reaction products for 3 different individuals. Peaks correspond to the 11 polymorphic sites screened (numbers above the peaks indicate their positions in the gene), and the genotypes are indicated in the top right-hand corners. The top electropherogram represents the CYP2D6*1/*4 genotype. Stars indicate the mutated positions defining the CYP2D6*4 allele. Two other samples were found to be duplication-positive by long-PCR detection. By comparing these electropherograms with the CYP2D6*1/*4 genotype and considering the ratio between the intensities of the 2 peaks at the same position, the duplicated allele can readily be identified as the one displaying higher signals (arrows). Electropherograms presenting genotypes CYP2D6*2/*4, CYP2D6*2xN/*4, and CYP2D6*2/*4xN are shown in Fig. 2 of the online Data Supplement.