RESUMEN
Conversion of clopidogrel (Plavix) to its active metabolite is catalyzed largely by the P450 enzyme 2C19 (CYP2C19). Numerous allelic variants of CYP2C19 exist. The *1 allele is considered wild type, whereas the *2 and *3 alleles have no in vivo enzymatic activity. Conversely, the *17 allele has increased expression, resulting in increased clopidogrel activation. Poor metabolizers (*2/*2 and *2/*3 genotypes) experience higher rates of therapeutic failure. For this reason, we have validated a CYP2C19 genotyping assay for the *1, *2, *3, and *17 alleles. Genomic DNA extracted from 30 deidentified EDTA whole-blood samples from patients was analyzed at 2 independent facilities using specific TaqMan realtime polymerase chain reaction primers and probes. Concordant genotypes were generated on all samples tested. Of the 30 samples, 15 were CYP2C19*1/*1, 8 were CYP2C19*1/*17, 5 were CYP2C19*1/*2, and 2 were CYP2C19*2/*17. There were no CYP2C19*3 alleles or *2/*2 homozygous genotypes detected. This CYP2C19 genotyping assay is appropriate for clinical testing, demonstrating excellent interlaboratory concordance, enabling the selection of the most effective clopidogrel treatment regimen for patients undergoing percutaneous coronary intervention.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/genética , Técnicas de Genotipaje/métodos , Polimorfismo Genético , Síndrome Coronario Agudo/tratamiento farmacológico , Síndrome Coronario Agudo/enzimología , Síndrome Coronario Agudo/genética , Hidrocarburo de Aril Hidroxilasas/sangre , Clopidogrel , Citocromo P-450 CYP2C19 , ADN/análisis , Genoma Humano , Genotipo , Humanos , Inhibidores de Agregación Plaquetaria/uso terapéutico , Ticlopidina/análogos & derivados , Ticlopidina/uso terapéuticoRESUMEN
Isolation of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue remains a laborious task for clinical laboratories and researchers who need to screen several samples for genetic variants. The objective of this study was to evaluate DNA isolation methods from FFPE tissues and to choose an efficient method with less hands-on time to obtain DNA of optimum concentration and purity for use in routine molecular diagnostic assays. Three methods were compared in this study: Gentra Puregene Tissue Kit, EZ1 DNA Tissue Kit and QIAamp FFPE Tissue Kit. Samples consisted of FFPE tissues of head/neck and lung tumor resections. Quality control for the extraction process end product included determination of the concentration and purity of isolated DNA and the ability to amplify a housekeeping gene, GAPDH, using real-time PCR assay. The hands-on-time required was less for the EZ1 protocol compared to the other methods. The average DNA concentration obtained was 112, 61 and 40 ng/µl, respectively, for the Gentra Puregene Tissue Kit, Qiagen EZ1 DNA Tissue Kit and QIAamp FFPE Tissue Kit. The purity and quality of samples obtained using the different DNA isolation methods were comparable. Comparative evaluation of three DNA isolation methods indicated that the Qiagen EZ1 method surpassed the other methods with reduced hands-on-time to produce optimum concentration of quality DNA for use in routine molecular analyses.