Balkan Journal of Medical Genetics

EFFECTS OF CYP2C19 AND P2Y12 GENE POLYMORPHISMS ON CLINICAL RESULTS OF PATIENTS USING CLOPIDOGREL AFTER ACUTE ISCHEMIC CEREBROVASCULAR DISEASE
Sen HM1,*, Silan F2, Silan C3, Degirmenci Y4, Ozisik Kamaran HI1
*Corresponding Author: Halil Murat Sen, M.D., Department of Neurology, School of Medicine, Çanakkale Onsekiz Mart Üniversity, Barboros Mah., Terzioðlu Kampüsü, Týp Fakültesi, Çanakkale, Turkey. Tel : +90-286-218-37-38. Fax : +90-286-218-00-18. E-mail: hmuratsen@gmail.com
page: 37

PATIENTS AND METHODS

Patients. The patient group comprised of patients who began prophylactic clopidogrel 75 mg/day as a result of acute ICVD in the previous 2 years. All patients were monitored by the Neurology Outpatient Clinic at Çanakkale Onsekiz Mart Üniversity Research Hospital, Çanakkale, Turkey. Those who had been monitored for at least 1 year were included in the study. Patients who stopped attending our clinic, or who did not take their medication regularly were not included in the study. The study was approved by the Institutional Review Board. Detection of the CYP2C19 Genotype. Venous blood samples (2 mL) were collected from each patient in EDTA tubes. Genomic DNA was extracted from the whole blood using a high-pure template preparation kit (Roche Diagnostics GmbH, Mannheim, Baden-Württenberg, Germany). CYP2C19 alleles were detected by specific probes in Lightmix for the detection of human CYP2C19*1 (wild type allele), CYP2C19*2 (rs4244285) and CYP2C19*3 (rs4986893). Detection reagent (TIBMOLBIOL GmbH, Berlin, Germany) by real-time polymerase chain reaction (RT-PCR) (LightCycler 2.0; Roche Diagnostics GmbH), according to the manufacturer’s recommendations. The G681A point mutation in exon 5 of CYP2C19*2 and G636A transition in exon 4 of CYP2C19*3 were detected. The genotypes were identified by running a melting curve analysis with specific melting points (Tm). The wild type CYP2C19*1 exhibits a Tm 54.4 °C at channel 530 and Tm 53.4 °C at channel 640. The allele variant CYP2C19*2 exhibits a Tm of 48.6 °C at channel 530 and the allele variant CYP2C19*3 exhibits a Tm of 60.8 °C at channel 640. Detection of the P2Y12 Genotype. The 52 (G>T) (rs6809699) and 34 (C>T) (rs17602729) polymorphisms of the P2Y12 gene was analyzed with the PCR-RFLP (restriction fragment length polymorphism) method. The primer sets used were: 5’-AAT AAT AAT TCA CCT CTG CGC CCG G- 3’/5’-CCG GAT TTG AAA GAA AAT CCT CA-3’ for the 52 (G>T) polymorphism, and 5’-TTT AGA GGA GGC TGT GTC CAA-3’/5’-AAT AAT GTT ACC AGG CGC AGA GGT GAA-3’ for the 34 (C>T) polymorphism. The PCR was performed with 25 μL DreamTaq Green PCR master mix (Thermo Scientific, Pittsburgh, PA, USA) with 5 μL (75 ng) DNA, 1 μm of forward and reverse primers and PCR grade water in a total reaction volume of 50 μL. An ABI PRISM™ 9700 thermal cycler (Applied Biosystems, Grand Island, NY, USA) was used for the PCR reactions. The thermal cycling conditions were: an initial denaturation step at 94 °C for 3 min. and 35 cycles at 94 °C for 20 seconds, 57 °C for 20 seconds, and 72 °C for 25 seconds; a final extension was performed at 72 °C for 3 min. An SmaI enzyme (Thermo Scientific) was used for digestion of the amplification product for the detection of the 52 (G>T) polymorphism. The PCR product used to detect the 34 (C>T) polymorphism was digested with Tsp509 I (synonime TasI) (Thermo Scientific). The products were sizefractionated on a 2.0% agarose gel. The study included patients monitored for acute ICVD in the previous 2 years and monitored by our clinic for at least 1 year. All patients included in the study began clopidogrel after acute ICVD. A total of 51 patients [21 males (41.17%) and 30 females (58.83%)] were included. Their average age was 66.4 ± 9.6 years. When the *1, *2, and *3 alleles of CYP2C19 were evaluated, two patients were homozygous for *2/*2, 13 patients were heterozygous for *1/*2 and 36 patients were homozygous for the wild type *1/*1 alleles. No patient carried the *3 allele. Three heterozygous patients, one for *2/*2 and two for *1/*2, stopped clopidogrel due to repeated strokes and began to take warfarin. These patients had no previous history of warfarin use. When the patients’ alleles were evaluated in the group without recurring ischemic stroke, the *2 allele frequency was 13.54%. In the recurring ischemic stroke group, the *2 allele frequency was 66.7%. In the relative risk calculation of the recurring ischemic stroke group, the odds ratio (OR) was identified as 13.23 [95% confidence interval (95% CI) 6.45-27.11], which was significant at p <0.0001 (Table 1). When the P2Y12 52 (G>T) and 34 (C>T) polymorphisms were evaluated, all alleles were of the wild type.

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