Evaluation of Polymorphisms of VKORC1 and CYP2C9 in – treated patients Iranian

Supervisor: Dr.Behzad Poopak Poopak By: Fariba Rad

1 Introduction

‰ Warfarin was introduced into clinical practice in the 1950s. ‰ Today Warfarin is widely prescribed by physicians for treatment and prevention of thrombotic diseases. ‰ The Appropriate dose of warfarin is important for each patient. ‰ Anticoagulant treatment with warfarin is regularly monitored according to the international normalized ratio (INR).

2 Introduction

‰ Its anticoaggypulant effect is widely variable between patients because of environmental and genetic factors. ‰ increasing evidence supports the association of genotype and bleedi ng t end ency f oll owi ng warf ari n th erapy. ‰ Genetic variants in CYP2C9 and VKORC1 strongly affect steady- state warfarin dose.

3 cytochrome P450 2C9 enzyy(me (CYP2C9)

‰ The human CYP2C9 is on 10 (10q24.2). ‰ CYP2C9 catalyzes (90%) the metabolism of S-warfarin into 6- hydroxy-S-warfarin and 7-hydroxy-S-warfarin. ‰ The most common variants of CYP2C9 are CYP2C9*2 and CYP2C9*3.

4 cytochrome P450 2C9 enzyy(me (CYP2C9)

‰ CYP2C9*2 andCYPd CYP2C9*3 alleli c vari ants diff er f rom the w ild type allele by a single nucleotide substitution: ¾CYP2C9*2 0> T in exon 3 → C 43 ¾ CYP2C9*3

‰ SNPs in the CYP2C9→ A 1075> gene C havein exon been 7 associated with lower dose requirements compared to the wild type allele (CYP 2C9*1). ¾ CYP2C9*1 (WT) – normal ¾ CYP2C9*2 (Arg144Cys) - low/intermediate ¾ CYP2C9*3 (Ile359Leu) - low

5 epppoxide reductase complex subunit 1 gene (VKORC1)

‰ VKORC1 is another gene affecting warfarin dose. ‰ VKORC1 gene is on (p11.2). ‰ A membrane protein located in hepatocytes. ‰ VKORC1 an enzyme responsible for the regeneration of vitamin K from its oxidized form.

6 vitamin K epoxide reductase complex subunit 1 gene (VKORC1)

‰ Vitamin K in its reduced form is essential for the gamma carboxylation of several clotting factors. ‰ Warfarin inhibits VKORC 1 activity . ‰ The VKORC1 –1639G>A polymorphism results in reduced promoter activity. ‰ The VKORC1 –1639G>A polymorphism have been associated with lower dose requirements compared to the wild type allele.

7 Aim Prescreening patients for their genotypes before prescribing the drug facilitates a faster individualized determination of the proper maintenance dose, minimizing the risk for adverse reaction and reoccurrence of thromboembolic episodes .

8 Methods

‰ Population in this study ¾ 52 patients with average 26 years old were selected (minimum 5 and maximum 74 years). ¾ 42.30% female and 57.70% male. ‰ SlSample collecti on ¾ 5 ml of peripheral blood was drawn from each subject and collected into K 2EDTA (for molecular typing) . ‰ Laboratory examination 9 PCR master mix ‰ PCR master mix are shown in Table 1.

TblTable 1.shows PCR mas ter mi x PN-Mix PN 2C9/VKORC1 15μL Polymerase buffer 10x 2.5 μL Mgcl2 2.5Mm 0.25 μL Taq DNA polymerase 1unit DNA 100ng

10 PCR Program ‰ PCR Program are shiTblhown in Table 2. Table 2.shows PCR Program. 25 cycle are done according to Table 2. . PCR Program Time Temperature (°c)

Denaturation 5´ 95 ºc Annealing 30'' 55 ºc Extension 30'' 72 ºc Final extension 8' 72 ºc

11 Results

‰ Patient characteristics: ¾ 52 patiihients with average 26 years old were sel ected (minimum 5 and maximum 74 years). ¾ 42.30% female and 57.70% male.

12 Results ‰ CYP2C9 genotypes and Daily Warfarin Dosage Mean (SD) mg/day are summarized in Table 3. Table 3. The Prevalence of CYP2C9 Genotypes and Mean Daily Warfarin Dosage

CYP2C9 genotype Prevalence (%) Daily Warfarin Dosage Mean (SD) mg/day

1*1* 55.77 4.58±1.57 1*2* 21.17 2.9±1.48 1*3* 17.30 2.72±1.26 2*3* 3.84 2.65±0.67 13 2*2* 1.92 2.20±0.43 Results ‰ VKORC1 genotypes and Daily Warfarin Dosage Mean (SD) mg/day are summarized in Table 4. Table 4. The Prevalence of VKORC1 Genotypes and Mean Daily Warfarin Dosage

VKORC1 genotype Prevalence (%) Daily Warfarin Dosage Mean (SD) mg/day

GG 44.22 4.5±1.32 GA 48.08 3.07±1.95 AA 7.7 2.7±1.16

14 Discussion

‰ This stdtudy was d esi gned dti to investi titgate th thfe frequency ofCYPf CYP2C9 and VKORC1 gene polymorphisms and their relation to warfarin dose in Warfarin – treated patients Iranian. ‰ The VKORC 1G/A geno type was the most common genot ype ( 48.08)f) found di in our subjects Table5.

Table5. Distribution of VKORC1 genotypes among different ethnic groups . Genotype Caucasian European Asian China African - Iranian American GG 25 27 0 0.6 73 44.22 GA 56 50 8 15.7 27 48.08 15 AA 19 23 92 83.7 0 7.7 Discussion ‰ It was also shown in our study population. Carriers of WT VKORC1 (G/G) required a higher warfarin dose as compared to (A/A) carriers (4.48±1.32 mg and 2.7±1.16 mg, respectively; P<0.01)Table 4. Table 4. The Prevalence of VKORC1 Genotypes and Mean Daily Warfarin Dosage

VKORC1 genotype Prevalence (%) Daily Warfarin Dosage Mean (SD) mg/day

GG 44.22 4.5±1.32 GA 48.08 3.07±1.95

AA16 7.7 2.7±1.16 Discussion

‰ The CYP2C91*1* genotype was the most common genotype (55.77%) found in our subjects Table6. Table6. Distribution of CYP2C9 genotypes among different ethnic groups. CYP2C9 Italy(%) Greek(%) China(%) India(%) Iranian

1*1* 79.4 76 36.7 35 55.77 1*2* 18.8 22 38.2 55 21.17 1*3* 1.6 0 5.8 0 17.30 2*2* 0 2 11 10 3.84 2*3* 0 017 1.4 0 1.92 Discussion ‰ WT CYP 2C9*1/*1 genotype was associated with a higher daily warfarin dosage (4.58±1.75 mg; P=0.02) as compared to other CYP2C9 genotypes (Table 3). Table 3. The Prevalence of CYP2C9 Genotypes and Mean Daily Warfarin Dosage

CYP2C9 genotype Prevalence (%) Daily Warfarin Dosage Mean (SD) mg/day

1*1* 55.77 4.58±1.57 1*2* 21.17 2.9±1.48 1*3* 17.30 2.72±1.26 2*3* 3.84 2.65±0.67 18 2*2* 1.92 2.20±0.43 Thanks