Background: Pharmacogenetic testing for drugmetabolizing enzymes is not yet widely used in clinical practice. Methods: In an attempt to facilitate the application of this procedure, we have compared two real-time PCRbased methods, the TaqMan_ and the LightCycler_ for the pharmacogenetic evaluation of CYP2C9*2/*3 polymorphisms. Results and Conclusion: Both procedures are suitable for pharmacogenetic studies. The TaqMan procedure was less expensive in terms of cost per sample, but the TaqMan apparatus is more expensive than the LightCycler apparatus

C. ESPOSITO

M. TORIELLO

MAZZACCARA, CRISTINA

P. MECCARIELLO

R. DI FIORE

SACCHETTI, LUCIA

English

Archivio della ricerca - Università degli studi di Napoli Federico II

Clin Chem Lab Med 2006;44(3):285–287  2006 by Walter de Gruyter • Berlin • New York. DOI 10.1515/CCLM.2006.049 2006/278Article in press - uncorrected proofShort CommunicationComparison of the TaqMan and LightCycler systems inpharmacogenetic testing: evaluation of CYP2C9*2/*3polymorphismsMario Toriello1, Pasquale Meccariello2, CristinaMazzaccara1,3, Rosanna Di Fiore1,3, CarmelaEsposito2 and Lucia Sacchetti1,3,*1 CEINGE–Biotecnologie Avanzate S.C.a r.l., Naples,Italy2 Dipartimento Clinico di Medicina Interna, Geriatria,Patologia Cardiovascolare e Immunitaria eCardiochirurgia,3 Dipartimento di Biochimica e BiotecnologieMediche, Università di Napoli Federico II, via S.Pansini 5, Naples, ItalyAbstractBackground: Pharmacogenetic testing for drug-metabolizing enzymes is not yet widely used in clini-cal practice.Methods: In an attempt to facilitate the application ofthis procedure, we have compared two real-time PCR-based methods, the TaqMan and the LightCyclerfor the pharmacogenetic evaluation of CYP2C9*2/*3polymorphisms.Results and Conclusion: Both procedures are suitablefor pharmacogenetic studies. The TaqMan procedurewas less expensive in terms of cost per sample, butthe TaqMan apparatus is more expensive than theLightCycler apparatus.Keywords: cytochrome P450 (CYP450); pharmaco-genetic testing; polymorphisms.Pharmacogenetic testing for drug-metabolizingenzymes is not widely used in clinical practice, prob-ably because of a poor evidence base and a slowtranslation of research results to the clinical setting(1). A major drug-metabolizing enzyme is cytochromeP450 2C9 (CYP2C9), which metabolizes over 100 cur-rently used drugs (2, 3). Over 50 single nucleotidepolymorphisms (SNPs) have been identified in theregulatory and coding regions of this gene (2). Thecoding variants CYP2C9*2 (Arg144Cys) and CYP2C9*3(Ile359Leu) reduce drug metabolism, which suggeststhat drug dosage should be appropriately tailored inpatients carrying these variants (4). CYP2C9 geno-types have been investigated mainly in relation to*Corresponding author: Lucia Sacchetti, Dipartimento diBiochimica e Biotecnologie Mediche, Università di NapoliFederico II, via S. Pansini 5, 80131 Naples, ItalyPhone: q39-081-7463532, Fax: q39-081-7462404,E-mail: sacchetti@dbbm.unina.itwarfarin, an anticoagulant widely used to preventthromboembolic events in cardiovascular disease (5).A simple, sensitive, rapid and accurate genotypetest would promote the use of pharmacogenetic test-ing in laboratory practice. The most widely usedgenotyping systems are: denaturing high-perform-ance liquid chromatography (dHPLC); polymerasechain reaction (PCR)-restriction fragment length poly-morphisms; matrix-assisted laser desorption/ioniza-tion time-of-flight mass spectrometry; pyrosequenc-ing; microarrays; and real-time PCR, each of whichhas advantages and limitations (6).We have compared the costs, analysis time, suc-cess rate and versatility of two real-time PCR-basedmethods, the TaqMan (Applied Biosystems, FosterCity, CA, USA) and LightCycler (Roche Diagnostics,Mannheim, Germany) procedures, in the identifica-tion of CYP2C9 polymorphisms. Using both instru-ments, we genotyped 114 subjects (65 patientsundergoing therapy with warfarin for over 10 yearsand 49 controls). All participants were recruited fromour University Hospital and gave informed consent tothe study. The study was approved by the EthicsCommittee of the School of Medicine of Naples Uni-versity ‘‘Federico II’’ and was conducted in accor-dance with the principles of the Helsinki II Declaration.Briefly, a fasted blood sample was collected fromenrolled subjects in the morning after an overnightfast. DNA was extracted with the Amersham Nu-cleon BACC 2 Kit (Amersham Biosciences, Europe, Lit-tle Chalfont, Buckinghamshire, England), and storedat q48C until required. Anamnestic and clinical datawere collected from all patients.The CYP2C9 *1 wild-type (wt), *2 and *3 polymor-phic alleles were genotyped with the TaqMan proce-dure (7, 8) and with the LightCycler Real Time PCR (9,10), two procedures widely used to identify SNPs. Thepre-developed TaqMan assay reagent kit contains onepair of PCR primers, one pair of fluorescent TaqManprobes, and control templates. The PCR is set up in a96-well plate with a 25-mL mix reaction and requires50 ng of genomic DNA per assay. The PCR and thesubsequent allelic discrimination were carried out onan ABI PRISM 7900 HT system (Applied Biosystems).SDS 2.1 software (Applied Biosystems) was used toanalyze the fluorescent PCR products and to genotypethe samples. The LightCycler-CYP2C9 Mutation Detec-tion Kit provides primers and hybridization probes forsequence-specific detection, pre-mixed reagents, asolution containing Fast Start Taq polymerase, and acontrol template. The PCR is set up in glass capillariesusing 20 mL of mix reaction and 50 ng of genomic286 Toriello et al.: Pharmacogenetic evaluation of CYP2C9 allelesArticle in press - uncorrected proofTable 1 Costs of analyzing CYP2C9*2/*3 polymorphisms using the LightCycler and TaqMan procedures.Cost/test, 7TaqMan LightCyclerConsumables (tips, well, 0.3661 1.2632 1.05cover or capillaries) (96-test manual kit) (384-test automated kit) (32-test kit)Reagent 6.25 1.25 13.06Total cost 6.6 2.51 14.111Cost for 96 tests in manual; 2cost for 384 tests in automation using the Biomek Workstation.Figure 1 Identification of CYP2C9 gene polymorphisms with the TaqMan (A) (PCR conditions: 508C for 2 min, 958C for 10 min,followed by 35 cycles at 928C for 15 s and 608C for 1 min) and LightCycler (B) (PCR conditions: 958C for 10 min followed by45 cycles at 958C for 10 s, 558C for 10 s, 728C for 10 s and 958C for 1 min, 408C for 1 min) systems.Toriello et al.: Pharmacogenetic evaluation of CYP2C9 alleles 287Article in press - uncorrected proofDNA, and is performed on the LightCycler system(Roche Diagnostics); the three CYP2C9 alleles areidentified based on fluorescence measurements. Thecosts and times required to isolate DNA and to set-upthe PCR mix were similar for both methods and aretherefore not included in the test comparison. In addi-tion, the times required to determine CYP2C9*2/*3genotypes after DNA isolation were comparable:106 min with the TaqMan procedure (up to 48 sam-ples) and 60 min with the LightCycler procedure (upto 32 samples). The cost per sample, including re-agents and consumables, was 76.61 for the TaqManset-up and 714.11 for the LightCycler system (seeTable 1). Moreover, the TaqMan procedure can beautomated using the 384-well apparatus and an auto-mated workstation, thereby further decreasing thecost/test. With the Biomek 2000 Workstation (Beck-man Instruments, Fullerton, CA, USA) the test/costwas 72.51. Both methods recommend a quality con-trol procedure, consisting of one negative and twopositive (mutated and wt) homozygous controls(TaqMan), or of one negative and one positive (het-erozygous mutated) sample (LightCycler).The advantages of the LightCycler were: (a) less riskof contamination because a single glass capillary isused; and (b) the cost per test is the same, irrespec-tive of the number of samples (from 1 to 32), whichis useful when dealing with a low number of samples.However, the glass capillaries are easily broken andthe cost per test is high. The advantages of theTaqMan were: (a) a low cost per test, particularly forhigh-throughput screening (from 96 to 384); and(b) possible automation that further decreases thecosts. The TaqMan procedure has the disadvantagethat it is not suitable for a low number of assays andthe open-well plate entails a risk of contamination.We tested the same sample 15 times to evaluatereproducibility of the CYP2C9 assay, and the resultsinvariably overlapped with the two instruments. Thesuccess rate, i.e., the number of runs successfullycompleted with each instrument, was 97% (36/37runs) with the TaqMan and 91% (29/32 runs) with theLightCycler method. Failures were due to non-ampli-fication of the positive control (TaqMan) and/or to thequenching of 1/2 fluorochromes (LightCycler).There was 100% concordance in the genotypingresults obtained with the two procedures. TheCYP2C9*1, CYP2C9*2 and CYP2C9*3 alleles werepresent in 80.2%, 13.1% and 6.6%, respectively, of thegenotypes, which agrees with data reported for otherItalian samples (11, 12). Both kits produced unambig-uous real-time sequence outputs (Figure 1A, B) andwere equally easy to use. The initial equipment costsvaried, depending on the model purchased, i.e.,between 738,500 and 7110,000 for the TaqMan sys-tem, and between 731,750 and 763,000 for theLightCycler system.In conclusion, the TaqMan and the LightCycler sys-tems appear to be suitable for the pharmacogeneticgenotyping of CYP2C9 gene polymorphisms. TheTaqMan procedure was less expensive in terms ofcost per sample, but the TaqMan apparatus is moreexpensive than the LightCycler apparatus.AcknowledgementsThis work was supported by grants from Centro IngegneriaGenetica (CEINGE)-Regione Campania (Convenzione-Del.G.R. 27/12/2002 No.6276), Regione Campania-AOU FedericoII, and Regione Campania/Ministero della Salute (L.229/99).We are grateful to Jean Gilder for editorial assistance andfor revising the English text.References1. Gardiner SJ, Begg EJ. Pharmacogenetic testing for drugmetabolizing enzymes: is it happening in practice? Phar-macogenet Genomics 2005;15:365–9.2. Rettie AE, Jones JP. Clinical and toxicological relevanceof CYP2C9: drug-drug interaction and pharmacogenet-ics. Annu Rev Pharmacol Toxicol 2005;45:477–94.3. Kirchheiner J, Brockmoller J. Clinical consequences of´´cytochrome P4502C9 polymorphisms. Clin PharmacolTher 2005;77:1–16.4. You JHS, Chan FWH, Wong RSM, Cheng G. The poten-tial clinical and economic outcomes of pharmacogenet-ics-oriented management of warfarin therapy – adecision analysis. Thromb Haemost 2004;92:590–7.5. Takahashi H, Wilkinson GR, Padrini R, Echizen H.CYP2C9 and oral anticoagulation therapy with acenocu-marol and warfarin: similarities yet differences. ClinPharmacol Ther 2004;75:376–80.6. Jannetto PJ, Laleli-Sahin E, Wong SH. Pharmacogeno-mics genotyping methodologies. Clin Chem Lab Med2004;42:1256–64.7. Yang JQ, Morin S, Verstuyft C, Fan LA, Zhang Y, Di XuC, et al. Frequency of cytochrome P450 2C9 allelic vari-ants in the Chinese and French populations. FundamClin Pharmacol 2003;17:373–6.8. Livak KJ. Allelic discrimination using fluorogenic probesand the 59-nuclease assay. Genet Anal 1999;14:143–9.9. Burian M, Grösch S, Tegeder I, Geisslinger G. Validationof a new fluorogenic real-time PCR assay for detectionof CYP2C9 allelic variants and CYP2C9 allelic distributionin a German population. J Clin Pharmacol 2002;54:518–21.10. Ririe KM, Rasmussen RP, Wittwer CT. Product differen-tiation by analysis of DNA melting curves during the pol-ymerase chain reaction. Anal Biochem 1997;245:154–60.11. Spreafico M, Peyvandi F, Pizzotti D, Moia M, MannucciM. Warfarin and acenocumarol dose requirementsaccording to CYP2C9 genotype in North-Italian patientswletterx. J Thromb Haemost 2004;1:2252–3.12. Scordo MG, Caputi AP, D’Arrigo C, Fava G, Spina E.Allele and genotype frequencies of CYP2C9, CYP2C19and CYP2D6 in an Italian population. Pharmacol Res2004;50:195–200.Received July 27, 2005, accepted December 1, 2005

Comparison of the TaqMan and LightCycler systems in pharmacogenetic testing: evaluation of the CYP2C9*2/*3 polymorphisms.

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Comparison of the TaqMan and LightCycler systems in pharmacogenetic testing: evaluation of the CYP2C92/3 polymorphisms.

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