HUMAN MUTATION 16:77�85 (2000)

© 2000 WILEY-LISS, INC.

METHODS

Rapid and Comprehensive Determination ofCytochrome P450 CYP2D6 Poor MetabolizerGenotypes by Multiplex Polymerase Chain ReactionRebecca Roberts,1 Patrick Sullivan,2 Peter Joyce,2 and Martin A. Kennedy1*1Department of Pathology, Christchurch School of Medicine, Christchurch, New Zealand2Department of Psychological Medicine, Christchurch School of Medicine, Christchurch, New Zealand

Communicated by Anne-Lise Borresen-Dale

The liver enzyme cytochrome P450 CYP2D6 (debrisoquine 4-hydroxylase) metabolizes numer-ous drugs, including many antidepressants, neuroleptics, antiarrhythmics, and antihypertensiveagents. Variability in the gene that encodes this enzyme is an important factor underlying variabledrug treatment responses. Some 5–10% of Caucasians lack functional CYP2D6, and the geneticbasis of most of these “poor metabolizer” alleles is now well defined. As the CYP2D6 status of apatient can have profound effects on response to drug treatment, it is important to devise methodsthat permit rapid and economical determination of CYP2D6 genotype. We have developed a ro-bust polymerase chain reaction method that simultaneously identifies the variants CYP2D6 *3,*4, *6, *8, *11, *12, *14, *15, *19, and *20. This constitutes most of the poor metabolizer allelesdescribed in Caucasian and Asian populations. Separate PCR reactions or Southern blots arerequired for *7, the*5 deletion, and the hybrid alleles *13 and *16. The multiplex assay wasvalidated on 100 individuals previously genotyped by specific polymerase chain reaction-restric-tion fragment length polymorphism analysis, and proved 100% accurate in this sample. The assayperformed consistently with Taq DNA polymerases from various suppliers, within a broad range oftemperatures and MgCl2 concentrations, and using genomic DNA prepared by a range of methodsincluding extraction from dried blood spots on card. This multiplexed, amplification refractorymutation system (ARMS) method is reliable, rapid, relatively cheap, amenable to automation, andoffers the advantages of minimal sample handling with no requirement for restriction enzymes asin earlier CYP2D6 assays. Hum Mutat 16:77–85, 2000. © 2000 Wiley-Liss, Inc.

KEY WORDS: CYP2D6; poor metabolizers; mutation detection; drug metabolism

DATABASES:

CYP2D6 – OMIM:124030; GDB:119832; HGMD:CYP2D6

Received 30 November 1999; accepted revised manuscript 5April 2000.

*Correspondence to: M.A. Kennedy, Ph.D., Department ofPathology, Christchurch School of Medicine, PO Box 4345, Christ-church, New Zealand. E-mail: martin.kennedy@chmeds.ac.nz

Contract grant sponsors: Health Research Council of NewZealand; Otago University.

Current address for Patrick Sullivan is Department of Psychia-try, Virginia Institute for Psychiatric and Behavioral Genetics, 800East Leigh Street, Suite 110, Richmond, VA 23219-1534.

INTRODUCTION

Genetic variability in liver enzymes that me-tabolize drugs and other substances is an impor-tant contributor to individual responses topharmaceutical drug treatment [Nebert, 1997]. Itis now clear that, in several circumstances, pre-scribing of drugs would be more effective and lesshazardous when an assessment of this genetic vari-ability is carried out for relevant enzymes [Kroemerand Eichelbaum, 1995; Linder et al., 1997]. Fur-thermore, the basis of some cases of unusual drugresponses can often be clarified by retrospectiveanalysis of genetic variation in key metabolic en-zymes [Bertilsson et al., 1993; Eichelbaum and

Evert, 1996; Johansson et al., 1993; Morgan et al.,1984; Shah et al., 1982; Spina et al., 1992].

The cytochrome P450 enzyme debrisoquine 4-hydroxylase, or CYP2D6 (MIM# 124030), medi-

78 ROBERTS ET AL.

ates the oxidative metabolism of many currentlyprescribed drugs, including various antidepres-sants, neuroleptics, β-adrenoreceptor blockers, andantiarrhythmics [Brosen and Gram, 1989;Fjordside et al., 1999; Heim and Meyer, 1990;Kroemer and Eichelbaum, 1995]. The CYP2D6gene is highly polymorphic, with at least 66 allelesnow described (http://www.imm.ki.se/cypalleles/),and this genetic diversity contributes to a widevariability in individual levels of CYP2D6 activity[Daly et al., 1996; Sachse et al., 1997]. About 5–10% Caucasians lack CYP2D6 activity, and theseindividuals are referred to as poor metabolizers(PM). Standard doses of some antidepressants andantiarrhythmics can lead to exaggerated drug re-sponse and side effects in PM individuals [Linderet al., 1997; Meyer et al., 1996], or a lack of effectsfor prodrugs, such as codeine, that are activatedby CYP2D6 [Poulsen et al., 1996].

Genetic analysis of CYP2D6 is an effectivemethod for predicting the majority of PM pheno-types, although it is currently less effective at pre-dicting intermediate or ultra-rapid metabolizerCYP2D6 phenotypes [Chen et al., 1996; Griese etal., 1998; Sachse et al., 1997]. Given the prevalenceof PM phenotypes and the wide impact of CYP2D6variability in drug responses, a simple blood test thatcan rapidly and economically evaluate virtually allCYP2D6 PM genotypes would be of considerableutility, particularly when investigating selected pa-tients with unusual drug responses.

MATERIALS AND METHODS

DNA Extraction

Peripheral blood.

Genomic DNA was routinely extracted fromperipheral blood using the method of [Ciulla etal., 1988]. Briefly, blood (10 ml in an EDTA tube)was mixed with 40ml lysis buffer (0.32M sucrose,10 mM Tris pH 7.5, 5 mM MgCl2, 1% Triton X-100) as soon as possible after drawing. Leukocyteswere recovered by centrifugation and resuspendedin lysis solution (4M guanidine isothiocyanate,25mM sodium acetate, 0.84% β-mercaptoethanol)to release DNA. An equal volume of isopropanolwas added to precipitate the DNA, which was re-covered by centrifugation and washed three timesin cold 70% ethanol. The DNA was then resus-pended and dissolved in 10 mM Tris pH 8.0-1mMEDTA (0.5 ml) and stored at –20° C.

In order to test the ability of the CYP2D6ARMS assay to function with DNA prepared indifferent ways, three other methods were used to

extract genomic DNA from peripheral blood.These methods were sodium chloride extraction[Lahiri and Nurnberger,1991], rapid sodium hy-droxide-boiling lysis [Dracopoli et al., 1994], andproteinase K-phenol-chloroform extraction[Kennedy et al., 1989].

Dried blood spots.

Peripheral blood was spotted directly onto anFTA GeneCard™ (Gibco BRL, Rockville, MD,USA) and allowed to dry overnight. Using clean,sterile surgical blades 2–3 mm2 samples were re-moved from the centre of the blood spots, placedin 1.5 ml microcentrifuge tubes, and each samplewas separately processed according to the cardmanufacturer’s instructions.

Buccal cells.

DNA was extracted from buccal cells using themethod of Richards et al. [1993].

Long PCR of Entire CYP2D6 Locus

The CYP2D6 gene is downstream of two highlyhomologous pseudogenes (CYP2D8 and CYP2D7).Of the mutations so far found in CYP2D6, threehave been also detected in CYP2D8 and nine inCYP2D7 [Heim and Meyer, 1992]. To avoid false-positive detection of mutations through co-amplifi-cation of pseudogene sequences, initial amplificationof the entire CYP2D6 gene was carried out usingprimers complementary to unique intronic sequencesof CYP2D6 (Fig. 1). The PCR method and primersof Sachse et al. [1997] were used with the followingmodifications. Reaction volumes were 25 µl, andcontained 2mM MgCl2, 1.25 µM of each primer, 2units of ELONGASE® Enzyme Mix (Gibco BRL)and ~100 ng of genomic DNA, or 2–3 mm2 of FTA

FIGURE 1. Location of oligonucleotide primers used togenotype CYP2D6. P100 and P200 are used in the pre-liminary long PCR amplification step of the entire locus.CYP2D6FC1-CYP2D6FC4 (here abbreviated to FC1-FC4)are forward primers, and mutation-specific primers areindicated below the map. Exons (numbered) and intronsof CYP2D6 are shown to scale, as indicated. For eachmutation-specific primer shown, a corresponding wildtype-specific primer also exists.

MULTIPLEX PCR FOR CYP2D6 79

GeneCard. Thermal cycling was performed withan initial denaturation of 30 sec at 94°C, followedby 35 cycles of 30 sec at 94°C, 6 min at 68°C, anda terminal extension of 7 min at 68°C. All longPCR products were checked on 1% agarose gels,then diluted in five volumes of TE (pH 8.0) beforeuse in the ARMS assay.

For FTA GeneCard-bound genomic DNAsamples, the PCR reaction mix was pre-heated to94°C prior to the addition of ELONGASE. This hot-start PCR strategy was not necessary for genomicDNA extracted directly from peripheral blood.

ARMS PCR Assay

The optimum conditions established in thiswork for the CYP2D6 ARMS assay are as follows.Two separate PCR reactions (ARMS1 andARMS2) containing a mixture of wild type ormutation-specific primers were performed for eachCYP2D6 long-PCR product (Table 1, Fig. 2). Re-actions consisted of a total volume of 25µl con-taining 200µM dNTPs, 2.0mM MgCl2, 2.5 U TaqDNA polymerase (Roche Molecular Biochemicals,Mannheim, Germany) and 1µl of CYP2D6 longPCR product. Optimal concentrations were de-termined separately for each primer. Primers

CYP2D6FC1, CYP2D6FC4, and both pairs ofCYP2D6*11 and CYP2D6*20 primers were usedat a concentration of 0.4µM, and all other primerswere used at 0.2µM. Temperature cycles (14 intotal) were 94 °C for 30 sec, 65°C for 20 sec, 72°Cfor 40 sec. PCR products (5 µl) were resolved byelectrophoresis on 3% agarose-TBE gels consist-ing of 3:1 NuSieve GTG (FMC, Rockland, ME,USA) and UltraPure agarose (Gibco BRL), stainedwith ethidium bromide (Fig. 2). Electrophoresiswas carried out in 1xTBE at 100V, and gels wererun until the bromophenol blue dye marker nearedthe end of the gel (usually about 2 hr for 9cm gels).A 25 base pair ladder (Gibco BRL) was used as asize marker. The CYP2D6*7 reaction was per-formed separately using the conditions describedabove, except annealing temperature was reducedfrom 65°C to 55°C (Fig. 2). Primers used in eachassay are indicated in Table 1.

Several commercial preparations of Taq DNApolymerase were tested in the ARMS assay. Thesewere: Taq DNA polymerase (Roche MolecularBiochemicals); Taq DNA polymerase (Promega,Madison, WI, USA); Taq DNA polymerase (Phar-macia, Uppsala, Sweden); Platinum® Taq DNApolymerase (Gibco BRL).

TABLE 1. Sequence and Location of Oligonucleotide Primers Used in This Study

Primers Sequencec Positiond Reaction Tm (C°)e

P100a 5′-GGCCTACCCTGGGTAAGGGCCTGGAGCAGGA-3′ –180 to –150 Long PCR 66P200a 5′-CTCAGCCTCAACGTACCCCTGTCTCAAATGCG-3′ +92 to +123 Long PCR 62CYP2D6FC1 5′-GAT GGT GGG GCT AAT GCC TTC ATG GCC ACG-3′ 1639 to 1668 ARMS1 & 2 74CYP2D6FC2 5′-CAA GAA CCT CTG GAG CAG CCC ATA CCC GCC-3′ –141 to –112 ARMS1 & 2 73CYP2D6FC3 5′-GCA AGG TCC TAC GCT TCC AAA AGG CTT TCC-3′ 2574 to 2603 ARMS1 & 2, *7 71CYP2D6FC4 5′-TCT CCT CCT TCC ACC TGC TCA CTC CTG GTA-3′ 849 to 878 ARMS1 & 2 68CYP2D6*3WT 5′-GGG GGG CTG GGC TGG GTC CCA GGT CAT CGT-3′ 2636 to 2665 ARMS1 74CYP2D6*3MU 5′-GGG GGG CTG GGC TGG GTC CCA GGT CAT CGG-3′ 2636 to 2665 ARMS2 76CYP2D6*4WT 5′-CAA GAG ACC GTT GGG GCG AAA GGG GCG TGC-3′ 1933 to 1962 ARMS1 78CYP2D6*4MU 5′-CAA GAG ACC GTT GGG GCG AAA GGG GCG TGT-3′ 1933 to 1962 ARMS2 76CYP2D6*6WT 5′-ACA AAG GCA GGC GGC CTC CTC GGT CAC CGA-3′ 1794 to 1823 ARMS2 78CYP2D6*6MU 5′-ACA AAG GCA GGC GGC CTC CTC GGT CAC CGC-3′ 1794 to 1823 ARMS1 79CYP2D6*7WTb 5′-GCT GCA CAT CCG GAT-3′ 3022 to 3036 *7 43CYP2D6*7MUb 5′-GCT GCA CAT CCG GAG-3′ 3022 to 3036 *7 46CYP2D6*8WT 5′-CGC TTT GTG CCC TTC TGC CCA TCA CCC AGC-3′ 1845 to 1874 ARMS1 76CYP2D6*8MU 5′-CGC TTT GTG CCC TTC TGC CCA TCA CCC AGA-3′ 1845 to 1874 ARMS2 75CYP2D6*11WT 5′-CTG AAC ACG TCC CCG AAG CGG CGC CGC ATC-3′ 970 to 999 ARMS2 81CYP2D6*11MU 5′-CTG AAC ACG TCC CCG AAG CGG CGC CGC ATG-3′ 970 to 999 ARMS1 82CYP2D6*12WT 5′-GAA GTC CAC ATG CAG CAG GTT GCC CAG CGC-3′ 211 to 240 ARMS1 76CYP2D6*12MU 5′-GAA GTC CAC ATG CAG CAG GTT GCC CAG CGT-3′ 211 to 240 ARMS2 74CYP2D6*14WT 5′-CGC TTT GTG CCC TTC TGC CCA TCA CCC AGC-3′ 1845 to 1874 ARMS1 76CYP2D6*14MU 5′-CGC TTT GTG CCC TTC TGC CCA TCA CCC AGT-3′ 1845 to 1874 ARMS2 74CYP2D6*15WT 5′-TGG TGT GTT CTG GAA GTC CAC ATG CAG CTG-3′ 223 to 252 ARMS1 70CYP2D6*15MU 5′-TGG TGT GTT CTG GAA GTC CAC ATG CAG CTA-3′ 223 to 252 ARMS2 69CYP2D6*20WT 5′-CCT CGC GCA GAA AGC CCG ACT CCT CCT TGA-3′ 2066 to 2095 ARMS2 75CYP2D6*20MU 5′-CCT CGC GCA GAA AGC CCG ACT CCT CCT TGG-3′ 2066 to 2095 AMRS1 76

aSachse et al. [1997].bStuven et al. [1996].c3′ allele-specific mismatches are in bold, and penultimate mismatches are underlined.dPosition according to Kimura et al. [1989].eCalculated according to Breslauer et al. [1986].

80 ROBERTS ET AL.

RESULTS

DNA Extraction

Genomic DNA was extracted by six differentmethods. DNA extracted from buccal cells provedsuitable only for PCR products of less than 500 bp,and we were unable to generate the long CYP2D6product, or obtain any result with the ARMS as-say using these DNA preparations. DNA extractedfrom peripheral blood by a rapid sodium hydrox-ide-boiling lysis method [Dracopoli et al., 1994]gave variable results in the long PCR reaction. Ofsix DNA samples prepared in this way, only threegave adequate long PCR products that yielded sat-isfactory ARMS assay results (data not shown). Inaddition to the guanidine isothiocyanate extrac-tion technique routinely used in our laboratory[Ciulla et al., 1988], two other methods yieldedgood quality genomic DNA from peripheral blood,suitable for generation of CYP2D6 genotypes bythe ARMS assay. These methods were a protein-ase K-phenol-chloroform extraction [Kennedy etal., 1989] and sodium chloride extraction [Lahiriand Nurnberger, 1991] (Fig. 3a, assay 6 and 7, re-spectively). Finally, DNA prepared by use of FTAGeneCards was of sufficient quality to produce theCYP2D6 long PCR product and successful ARMSresults (Fig. 3a, assay 2). A hot-start strategy wasessential when card-bound templates were used forthe long PCR, although this was not necessary fortemplate DNA prepared directly from peripheralblood by the other methods.

Development of Multiplex ARMS

The CYP2D6 PM ARMS assay was developedusing as template the CYP2D6-specific long PCRproducts generated with primers P100 and P200[Sachse et al., 1997] (Table 1). The final design ofthe assay includes four forward (common) prim-ers and 10 pairs of allele-specific reverse primers(Fig. 1 and Table 1). The choice of primers wasrestricted by the desire to obtain products of anappropriate size for the multiplex format, andwhere necessary selection of primers was aided byuse of the Wisconsin Sequence Analysis Package(GCG, Madison, Wisconsin). The positions of thecommon primers were chosen so all products wereless than 500 bp, and where possible were sepa-rated by more than 50 bp for ease of resolution ona 3% agarose gel (Fig. 1, Table 1, and Table 2).Penultimate 3′ mismatches were incorporated inthe allele-specific primers to ensure allele-specificamplification [Kwok et al., 1990].

Each allele-specific PCR reaction was optimized

FIGURE 2. Outline of the approach developed here forCYP2D6 PM genotyping. Methods are described in thetext. The left gel panel illustrates the pattern obtained af-ter PCR on wild type (*1/*1) DNA with ARMS1 (left lane)and ARMS2 (right lane). The right gel panel illustrates thepattern obtained with the *7 ARMS PCR reactions for anindividual who is heterozygous for the *7 allele (*1/*7).All bands illustrated in ARMS1 and ARMS2 are for wildtype alleles. Mutations would appear as bands at the cor-responding position in the other lane (e.g., a *4 mutationwould appear in the ARMS2 lane; see Fig. 3b). *19 allelesare detected as a downward displacement of the *3 wildtype fragment detected by ARMS1 (see Fig. 3b). Electro-phoresis was carried out at 100V, in 3% agarose-TBE gelsconsisting of 3:1 NuSieve GTG and UltraPure agarose,stained with ethidium bromide.

Generation of CYP2D6*14 and CYP2D6*20

Positive Control Templates

We were unable to obtain patient DNA con-taining the rare *14 or *20 alleles. In order to testthe specificity of our ARMS assay for these alle-les, we generated DNA templates containing eachof these mutations. This was achieved by carryingout PCR on a wild type CYP2D6 long PCR prod-uct with the mutation-specific primers (eitherCYP2D6*14MU or CYP2D6*20MU) and forwardprimer CYP2D6FC1, under conditions that en-couraged mispriming on the wild type templateDNA by the mutation-specific primers. An an-nealing temperature of 55°C was used for bothPCR reactions, with 25 cycles, to produce tem-plates that mimicked the heterozygous conditionfor both mutations. We refer to these as “mock”templates. The resulting products were diluted inTE (pH 8.0) and used as templates in the ARMSassay.

Ethical approval to carry out all assays on pa-tient material was obtained from the SouthernRegional Health Authority Ethics Committee(Canterbury, New Zealand).

MULTIPLEX PCR FOR CYP2D6 81

FIGURE 3. a: Effectiveness of the ARMS assay with Taq DNA polymerase from various suppliers, and different DNA prepa-rations. Each pair of lanes contains the products of ARMS 1 (left lane) or ARMS 2 (right lane). Assays 1, 2, 6, and 7, TaqDNA polymerase (Roche Molecular Biochemicals); assay 3, Taq DNA polymerase (Promega); assay 4, Taq DNA polymerase(Pharmacia); assay 5, Platinum® Taq DNA polymerase (Gibco BRL). The DNA used for assay 2 was extracted from FTAGeneCard™. DNA for assays 1, 3,4, and 5 was extracted by the guanidine isothiocyanate method [Ciulla et al., 1988]; forassay 6, by proteinase K-phenol-chloroform extraction [Kennedy et al., 1989]; and assay 7, by sodium chloride extraction[Lahiri and Nurnberger, 1991]. The same homozygous wild type (*1/*1) template was used in all reactions. b: Use of theARMS assay to detect a range of mutations. Each pair of lanes contains the products of ARMS 1 (left lane) or ARMS 2 (rightlane). The genotype of each individual is shown above the lanes, and bands specific for mutations are indicated by arrows.White arrows indicate the *4-specific band in compound heterozygotes. The patterns obtained with *14 and *15 alleles arenot shown as these are identical to the *8 and *12 alleles respectively. The gel used to resolve the 3 bp deletion characteristicof *19 was run for approximately 10–15 minutes longer than the other gels in this figure. Approximate position of productscorresponding to each allele are indicated to the right of the gels. Large PCR products indicated on all gels result fromamplification between reverse primers and more than one forward primer, as described in the text. Electrophoresis wascarried out at 100V, in 3% agarose-TBE gels consisting of 3:1 NuSieve GTG and UltraPure agarose, stained with ethidiumbromide. Molecular weight markers (M) are a 25 bp DNA molecular weight ladder (Gibco BRL).

TABLE 2. Details of the Multiplex ARMS Assay, and Reported CYP2D6 Allele Frequencies

PCR Frequency inAllele Mutation Primer pair product (bp) Caucasians (%) Reference

CYP2D6*3 2637delA FC3 and 3WT/3MU 92 1.0–3.0 Sachse et al. [1997]CYP2D6*4 1934G>A FC1 and 4WT/4MU 324 18.0–23.0 Sachse et al. [1997]CYP2D6*6 1795delT FC1 and 6WT/6MU 185 0.5–2.0 Sachse et al. [1997]CYP2D6*7 3023A>C FC3 and 7WT/7MU 463 0.0–0.5 Sachse et al. [1997]CYP2D6*8 1846G>T FC1 and 8WT/8MU 236 0.0–0.3 Sachse et al. [1997]CYP2D6*11 971 G>C FC4 and 11WT/11MU 151 0.0–0.3 Sachse et al. [1997]CYP2D6*12 212G>A FC2 and 12WT/12MU 382 0.0–0.3 Sachse et al. [1997]CYP2D6*14 1846G>A FC1 and 14WT/14MU 236 0.0–0.3 Sachse et al. [1997]CYP2D6*15 226-227insT FC2 and 15WT/15MU 394 0.0–0.5 Sachse et al. [1997]CYP2D6*19 2627-2630del FC3 and 3WT/3MU 89 0.0–0.7 Marez et al. [1997]CYP2D6*20 2067T>C FC1 and 20WT/20MU 457 0.0–1.0 Marez-Allorge et al. [1999]

separately for MgCl2 and annealing temperature.The multiplex assay was established by choosingconditions that represented the best compromisefor all reactions. In addition, the amount of TaqDNA polymerase in each reaction was increasedto 2.5 U, and the extension time was increased to40 sec to ensure amplification of the larger frag-

ments. The specificity of the reactions was im-proved by reducing the number of cycles to 14.Once the individual reactions were multiplexed itwas necessary to increase the relative yield of theCYP2D6*11 and CYP2D6*20 allele PCRs by dou-bling the concentration of both forward primers(CYP2D6FC4 & CYP2D6FC1) and the four

82 ROBERTS ET AL.

CYP2D6*11 and CYP2D6*20 reverse primers.Primers of several lengths were tested (data notshown), and those of less than 28 bases generatednon-specific products in the multiplex ARMS for-mat. In contrast, primers of 30 bases containing a3′ penultimate base mismatch were found to havehigh specificity (Figs. 2 and 3). Use of less than 2.5U of Taq DNA polymerase resulted in loss of the*4 and *20 products (data not shown).

Assembly of the multiplex reactions was de-signed to ensure that both PCR reactions (ARMS1and ARMS2) contained internal positive controls.To achieve this, primers specific for wild type alle-les were distributed between both reaction tubes(Table 1), ensuring that each multiplex PCR al-ways gave a characteristic pattern of bands thatvaried only slightly depending on the presence ofalleles detected by the mutation-specific primers(Figs. 2 and 3). Primers specific for *19 were notrequired, as this mutation is a 3 bp deletion whichcharacteristically alters the mobility of the *3 frag-ments (Fig. 3b).

DNA products of greater than 1 kb (indicated onfigures as “large PCR products”) occur in bothARMS1 and ARMS2, presumably because of read-through of some reverse primers to the more upstreamforward primers (Figs. 2 and 3). These products oc-cur consistently and, because they are significantlylarger than any of the true ARMS products, they donot interfere with the assay readout.

Despite repeated attempts with various prim-ers the allele-specific PCR reaction for theCYP2D6*7 allele could not be incorporated intothe multiplex system. All primer combinationstested, regardless of primer and product lengths,produced non-specific bands or did not distinguishbetween the wild type sequence and the mutantCYP2D6 sequence. Therefore, screening forCYP2D*7 must be carried out independently ofthe ARMS assay (Fig. 2), using the common primer(CYP2D6FC3) and the allele-specific primers pre-viously described [Stuven et al., 1996].

ARMS Assay Specificity and Reliability

The specificity of each reaction in the multi-plex ARMS assay was tested using a panel of ge-nomic DNA samples from patients containingvarious genotypes, some of which are illustratedin Figure 3b. Because patient DNA containing ei-ther *14 or *20 was not available, we used mocktemplates (generated as described above) to testthe ability of the ARMS assay to detect these mu-tations (Fig. 3b).

The accuracy of the multiplexed assay was

tested on 100 patient DNA samples previouslygenotyped by established PCR-RFLP analyses[Sachse et al., 1997]. Of the CYP2D6 genotypescapable of ascertainment with the ARMS assay,the distribution in this sample was *1/*4 (17%),*4/*4 (5%), *1/*3 (2%), and *1/*6 (3%). Singlecopies of *3, *4, and *6 were present in a further16% of patients, who were also heterozygous forthe common *2 allele. Our multiplex ARMS as-say detected all *3, *4, and *6 alleles in this samplewith 100% accuracy (representative data shownin Fig. 3b). One *10 and seven *5 alleles were de-tected by followup PCR-RFLP and Southern blotanalysis in this sample. The remaining patientswere all extensive metabolizers, either heterozy-gous or homozygous for the *2 allele, detected byfollowup PCR-RFLP analysis.

The ability of the multiplexed assay to functionin a range of PCR conditions was tested (data notshown). We found that adequate results were ob-tained with annealing temperatures ranging from55–75°C, and in MgCl2 concentrations of 1.4–2.0mM. However, below 1.4mM, MgCl2 productsfor CYP2D6*3 and *4 were lost.

We also tested the ability of the assay to func-tion with a range of different commercially avail-able enzymes. Trials of four commercially availableTaq DNA polymerase systems were conducted inthe multiplex ARMS assay. These were Taq DNApolymerase (Roche Molecular Biochemicals), TaqDNA polymerase (Promega), Taq DNA poly-merase (Pharmacia), and Platinum® Taq DNApolymerase (Gibco BRL). ARMS1 and ARMS2were carried out as described in methods and ma-terials, substituting only the polymerase and thebuffer supplied by each enzyme manufacturer. Allthermostable DNA polymerases tested producedreadable, reproducible results under the same PCRconditions (Fig. 3a).

DISCUSSION

Accurate and reliable detection of CYP2D6poor metabolizers is a valuable tool for pin-point-ing individuals who are likely to have either ad-verse reactions or receive no therapeutic benefitfrom a particular drug. In addition, retrospectivegenotype analysis of individuals who show atypi-cal drug reactions can be informative and useful.The ability to apply a simple, robust, rapid, andeconomical assay would facilitate investigation ofCYP2D6 genotypes in clinical practice.

Currently, 14 PM alleles have been found inCaucasians. Eleven of these variants consist ofpoint mutations or small deletions that can be

MULTIPLEX PCR FOR CYP2D6 83

readily detected using allele-specific PCR orARMS. Here we present a multiplex ARMS assayconsisting of two PCRs that simultaneously iden-tifies 10 of these PM alleles. This assay, used incombination with an additional allele-specific PCRand a Southern blot, detects all PM alleles de-scribed in Caucasians. This approach obviates theneed for RFLP analysis for all except the CY-P2D6*5, *13, and *16 alleles, and reduces thenumber of required PCRs and electrophoreticanalyses substantially. Furthermore, the design ofthe assay ensures that both multiplex PCRs arecontrolled internally against failure of the reaction,as each detects a proportion of the wild type alle-les in addition to specific mutations (Fig. 3b).

The quality of the patient DNA proved to be akey limiting factor in reliably assessing CYP2D6genotype. The long PCR step proved very sensi-tive to DNA quality, and required DNA that wasnot degraded. We found that poor genomic DNApreparations capable of giving excellent results withshort (less than 900 bp) PCR products often failedto give a long CYP2D6 PCR product. DNA ex-tracted from buccal samples could not be used suc-cessfully in the assay, and DNA samples preparedby a rapid sodium hydroxide-boiling lysis method[Dracopoli et al., 1994] gave variable results, withonly three of six samples proving of sufficient qual-ity to generate satisfactory assay products. How-ever, DNA prepared by guanidine isothiocyanateextraction [Ciulla et al., 1988] (Fig. 3a, assays 1,3, 4, 5), proteinase K-phenol-chloroform extrac-tion [Kennedy et al., 1989] (Fig. 3a, assay 6), andsodium chloride extraction [Lahiri and Nurn-berger, 1991] (Fig. 3a, assay 7) all gave high qual-ity DNA very suitable for use in the ARMS assay.In addition, DNA extracted from blood spots onFTA GeneCard™ was perfectly acceptable, evenafter nine months storage at room temperature(Fig. 3a, assay 2).

An earlier report [Stuven et al., 1996] describedan ARMS assay which detected the five most com-mon CYP2D6 alleles (*3, *4, *6, *7, *8). In ourhands, this assay was somewhat prone to give non-specific bands, and did not seem particularly ro-bust. We found that increasing the length of theprimers to 30 bases and introducing a 3′ penulti-mate mismatch into each primer achieved substan-tially improved specificity and performance. Theassay we developed consists of a pair of multiplexPCR reactions called ARMS1 and ARMS2:ARMS1 detects *6, *11, *19, and *20 mutations,and ARMS2 detects *3, *4, *8, *12, *14, and *15mutations (Figs. 2 and 3). This ARMS assay func-

tions well with different preparations of Taq DNApolymerase, over a range of annealing tempera-tures (55–75°C) and MgCl2 concentrations (1.4–2.0mM), suggesting it should be readily adaptableto other laboratory settings.

The allelic variants CYP2D6*8 and CY-P2D6*14 are defined by different base substitu-tions that occur at nucleotide number 1846.Specific primers for both variants are included inthe multiplex ARMS assay reactions, and eithervariant will generate a band of 236 bp (Fig. 3b). Inorder to distinguish the variants, separate PCRsspecific for CYP2D6*8 and *14 can be performedfor any DNA sample that generates the 236 bpband. As both of these alleles are rare (Table 2),this is an infrequent occurrence.

The CYP2D6*12 and *15 mutations are sepa-rated by only 12 bp, making it difficult to distin-guish between a sample carrying either of thesealleles (Fig. 3b). When a band of this size occurs,the specific reactions for these two mutations canalso be repeated separately to identify which ispresent. Again, these alleles are rare (Table 2) andthis is only occasionally necessary.

The CYP2D6*7 allele is defined by the singlepoint mutation 3023A>C. Attempts to incorpo-rate this assay in the multiplex ARMS format ledto non-specific bands, despite trying three differ-ent mutation-specific primers. The reason for thisintractability is unclear. The CYP2D6*7 primersand product show no obvious unusual features suchas internal complementarity, 3′ end overlap, orhomology with known human repetitive DNA se-quences. Therefore, we screen for CYP2D6*7 al-leles in a separate ARMS PCR using the commonprimer CYP2D6FC3 and the allele-specific reverseprimers previously described [Stuven et al., 1996].

Allele-specific primers were not required forCYP2D6*19 because the 3 bp deletion that de-fines this variant is contained within the CY-P2D6*3 product. When visualised on a 3%NuSieve gel, presence of the CYP2D6*19 allelecan be distinguished as altered mobility of the *3product (Fig. 3b). On routine gel analysis, this al-lele shows up as a “fuzzy” or larger band when com-pared with ARMS assays of patients that lackCYP2D6*19. The two bands can be clearly re-solved by continuing electrophoresis for an addi-tional 10–15 minutes.

In practice then, for each DNA sample we ap-ply the two multiplex ARMS reactions (ARMS1and ARMS2), and the additional two ARMS PCRreactions for CYP2D6*7 (Fig. 2). The three re-maining PM alleles not detected in these assays

84 ROBERTS ET AL.

(CYP2D6*5, CYP2D6*13, and CYP2D6*16) in-volve major gene deletions or rearrangementswhich are detected using Southern blots or longPCR methods [Skoda et al., 1988].

In conclusion, we have improved and consoli-dated the range of existing genetic assays forCYP2D6 PMs, allowing the detection of a widerange of alleles in a simple, robust, and economicaltest that is amenable to automation. The assay caneasily be completed in one working day, once DNAhas been prepared. Although many of the allelesdetected by this assay are quite rare, we anticipatewidespread application of the assay in the followupof patients who show unusual drug responses. Insuch selected groups, it will be advantageous to de-tect the widest possible range of PM mutations withthe minimum of effort and expense.

ACKNOWLEDGMENTS

We thank Dr. Christopher Sachse for providingCYP2D6*15 control DNA, Dr. Franck Broly forCYP2D6*8, *11, *12, *19 control PCR samples,and Dr. Ulrich Griese for CYP2D6*7 control DNA.Buccal DNA samples were kindly provided by Dr.Peter Crossen and Mary Morrison. Rebecca Rob-erts is a Leslie Averill Fellow of the CanterburyMedical Research Foundation, and Dr. Kennedyis a Senior Research Fellow of the Health ResearchCouncil of New Zealand.

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