Evaluation of Polymorphisms of VKORC1 and CYP2C9 Genes in Warfarin – 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 gene is on chromosome 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 vitamin K epppoxide reductase complex subunit 1 gene (VKORC1)
VKORC1 is another gene affecting warfarin dose. VKORC1 gene is on chromosome 16 (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