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Dalal Nasser Eldin ElKaffash,Aymen Abdel Hay,Nermine Hossam Zakaria & Mounir Elhag. Role of CYP2C9 and VKORC1 polymorphism in dose dependent warfarin therapy management. Journal of Pharmaceutical and Biomedical Sciences (J Pharm Biomed Sci.) 2013 August; 33(33): 1468-1485.

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ISSN NO- 2230 – 7885 CODEN JPBSCT NLM Title: J Pharm Biomed Sci.

Dalal Nasser Eldin ElKaffash,Aymen Abdel Hay, Nermine Hossam Zakaria & Mounir Elhag

Research

article

Role of CYP2C9 and VKORC1 polymorphism in dose dependent warfarin therapy management Dalal Nasser Eldin ElKaffash1 , Aymen Abdel Hay2 ,Nermine Hossam Zakaria1 * & Mounir Elhag4 Affiliation:1 Clinical

Pathology Department, Faculty of Medicine, Alexandria University, Faculty of Medicine, Alexandria University, Egypt 2 Cardiology Department Faculty of Medicine, Alexandria University, Egypt 3 Clinical Pathology Department, Faculty of Medicine, Alexandria University Faculty of Medicine, Alexandria University, Egypt 4 Resident in Clinical Pathology Department, Faculty of Medicine, Alexandria University, Egypt Author’s contributions-All the authors contributed equally to this paper. *Correspondence to:NERMINE HOSSAM ZAKARIA. Clinical Pathology Department, Faculty of Medicine, Alexandria University Faculty of Medicine, Alexandria University, Egypt Contact no: - 00966507901308 E mail address: [email protected]

Abstract: Warfari n is the therapeutic drug of choice for treatment and maintenance of anticoagulation therapy. The dosage requi red to achiev e the therapeutic effect vari es between individuals. These differences in drug response and the narrow therapeutic window lead to increas ed risk of life threatening hemorrhagic adv erse events. The standard treatment monitoring is the international normalized ratio (INR).Warfarin activity is determined by polymorphisms in CYP29C and VKORC1 genes. The CYP2C9 gene encodes enzyme that catalyzes the conversion of Warfari n to inactive metabolites. Polymorphisms of CYP2C9 includes the variant alleles *2 and *3; which have decreas ed enzymatic activity than the wild type CYP2C9*1. When Warfarin is given to pati ents with*2or*3vari ants it will be metabolized less efficiently and will remain in ci rculation longer, so lower Warfarin dos es will be needed to achieve anticoagulation.VKORC1gene encodes the molecular target of coumarin type anticoagulant,

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vitamin Kepoxide reductase (VKORC1).It recycles vitamin K 2, 3 epoxide to vitamin K hydroquinone, which functions as the essenti al cofactor for the carboxylation of coagulation factors II, VII, IX, and X; proteins C, S. In the VKORC1 1639 single nucleoti de polymorphism, the common G allele is repl aced by the A allele. Because A allele produce less VKORC1 than do thos e with the G allele, lower Warfari n doses are needed to inhibit VKORC1 and to produce an anticoagul ant effect. The aim of this study was to assess CYP2C9 and VKORC1 polymorphisms among Egyptian patients on chronic Warfarin therapy and determine its relation to Warfarin dosing protocol; study the CYP2C9 and VKORC1 alleles and genotypes frequency among sample of Egyptian patients. Subjects and Methods : The study was conducted on forty Egyptian male patients on stable Warfarin dose with a stable INR within the therapeutic range of 2.0 - 3.0.The CYP2C9 and VKORC1 genotypes were determined by PCR amplification reverse hybridization technique. Results: The frequency of CYP2C9 genotypes were; 65% for *1/*1, 10% for*1/*2. 15% for *1/*3, 5% for *2/*2 and 5% for*3/*3, genotype *2/*3 was not detected among studied patients. CYP2C9 alleles frequencies were; 77.5% for *1 allele, 10% for *2 allele and 12. 5% for *3 allele. VKORC1 AA frequency was 7.5%, AG 65% and GG 27.5%. VKORC1 allele frequency: VKORC1 G allele 60% and VKORC1 A allele 40%. For CYP2C9 and Warfari n dose; the majority of low dose responders 76. 9% was made by CYP2C9* 3 and CYP2C9*2 while CYP2C9*1 made the rest 23.1%. In the intermedi ate dose responders, CYP2C9*1made 78. 6% and CYP2C9*2 made 21.4%, and in hi gh dose res ponder the CYP2C9*1 made 100%. As VKORC1 genotypes and Warfarin dose; the majori ty of low dose responders 76.9% was made by AG, while VKORC1 AA made 23,1%. In the intermediate dose responders, VKORC1 GG made 28.6%, AG made71.4% and AA not detected .in high dose responder the VKORC1 GG made 54.6 %, AG made 45. 4% and AA not detected. For CYP2C9/VKORC1 haplotypes and Warfarin dos e



our findings was as follows: In group I low dos e responder (Warfarin dose < 4 mg / day) *1/*3/AG haplotype made 38.5% ,*3/*3/AG haplotype made 15.4% ,*1/*1/AG haplotype made 15.4% and haplotypes *1/* 1/AA, *1/*2/AG, *1/* 3/AA and*2/*2/AA made 7.7% for each. In group II intermediate dose responders *1/*1/AG made 57.1%,*1/*1/GG 21.4%,*1/*2/AG 14.3% and *1/*2/GG made 7.1%. In High dose responders Haplotype *1/*1/GG made 63.6% and *1/*1/AG 36.4%. Conclusions: CYP2C9 common vari ant alleles CYP29C*2 and CYP29C*3 were detected in this study sample. CYP29C*2 frequency was comparable wi th Caucasians, while CYP29C*3 was higher. CYP29C*2 and CYP29C*3 were associated with lower mean daily doses of Warfarin than

CYP2C9*1 wild type. VKORC1 mutant A and wild type G alleles were detected in a frequency similar to Caucasian. Peopl e with VKORC1 mutant allele A in the pres ent study was associated with lower Warfari n dos es than wild type G. Polymorphism in CYP2C9 and VKORC1 genes significantly affect individual response to Warfarin therapy, and could explai n some of interi ndividual vari ations in Warfari n therapy.

Key

words : Warfarin, vitamin Kepoxide reductase (VKORC1), INR; International Normalized Ratio, PCR amplification revers e hybridization technique.

Article citation:Dalal Nasser Eldin ElKaffash,Aymen Abdel Hay, Nermine Hossam Zakaria & Mounir Elhag. Role of CYP2C9 and VKORC1 polymorphism in dose dependent warfarin therapy management. Journal of Pharmaceutical and Biomedical Sciences (J Pharm Biomed Sci.) 2013 August; 33(33): 1468-1485.Available at http://www.jpbms.info

INTRODUCTION

W

arfarin (Coumadi n®) as Vitami n K antagonists is the most commonly oral anticoagulant worldwide1. The aim of the anticoagulation is to administer the appropriate anticoagulant in the correct dose to produce the desired therapeutic effect with minimum toxicity 2. The dosage requi red to achieve the desired therapeutic effect varies up to 120 folds between individuals. Currently the choice of the drug and dose are based on the trial and error basis with a wide range of efficacy and side effects. In addition, the management therapy is complicated with inter-i ndividual differences in drug respons e, delayed onset of action and narrow therapeutic window leading to increas ed risk of life threatening hemorrhagic adverse events or thromboembolism 2. Furthermore, the determination of safe and effective loading dose during the early phas e of therapy and maintenance doses, require frequent laboratory moni toring and adjus tment to compens ate for changes in patients' age, Body Mass Index (BM I), dietary vitamin K intake, disease status, comorbidi ties, concomitant use of other medications, and patient specific genetic factors 3. While treatment monitori ng remains the international normalized ratio (INR), individual patient variabilities and responsiveness to treatment remain a challenge. Cytochrome P4502C9 (CYP2C9) and Vitamin K Epoxide Reductase complex 1 (VKORC1) gene polymorphisms are associated with v ariable drug respons es 4. These two genes, (CYP2C9 and VKORC1) encode enzymes involved i n Warfarin metabolism and regulation. Current evidence is clear that polymorphisms in either CYP2C9 or VKORC1 genes affect Warfarin sensitivity. Different studies aimed to use pharmacogenetic information’s in the clinical practice which may lead to rapid, efficient, optimum and safe Warfarin dosing. Warfarin ph armacokin etics Warfari n is a synthetic derivative of Coumarin, a compound found naturally in many plants, notably woodruff (Galium odoratum,

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Rubiaceae), and at lower levels in licorice, lavender5. D espite its effectiveness, treatment wi th Warfari n has several shortcomings. Many commonly used medications interact wi th Warfari n, as do some foods, and its activity has to be monitored by frequent blood tes ting for the INR to ensure that an adequate safe dose is taken6. The av erage half life of Warfarin was found to be 36 hours in heal thy volunteers. A range of 20 to 80 hours has been reported3. Warfari n is metabolized in the liver, but the liver enzymes mediating its metabolism differ completely between the two Warfari n enantiomers. While SWarfari n is almost exclusively metabolized by the CYP2C9 enzyme, R-Warfarin is metabolized by a wide range of cytochrome P450 enzymes, including CYP3A4, CYP2C19 and CYP1A27. Following transformation into a number of hydroxylated or reduced watersoluble metabolites, Warfarin is excreted i n urine (80%) and faeces (20%), while the excretion



of non metabolized Warfarin is negligible8. Mechanism of action of Warfarin: Warfari n is an antagonist of vitamin K which is a necessary element in the synthesis of clotti ng factors II, VII, IX and X, and proteins C and S. These factors are biologically inactive wi thout the carboxylation of certain glutamic acid residues. This carboxylation process requires a reduced vitamin K as a cofactor. Antagonism of vitamin K by Warfarin or deficiency of this vitami n reduces the rate at which these factors and proteins are produced, thereby creating a state of anticoagulation9. Warfarin ph armacodynamics: 1. Anticoagulant Activity: The anticoagulant activity of Warfari n depends on the clearance of functional clotting factors from the systemic circulation after administration of the dos e (10). The clearance of these clotting factors is determined by their half-lives. The earliest changes in the INR are ty pically noted 24 to 36 hours after a dose of Warfari n is administered. Thes e changes are due to the clearance of functional factor VII, which is the vitamin K-dependent clotti ng factor wi th the shortest half-life (six hours)10. 2. Antithro mbotic Effect: The antithrombotic effect of Warfarin is not present until approximately the fifth day of therapy. This effect depends on the clearance of functional factor II (prothrombin), which has a half -life of approximately 50 hours in pati ents with normal hepatic function. Becaus e antithrombo tic effect depends on the clearance of prothrombin (which may take up to five days), loading doses were of limited value.;because Warfarin has a long half-life, increases in the INR may not be noted for 24 to 36 hours after administration of the firs t dose, and maximum anticoagulant effect may not be achieved for 72 to 96 hours 10. Warfarin dose adjustment (Loading and mainten ance doses of Warfarin): Dosing of Warfarin is complicated by the fact that it is known to interact with many commonly used medications and even with chemicals that may be present in certain foods 11. These interactions may enhance or reduce Warfari n's anticoagulation effect. In order to optimize the therapeutic effect wi thout risking dangerous side effects such as bleeding, close monitori ng of the degree of anticoagulation is requi red by blood tes ting of the INR. During the initial stage of treatment, checking may be required daily; intervals between tests can be lengthened if the pati ent manages stable therapeutic INR levels on an unchanged Warfarin dose. The target INR level will vary from case to case depending on the clinical indicators, but tends to be 2–3 in most conditions.

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The usual loading doses of Warfari n are 10 mg per day depending on the clinical indication. This may increase the pati ent's risk of bleeding episodes early in therapy by eliminating or severely reducing the production of functional factor VII. The administration of loading doses is a possible source of prolonged hospitalization s econdary to dramatic rises in INR that necessitate increas ed monitori ng12.Administration of loading doses has also been hypothesized to potentiate a hypercoagulabl e state because of severe depletion of protein C (approximate half-life of eight hours) during the first 36 hours of Warfarin therapy 13. Adjusting the maintenance dose of Warfarin is recommended by many international bodies includi ng the American College of Chest Physicians which hav e been distilled to help manage dose adjustments. Although Warfarin has poorly predictable pharmacoki netics, many physicians continue to us e clinical judgment alone as the basis for initiating and adjusti ng the dose. A number of studies have validated approaches to initiate anticoagulation that provide more rapid effect wi th less chance



of complications. Algorithms for commencing Warfarin treatment have been proposed by several researchers, for exampl e: 1.The Kov acs 10 mg algorithm 14. 2.The Fennerty 10 mg regimen for urgent anticoagulation 15 3. The Tait 5 mg regimen for "routine" (low-risk) anticoagulation16. 4. Derived and prospectively validated a model including CYP29C and VKORC1 genotypes. This model could predict 70% of the variation in Warfarin doses in a v alidation cohort (versus 48% without genotype). The pharmacogenetics protocol leads to a significant reduction in out of range INR values 17. Warfarin laboratory monitorin g: Laboratory monitori ng falls into two broad categories: General monitori ng and specific monitori ng. General monitoring is directed toward the assessment of bleeding or other unwanted effects of therapy, the tests i nclude hematocrit, hemoglobin, platel et count, occult blood. Specific monitori ng is di rected toward the assessment of the specific anticoagulant effects, these tests include the prothrombin time (PT) and the International Normalized Ratio (INR). Now testing the genoty ping of CYP2C9 and VKORC1 had been recommended. Why to perform Warfarin lab monitoring: The rationale for Laboratory monitoring of Warfarin is requi red for several reasons: 1.Warfarin has a relatively narrow therapeutic window. Underanticoagulation greatly reduces Warfarin’s therapeutic efficacy, while over-anticoagulation greatly increases the risk of bleeding. Severe bleeding episodes may be fatal or lead to severe morbidi ty 18. 2 .The dose-respons es between individuals are highly variable and may be quite v ariable in the same individual over time19. 3. Warfarin’s effect may be either potentiated or inhibited by a large number of medications. For example, potentiators i nclude acetaminophen, erythromycin, miconazole, propranolol, and cimetidine, and inhibitors include barbi turates, prednisone, carbamazepine, nafcillin, and cholestryramine20. 4. Warfarin’s effect is influenced by fluctuations in dietary vitamin K intake. Dietary vitamin K is obtained principally from green leafy vegetables, olive oil, soybean oil, cottons eed oil, and canola oil and to a lesser extent from butter, margarine, liver, milk, ground beef, coffee, and pears. Multivitamins and herbal remedies are additional sources of vitamin K. The half-life of vitamin K is only about 1.5 days, so continual intake is required, and changes in vitamin K intake affect the anticoagulant activity of Warfarin within days 18. 5. Coexisting diseases or illnesses may affect the absorption and metabolism of Warfarin and vitamin K and the synthesis of clotting factors. 6. The Warfarin activity is determined by genetic factors especially polymorphism in CYP29C and VKORC1 gene. The mentioned factors combine to make the laboratory monitori ng vital for the safe management of Warfari n therapy. PT and INR:

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The PT tes t is the primary assay used i n monitori ng Warfarin therapy. The prolongation of PT depends on reductions i n three of the vitamin K¬ dependent clotting factors (II, VII and IX). Changes in the PT noted in the first few days of Warfarin therapy are primarily due to reductions in factors VII and IX, which have the shortest half-lives (6 and 24 hours, respectively) .The early changes in PT vary based on the responsiveness of the particul ar thrombopl astin that a laboratory uses to perform the PT test. The International Sensitivity Index (ISI) is used to measure and compare the vari ability in thrombopl astin responsiveness. Becaus e of the variations in thrombopl astin s ensitivity and the different ways of reporting PT, information about patients treated with oral anticoagulants was not interchangeable among laboratories until 1982, when the World Heal th Organization Expert Committee on Biologic Standardization developed the INR21.When a patient is started on an oral anticoagulant, INR monitori ng should be performed on a daily basis until the INR is within the therapeutic range for at l east two consecutive days. Then INR monitoring should be performed two to three times a week for one to two weeks. If the pati ent remai ns stable, this interval can be widened to a monitori ng frequency of once every four to six weeks. If dosage adjustments are necessary, INR monitori ng should be performed more often until a new state of stability is achieved. Unexpected fluctuations of the INR in an otherwise stable pati ent should be investi gated. Often, it is possible to identify one or more causes, such as change in diet, poor compliance, undisclosed drug use, alcohol consumption and/or self-medication. If none of



thes e causes can be i dentified, l aboratory error should be considered. When no cause for INR fluctuations can be determined, weekly dosage adjus tment should be tried. The reduction or withholdi ng of a single dose or an increase in that day's dos e is often sufficient to res tore a therapeutic INR in a pati ent who is otherwise medically stable22. Pharmacogenomics CYP2C9 and VKORC1 polymorphisms: Warfari n activity is determined partially by genetic factors. The American Food and Drug Administration (FDA) highlights the opportunity for healthcare providers to use genetic tests to improve thei r initial estimate of what is a reasonable Warfari n dose for individual patients. Polymorphisms in two genes are particularly important: CYP2C9 and V KORC23. Cytochrome P450 2C9 (CYP2C9): It is evident that knowledge about polymorphic drug metabolizing enzymes may provide important i nformation for choice of drug therapy and drug dos age. Among these, cytochrome enzymes (CYPs) some of them have potential role to improve drug therapy

and achieve higher response rates and reduce adverse effects 24.The cytochrome P450 complex is a group of hepatic microsomal enzymes res ponsible for the oxidative metabolism of various subs trates and the synthesis of cholesterol and other lipids. The CYP2C9 isoenzyme as a type of CYPs is primarily responsible for the metabolism of a number of important drugs, including Warfarin, Phenytoin, Losartan, Tolbutamide, Glipizide, and Diclofenac. The gene coding for CYP2C9 has been mapped to the long arm of chromosome 10 (10q24.2), wi thin a cluster of cytochrome P450 genes 24.

Figure 2. CYP2C9 gene: Cytogenetic Location (10q24.2)

Around 12 CYP2C9 vari ants hav e now been identified (Sanderson S et al. 2007). The most common variant is considered to be the wildtype allele, CYP2C9*1. Two other vari ants are known to reduce the metabolism of Warfarin: CYP2C9*2 and CYP2C9*3. The nomenclature for the CYP2C9 single nucleotide polymorphisms (SNPs) is unique: CYP2C9*1*1 (Arg144/Ile359) is the reference sequence or wild type. The two CYP2C9 polymorphisms that have been identified in humans most frequently are CYP2C9*2 (Cys144/Ile359) and CYP2C9*3 (Arg144/Leu359). Both of these variants are associated with decreased enzymatic activity and hence impaired Warfarin clearance25.Each of these polymorphisms can occur in a heterozygous or homozygous form, and the pres ence of both polymorphisms resul ts in a compound heterozygote (CYP2C9*2*3). The normal, or wild-type, variant is referred to as *1 (star 1), the two polymorphic versions are *2 (star 2) and *3 (star

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3) and each person can carry any two versions of the SNP. For example, a person with two normal copies would be *1/*1, a person with only one polymorphism could be *1/* 2 or *1/*3 and a person with both polymorphisms is *2/*3. The prev alence of each variant varies by race; 10% and 6% of Caucasians carry the * 2 and *3 variants, res pectively, but both variants are rare (A, CYP2C9 430 C>T (2C9*2), CYP2C9 1075 A>C (2C9*3).

Interpretation of results: Test strips were aligned i nto the schematic drawing in the enclosed Collector TM sheet. Genotyping of each sampl e was determined online using the strip evaluator. http://viennalab. scidesign. at/webevaluator25/evaluate.php ? assay=4-730_20080723). For each polymorphic position on the strip, one of the following staining patterns were obtained:

Figure 4. Staining patterns for VKORC1

Figure 5.Staining patterns for CYP2C9

Statistical analysis The statistical analysis of the data obtained in the present study was carried out using SPSS 16 (Chicago, IL, USA). D ata was expressed in frequency and percentages; (Hardy Weinberg equation applied for allele frequency. The rel ation between genotypes and Warfari n dose was evaluated by fisher and chi square tests. P-value 6 mg/day) • CYP2C9*1 made 100%. • CYP2C9*2 and CYP2C9*3 were 0. 0% The difference between the three groups was statistically significant (p value =0. 01). Table 4. Genotypes for CYP2C9 and Warfarin dose. Warfarin Dose groups

Genotype for CYP2C9

*1/*1

*1/*2 & *2/*2

*1/*3 & 3*/3*

Count % of cases in each group Count % of cases in each group Count % of cases in each group

Group I < 4 mg/day n=13 cases (34.3%)

GROUP II 4 – 6 mg/day n =14cases (36.8%)

GROUP III > 6 mg/day n =11 cases (28.9%)

3

11

11

23.1%

78.6%

100.0%

2

3

0

15.4%

21.4%

0.0%

8

0

0

61.5%

0.0%

0.0%

Total n.=38 cases (100%)

25 (65.8%)

5 (13.2%)

8 (21.0%)

P value =0.01 5.Dose reduction between CYP2C9 wild type and variants in the present study: • For CYP2C9*1/*1 (wild type) the mean daily dose was 6.46 mg/ day • For CYP2C9*1/*2 the mean daily dose was 4.62 mg/ day, reduced by 28.17% from CYP2C9*1/*1. • For CYP2C9*1/*3 the mean daily dose of Warfarin was 2.41 mg/ day reduced by 62.69% from the wild type. • For CYP2C9*3/*3 the dose was 1.5 mg/ day, reduced by 76.78% from the wild type. Table 5. Mean daily dose reduction between CYP2C9 wild type and variants CYP2C9 genotype *1/*1 *1/*2 *1/*3

*3/*3

Warfarin mean daily dose

6.46

4.62

2.41

1.5

Dose reduction

0.0%

28.17%

62.69%

76.78%

6.VKORC1 genotypes and Warfarin dose: In group I (low dos e responders’ patients) • GG (wild type) made 0.0% • AG (heterozygote) made 76.9%. • VKORC1 AA (homozygote mutant) made 23.1%. In group II (intermediate dose responders) • GG made 28. 6%. • AG made71.4%. • AA made 0. 0%. In group III (high dose responders) • GG made 54. 6 % • AG made 45.4% • AA made 0. 0%

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The difference between the three groups was statistically significant (p value =0. 0136). Table 6. Genotypes for VKORC1 and Warfarin dose Warfarin dose Genotype for VKORC1

AA AG GG X2 =9.85

Count % of cases in each group Count % of cases in each group Count % of cases in each group P value =0.0136*

Group I < 4 mg/day n=13 cases (34.3%)

GROUP II 4 – 6mg/day n=14 cases (36.8%)

GROUP III > 6 mg/day n. =11 cases (28.9%)

3 23.1% 10 76.9% 0 .0%

0 0.0% 10 71.4% 4 28.6%

0 0.0% 5 45.4% 6 54.6%

The mean daily dose reduction between VKORC1GG (wild type) and other variants: • Mean daily dose for VKORC1GG (wild type) was 8.37 mg / day. • For VKORC1 AG the dose was 4.33 mg / day, reduced by 48.26% from wild type. • For VKORC1 AA the dos e was 2. 41 mg / day, reduced by 71.20% from wild type. Table 7. Mean daily dose reduction between VKORC1 wild type and variants VKORC1 genotype

GG

AG

AA

Warfarin mean daily dose

8.37

4.33

2.41

Dose reduction

0.0%

48.26%

71.20%

7.CYP2C9/ VKORC1 haplotypes and Warfarin dose: In group I low dose responder (Warfarin dose < 4 mg / day) • *1/*3/AG haplotype made38.5% • *3/*3/AG haplotype made 15.4% • *1/*1/AG haplotype made 15.4% • Haplotypes *1/*1/AA, *1/*2/AG, *1/*3/AA and*2/*2/AA made 7. 7% for each. In group II intermediate dose responders • *1/*1/AG made 57.1% • *1/*1/GG 21.4%, • *1/*2/AG14. 3% • *1/*2/GG 7.1%. In Group III High dose responders • Haplotype *1/*1/GG made 63.6% and • *1/*1/AG 36. 4%. Table 8. CYP2C9/VKORC1 haplotypes and Warfarin dose< 4 mg /day CYP2C9/VKORC1 haplotype NO. of cases Frequency *1/*1/AA 1 7.7% *1/*1/AG 2 15.4% *1/*2/AG 1 7.7% *1/*3/AA 1 7.7% *1/*3/AG 5 38.5% *2/*2/AA 1 7.7% *3/*3/AG 2 15.4% Total 13 100.0% Table 9. CYP2C9/VKORC1 haplotypes and Warfarin dose< 4-6 mg /day CYP2C9/VKORC1 haplotype No. of cases Frequency *1/*1/AG

8

57.1%

*1/*1/GG *1/*2/AG

3 2

21.4% 14.3%

*1/*2/GG

1

7.1%

Total

14

100.0%

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Total n=38 cases (100%)

3 (7.9%) 25 (65. 8%) 10 (26.3%)


Dalal Nasser Eldin ElKaffash,Aymen Abdel Hay, Nermine Hossam Zakaria & Mounir Elhag Table 10. CYP2C9/VKORC1 haplotypes and Warfarin dose >6 mg /day CYP2C9/VKORC1 haplotype No. of cases Frequency *1/*1/AG

4

36.4%

*1/*1/GG Total

7 11

63.6% 100.0%

DISCUSSION Warfari n treatment is problematic because the dose requirement for Warfari n is hi ghly vari able, both inter-individually and interethnically 32. Physician fear of Warfarin overdosing leads to either under-dosing or failure to prescribe Warfari n at all, resulting in 60% of all patients pres enting with stroke being inadequately anticoagulated33. Among patients in the Registry of the Canadian Stroke Network, only 40% had been prescribed Warfarin; 30%, combined antiplatelet therapy ; and 29%, no antithrombotic drug. Moreover, three-fourths of thos e taking Warfarin had a sub therapeutic INR (INR 2.0) at admission. Much effort has been devoted to moni tor the s afety of this oral anticoagulant. Currently, the s election of Warfarin dos age is relying upon gradual change in daily dosage until the desired therapeutic effect is achieved. The dose has to be closely monitored by serial determinations of blood prothrombin time using INR. During this process of change towards an optimal INR, the patients are still at risk from thromboembolism and bleeding, some of which could be fatal 33. Two gene products known to i nfluence Warfari n dose are the enzymes Cytochrome P 450 subtype 2C9 (CYP2C9) and the Vi tamin K Epoxide Reductase 1 (VKORC1) 34 which are involved in drug metabolism and vitamin K activation, respectively. Common gene polymorphisms exist for both enzymes, resulting in marked alteration of enzyme activity, and sev eral studies have characterized the role of thes e polymorphisms in explaining a substantial part of the variation in Warfarin dosage requirement. As expected, using standard dosing algorithms in patients with thes e variants leads to adverse clinical and laboratory outcomes because of their genetically mediated s ensitivity to the drug. In particular, standard dosing algorithms lead, on average, to a 2 to 3 folds increased risk of serious or life threatening bleeding or an out-of-range INR (>4.0) in carriers of the *2 or *3 alleles of CYP2C935. Similarly, carri ers of the VKORC1 A allele are also at a 2 to 3 folds higher risk of an INR >4.0 during initiation of Warfarin therapy when standard dosing algorithms are us ed35. Finally As a result of the sensitivity of these patients to Warfarin and the addi tional dose adjustments required, the time required to achieve a stable INR between 2.0 and 3.0 is significantly delayed in carriers of all three single nucleotide polymorphism 36. These facts make Warfari n pharmacogenetics a case s tudy for personalized medicine. Algorithms incorporating selected SNPs of these two genes, CYP2C9 and VKORC1, show improved dose prediction compared with algori thms bas ed solely on clinical and demographic factors 37,38. In this genetic study we strictly select stable pati ents on different Warfari n dos es, with an INR between 2 -3 that was fixed for at l east

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three clinical visits within a minimum period of three months. To ensure that selected s ubjects were receiving accurate maintenance doses, and therefore we believe that the data allow one to determine in a preliminary way the ability to assess the extent to which CYP2C9 and VKORC1 variant genoty pes alter the patients’ odds of being low or high dos e responders to Warfari n. CYP2C9 alleles CYP2C9 *1 allele frequency in the study sample was less than the results of another s tudy among 247 Egyptian subjects 40., where the author reported a frequency of 82% .Our result was very close to those found in Caucasian study done by Scott et al.41 who had reported frequency of 78.8%, 82.2% in Hispanic as stated by Scordo et al. 42 in study of 93 Italian 75% while Takahashi et al.43 stated a percentage of 97% among Japanese. The frequency of the CYP2C9*2 allele in this study sample was in a range comparable with the frequency in Caucasian populations; Taube et al. reported a frequency of 10.6% in British44. Limdi et al. 45 stated a frequency of 8 % in American-Caucasian and Rosemary J et al., stated the frequency was 11. 4% among Russian46 Scordo et al. in study for Italians reported 12% 42. Moreover our results di d not show greater difference from the result of another larger size Egyptian population study (n=247) which gave frequency of 12% 40. By contrast, the CYP2C9*2 allele frequency is zero or at least



very rare in the East Asian populations and very low in African Americans 1% according to s tudi es of lee et al. and Takahashi et al.37who give a 0% among Japanese(n=90) 0,Caucasian(n=47) 22% 41 , Loebstein et al.32 stated a frequency of Jewish 10%. Taube et al. stated a frequency among British 10.6% 44, and in Italians 17.8% 43. The frequency of CYP2C9*3 allele in this study was higher than the result of the previously mentioned Egyptian study (6.0%) 40. This variation may be due to the difference in the size of the study sample between the present study (n=40) and the other study (n=247). Our result was comparable wi th that found in Turkish study done by Borgiani 10.0 % 47. Even so, it was higher than other Caucasian populations; which were 6.0% in American according to Limdi et al. and 8.5% among British in study of Taube et al. 44. Also It is much greater than what Takahashi et al. found in African American 2. 0% and Asian 3.9% 50. Scordo et al. in study of 93 Italian reported a frequency of 13% 42, while Takahashi et al. reported a frequency among Japanese (n=90) of 0.03%, Caucasian (n=47) 5% 43, Taube et al.44 reported a frequency of 5.3% among British, Margaglione et al., Caucasian17.8% 43, Rosenary et al. stated a frequency in British of 14.2% 48. VKORC1 alleles The frequency of VKORC1 wild ty pe G allele found in our study was similar to Caucasian. It was higher than Asian but far less than African American. In Stuart A Scott review41, the frequency among Caucasian was 59.4%, Asian 33.3% and African American 89.2 % 41.The VKORC1 A allele frequency was similar to Caucasian. 40.6%. It was less than Asian 66.7%.Yet; it was much higher than African American 10.8% 50. CYP2C9 and VKOR C1 genotypes As regards to genoty pe frequency our results also shows that: CYP2C9 wild type *1/*1 frequency in this study was similar to Caucasians, but it was less than both Asian and African American. In a s tudy of 189 Caucasians Thomas P. Moyer et al. 51 found the frequency was 66.0%, Borgi ani P et al. in study of 148 Caucasians reported 67% 47 and Yuan HY et al. in study of 92 Caucasian found the frequency was 66% 52. On the other hand, Yuan HY et al. reported the frequency was 93% in study of 95 Asians 52. Obayashi K et al. reported higher frequency 98 % among 239 Asians 49. Limdi NA et al. in s tudy of 226 African descent found the frequency was 90 % 45. In comparison with the above mentioned studies, CYP2C9 *1/*3 frequency in our study was in a range comparable with Caucasian. But it was higher than both African American and Asian. In a study of 189 Whites Thomas P. Moyer et al. 51 found the frequency was 8%. Borgi ani P et al reported 17 % in 148 Caucasians, Yuan HY et al. in study of 92 Caucasians found the frequency was 12% 47. On the other hand, Yuan HY et al. reported the frequency was 7% in study of 95 Asians 52. However, Obay ashi K et al. reported lower frequency2 % among 239 Asians 49. Limdi NA et al. in study of 226 African descent found the frequency was 3 % 45. The frequency of CYP2C9 *1/*2 found in this study was lower than Caucasian. In a s tudy of 189 Whites Thomas P. Moyer et al .,51.

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Found the frequency was18%. Borgi ani P et al. reported 33% in 148 Caucasians 47, Yuan HY et al. in study of 92 Whites found the frequency was 20% 52. The frequency among Asian and African American was v ery low. Both Yuan HY et al.52. and Obayashi K et al.49, reported the frequency was 0% among Asians. Limdi NA et al. in study of 226 African descent found the frequency was 2 % 45. CYP2C9 *2/*2 frequency in our study was higher than some studies results among Caucasian. In a study of 189 Caucasians Thomas P. Moyer et al. 51 found the frequency was1%. Borgiani P et al. reported 1% in 148 Caucasians 47, Yuan HY et al. in study of 92 Caucasians found the frequency was 1% 52. Scott SA et al. reported a frequency of 6.6% among Caucasian41. The frequency among Asian and African American was v ery low. Both Yuan HY et al.,52. and Obayashi K et al.49 reported the frequency was 0% among Asians. Limdi NA et al. in study of 226 African descent found the frequency was 0 % 45. CYP2C9*3/*3 frequency in this study sample was comparable with what Hamdy S et al. found among Egy ptian 4% 40. Both results were higher than all three ethnic groups. In the study of 189 Caucasians Thomas P. Moyer et al.51 did not detect this genotype. Borgi ani P et al reported 1% in 148 Caucasians 47, Yuan HY et al. in study of 92 Caucasians found the frequency was 0.4% 52. Stuart A Scott et al reported a frequency of 0.0% in Caucasian, Asian and African American41. CYP2C9*2/*3 was not detected in our s tudy. Similarly, Hamdy S et al. did not record its presence among Egyptian40. In a study of 189 Caucasians Thomas P. Moyer et al. found the frequency was2% 51. Borgiani P et al. did not



detect this genotype in 148 Caucasians 47, Yuan HY et al. in study of 92 Caucasians found the frequency was 0% 52. Stuart A Scott et al. also found CYP2C9*2/* 3 were: missing from Asian, very rare in African American 0.3% and rare in Caucasian1.9 % 41. In the analysis of VKORC1 genotype frequency we found that: The wild ty pe GG frequency in our study sampl e is comparable with Hispanic and in the mid way between the frequency in Caucasian and Asian. African American had the highes t frequency. In the study of Scott SA et al. and the review of Stuart A Scott et al, the frequency in Caucasian was 36.8%, 22.5% in Asian and 80.3% i n African American41. Heterozy gote AG frequency in this study was hi gher than Caucasian. Besides, it is much higher than both Asian and African American. The reported AG frequency among Caucasian was 45.3%, 21.6% in Asian and 17.7% in African American41. Homozygote mutant AA was higher than African American; lower than Caucasian and much lesser than Asian. The frequency among African American was 2.0%, Caucasian 17.9% and Asian 55.9%. Genotype an d Warfarin dose relationship: As mentioned abov e, CYP2C9 common vari ants*2 and *3 were detected in our studied sampl e. The variant CYP2C9*3 was detected only in Warfarin low dos e responder patients. While CYP2C9*2 was present in both low and intermedi ate dose responders groups. However, both CYP2C9*3 and CYP2C9*2 are completely absent from the high dose res ponders group. On the other hand, the wild type CYP2C9*1 was detected in all three groups. Therefore, mutations in CYP2C9 account for some of Warfari n sensitivity in our study s ample and did not account for all Warfari n sensitivity. Our findings are consistent with the previous reports of Xi e et al., Takahas hi et al. and Rosemany et al.; that variants discovered in CYP2C9 can only parti ally explain some of the inter-i ndividual differences in Warfarin dosage48,50,53. Our study also shows that the mean daily Warfari n dose requirement for CYP2C9 variants was greatly reduced from CYP2C9 wild type. The reduction was 28.17% for CYP2C9*1/*2, 62.69% for CYP2C9*1/*3 and 76.78% for CYP2C9*3/*3.the dose reduction in our findi ngs are hi gher than the results of Sconce et al.54. Who found in a study of 176 patients that patients with CYP2C9*2 and CYP2C9*3 required 17% and 34% less Warfarin than those wi th the normal wild ty pe CYP2C9.also our results are also greater than what Sanderson et al.55 reported In a review of 9 studies. When they compared Warfari n dose i n patients wi th the wild type CYP2C9 and those with CYP2C9*2 and CYP2C9*3, and found a reduction in Warfarin dose by 0.8 mg daily (17%) in those with CYP2C9*2 and 1.92 mg daily (37%) in thos e with CYP2C9*3. In addition, this work also provides the evidence that ch anges in the VKORC1 gene could alter Warfari n dosage requirements interindividually in this study sample. It demonstrated that pati ents with the AA genoty pe had the lowest dos e requi rements than compared with AG and GG genotypes. This finding goes with m any previous published results. Mark JR et al.56 showed in a retrospective study that patients wi th GG genotype spent more time below therapeutic INR compared with

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pati ents’ carriers of A allele. Sonce et al. 54 also showed that the mean Warfarin dose was the highest in patients with GG genotype compared with the GA and AA genotype patients. G to A substitution at position –1639 in the VKORC1 promoter appears to diminish the Warfari n dose requirement56,57. The −1639 G>A single nucleotide polymorphism pres ented in the homozygous form (genotype AA) was found in pati ents who requi red lower doses than pati ents with ei ther AG or GG genotypes in the HanChinese population. This was also found to be true in other Asian and Caucasian populations 57,58. The fact that peopl e with the −1639 GG genotype requi re higher Warfarin dose can be expl ained by that when VKORC1 promoter activity is increased, the VKORC1 mRNA expression would raise as a result. This would translate into an increas ed translation of the VKORC1 protein which lead to a hi gher VKOR activity 59 and thus enhance the efficiency of the regeneration of the reduced vitami n K which ultimately would produce more gamma-carboxylation of the vitamin K dependent clotting factors. Having more active clotting factors would render the requirement of Warfari n to be high. Comparison of Warfarin mean daily dose for different VKORC1 and CYP2C9 genotypes in our study with some other results: CYP2C9: The mean dose of Warfarin for CYP2C9*1/*1 in this s tudy was higher than the mean of similar studies. Kamali F et al. 58 found in a study of 121 caucasian the mean daily dose was 4.1 mg / day. Higashi et al. 59 in a study of 185 caucasian reported the mean dose was 5.6 mg /day. In a s tudy



of 189 caucasian Thomas P. Moyer et al. 51 reported 5mg/day. Borgi ani P et al reported 4.1mg in 148 caucasian47. Obayashi K et al in a study of 239 Asian reported the mean dose was 4.7 mg / day 49. For genotype CYP2C9*1/*2 mean daily dose of warf ari n in our study was lower than the mean dose(4.9 mg / day) reported by Higashi et al. for this genotype in the above mentioned study. Yet, it was higher than what Thomas P. Moyer et al.51 reported in a study of 189 caucasian (4. 1mg/day) and Borgi ani P et al. in 148 caucasian (2.6 mg / day)47. CYP2C9*2/*2 mean dose in our findings was similar to the mean dose reported by Higashi et al in the abov e mentioned study (4.1 mg / day). Ev en so, it was higher than the findings of both Thomas P. Moyer et al. 3.4mg/day 51, and Borgiani P et al. 0.6mg/day in their above mentioned studies 47. The dose for CYP2C9*1/*3 in this study was higher than that required by Asian ethnic group but lower than caucasian. Obay ashi K et al. in a study of 239 Asian subjects reported that, the mean daily dose was 1.9 mg /day(4). Thomas P. Moyer et al. 51, reported lower average mean dose for this genoty pe (3.2 mg / day) in the above mentioned six studies among caucasian. The mean dose for CYP2C9*3/*3 in our study was same as the average of mean daily doses of four unrelated studies(1.5 mg / day) among caucasian reported by Thomas P. Moyer et al. 51. VKORC1 The mean daily dose for VKORC1 AA in our results was lower than the average mean daily doses of several studies in Caucasian and Asian. In the review of Thomas P. Moyer et al., the av erage mean of four unrel ated studies in Caucasian was 3.1 mg / day 51. Similarly, the average mean of another four unrelated s tudi es among Asian was also 3.1 mg / day 60. However, Ri eder MJ et al. in a study of 96 African descent pati ents reported higher mean daily dose 3.7 mg / day 61. The mean dose for VKORC1 AG in our s tudy was lower than the mean dose reported by Vacis Tatarūnas et al. in their study of 85 Lithuanian patients (5. 6 mg / day)62. For VKORC1 GG, we found the mean daily dose in our s tudy s ample was higher than the mean dos e (6.20 mg / day) reported by Vacis Tatarūnas et al. for the same genotype i n their abov e mentioned study 62. Moreov er, Gan et al. in a s tudy among M alaysian’s subjects reported lower mean Warfarin dose for patients with GG genotype 4.9 mg/ day 63. CYP2C9/ VKORC1 haplotypes: CYP2C9 wild type /VKORC1 heterozygote haplotype *1/*1/AG was the commones t haplotype in this study 35.0%, it also made the majority of cases 57.1% in intermedi ate dose res ponder group (Warfari n dose 4-6 mg / day) and s econd haplotype in both low and hi gh dos e responder patients( Warfarin dose < 4 and > 6 mg / day ) 15.4% ,36.4% respectively. The second haplotype in frequency was CYP2C9 wild type/ VKORC1 wild type *1/*1/GG making 27.5%, i t was the major haplotype in the group of hi gh dose responder patients 63.6% and the second i n intermediate dose responders ,but was totally absent from low dose responders group.

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Haplotypes containing CYP2C9 *2 variant were present in both low and intermedi ate dos e responder group, while all haplotypes contai ning CYP2C9 *3 variant or combined mutant VKORC1 AA were present only in low dose responder group. We had excluded 2cases from statistical analysis of this study (the fi rst one with phenoty pe *1/*1/ GG and Warfarin dose 17 mg / day, the other wi th phenotype *2/*2/ AG and Warfari n dos e 9 mg /day).The first case although i t is located among high dose res ponder yet, the dose value is higher compared to the mean dose in this group. For the second case, the dose value di d not match genotype dose rel ationshi p, and is extremely high in comparison to our patients’ dose range. These results may be due to the pres ence of different subs ets of the VKORC1 SNPs which were reported as mi nor contributors to Warfari n dose, and were not included in our study Or due to rare coding variants in VKORC1 that produce extreme Warfarin resistance, the frequency of which may vary between racial groups. Also high di etary intake of vitamin K could alter individual response. Population differences with respect to thes e factors could explain some of the differences in Warfarin dosing across raci al groups. Our Justifications were supported by similar comments from other researchers; H Takahashi and H Echizen64 declare that, previously recognized genetic polymorphisms of CYP2C9 cannot adequately account for the observed population differences in the in vivo CYP2C9 activity at least between Caucasians and Japanes e patients. Nita A. Limdi et al.65 also show same obs ervation regarding VKORC1; they commented that “although



possession of the VKORC1−1639A allele is associated with a similar decreas e in the individual Warfarin dose requirement irres pective of race, the variability in dose explai ned by VKORC1 at a population level varies by race. They found that as the minor allele frequency (MAF) increases, the percentage of variation in dose explained by−1639A allele increases, with the hi ghes t variance expl ained at MAF of 60% to 70%. These res ults show that the differences in the percentage of variation explained by VKORC1 across race are driven by the MAF“. Knowledge of the variant genotype may influence the clinician’s decision to use Warfarin, particularly in the high risk elderly pati ents 66,67,or in pati ents with multiple genetic abnormalities. Genotype information may also help the clinician decide to choose other anticoagulant agents, the metabolism of which is not influenced by the CYP2C9 gene. Finally, anticoagulant regimens based on fi xed low dos es of Warfarin administered with little laboratory moni toring should be considered wi th caution i n pati ents who are high res ponders to Warfarin68,69.

REFERENCES 1.Hirsh J, Dalen JE, Anderson DR, Poller L, Bussey H, Ansell J, et al. Oral anticoagulants: mechanism of action, clinical effectiveness, and optimal therapeutic range. Chest. 2001;119(1 suppl):8S.Pubmed 2.Bernard JM BJ, Kokseng CU.et al. Warfarin sodium.Practitioner beware. AmPodiatr Med Assoc. 1992;82:345.Pubmed 3.Brummel KE, Paradis SG, Branda RF, Mann KG. Oral anticoagulation thresholds. Circulation. 2001;104(19):2311.Pubmed 4.Schelleman H, Limdi NA, Kimmel SE. Ethnic differences in warfarin maintenance dose requirement and its relationship with genetics. Pharmacogenomics.2008;9(9):1331-46.Pubmed 5.Chow W , Chow T , Tse T , Tai Y , Lee W . Anticoagulation instability with life threatening complication after dietary modification. Postgraduate medical journal . 1990; 66(780):855.Pubmed 6.Lee SH, Ahn YM, Ahn SY, Doo HK, Lee BC. Interaction between warfarin and Panax ginseng in ischemic stroke patients. The Journal of Alternative and Complementary Medicine. 2008;14(6):715-21.Pubmed 7.Zhao F, Loke C , Rankin S.C , Guo J.Y, Lee H.S , Wu T.S , et al. Novel CYP2C9 genetic variants in Asian subjects and their influence on maintenance warfarin dose. Clin Pharmacol Ther. 2004;76:210–9.Pubmed 8.Rane A, Lindh JD. Pharmacogenetics of anticoagulants. Hum Genomics Proteomics. 2010 Sep 13;2010:754919. doi: 10.4061/2010/754919.Pubmed 9.Krynetskiy E, McDonnell P. Building individualized medicine: prevention of adverse reactions to warfarin therapy. Journal of Pharmacology and Experimental Therapeutics. 2007;322(2):427.Pubmed 10.Majerus P, Broze G, Miletich J, Tollefsen D. Anticoagulant, thrombolytic, and antiplatelet drugs. Goodman and Gilman’s The pharmacological basis of therapeutics. 1995:1341-59. 11.Zhang K, Young C, Berger J. Administrative claims analysis of the relationship between warfarin use and risk of hemorrhage including drug-drug and drug-disease interactions. Journal of Managed Care Pharmacy. 2006;12(8):640.Pubmed 12.Harrison L, Johnston M, Massicotte MP, Crowther M, Moffat K, Hirsh J. Comparison of 5-mg and 10-mg loading doses in initiation of warfarin therapy. Annals of Internal Medicine. 1997;126(2):133.Pubmed 13.Lamb GC. Loading Dose and Monitoring of Warfarin Therapy-Reply. JAMA: The Journal of the American Medical Association. 1997;278(7):548.Pubmed 14.Kovacs MJ, Rodger M, Anderson DR, Morrow B, Kells G, Kovacs J, et al. Comparison of 10-mg and 5-mg warfarin initiation nomograms together with lowmolecular-weight heparin for outpatient treatment of acute venous thromboembolism. Annals of Internal Medicine. 2003;138(9):714.Pubmed 15.Fennerty A, Campbell I, Routledge P. Anticoagulants in venous thromboembolism. British Medical Journal. 1988;297(6659):1285.Pubmed

1483

16.Smellie WSA, Hampton K, Bowlees R, Martin S, Shaw N, Hoffman J, et al. Best practice in primary care pathology: review 8. Journal of clinical pathology. 2007;60(7):740.Pubmed 17.Lenzini PA, Grice GR, Milligan PE, Dowd MB, Subherwal S, Deych E, et al. Laboratory and clinical outcomes of pharmacogenetic vs. clinical protocols for warfarin initiation in orthopedic patients. Journal of Thrombosis and Haemostasis. 2008;6(10):1655-62.Pubmed 18. Bennett S. Monitoring Anticoagulant Therapy. Laboratory Hemostasis. 2007:167-205.Link 19.Schulman S. Care of patients receiving long-term anticoagulant therapy. N Engl J Med. 2003 Aug 14;349(7):675-83.Pubmed 20.Greenblatt DJ, von Moltke LL. Interaction of warfarin with drugs, natural substances, and foods. The Journal of Clinical Pharmacology. 2005;45(2):127.Pubmed 21.Horstkotte D, Piper C, Wiemer M. Optimal frequency of patient monitoring and intensity of oral anticoagulation therapy in valvular heart disease. Journal of thrombosis and thrombolysis. 1998;5:19-24.Pubmed 22.Ansell J, Jacobson A, Levy J, Völler H, Hasenkam JM. Guidelines for implementation of patient self-testing and patient self-management of oral anticoagulation. International consensus guidelines prepared by International SelfMonitoring Association for Oral Anticoagulation. International journal of cardiology. 2005 Mar 10 ;99(1):3745.Pubmed 23.Sconce EA, Khan TI, Wynne HA, Avery P, Monkhouse L, King BP, et al. The impact of CYP2C9 and VKORC1 genetic polymorphism and patient characteristics upon warfarin dose requirements: proposal for a new dosing regimen. Blood. 2005 Oct 1;106(7):2329. Pubmed 24.Yin T, Miyata T. Warfarin dose and the pharmacogenomics of CYP2C9 and VKORC1--Rationale and perspectives. Thrombosis research. 2007;120(1):110.Pubmed 25. Sanderson S, Emery J, Higgins J. CYP2C9 gene variants, drug dose, and bleeding risk in warfarin-treated patients: A HuGEnet (TM) systematic review and meta-analysis. Genetics in Medicine. 2005;7(2):97.Pubmed 26.Takahashi H, Echizen H. Pharmacogenetics of warfarin elimination and its clinical implications. Clinical pharmacokinetics. 2001;40(8):587-603. 27.Li T, Chang CY, Jin DY, Lin PJ, Khvorova A, Stafford DW. Identification of the gene for vitamin K epoxide reductase. Nature. 2004;427(6974):541-4.Pubmed 28.Rost S, Fregin A, Ivaskevicius V, Conzelmann E, Hörtnagel K, Pelz HJ, et al. Mutations in VKORC1 cause warfarin resistance and multiple coagulation factor


Dalal Nasser Eldin ElKaffash,Aymen Abdel Hay, Nermine Hossam Zakaria & Mounir Elhag deficiency type 2. Nature. 2004;427(6974):537-41.Pubmed 29.Oldenburg J. VKORC1: the little big protein. Blood. 2005;106(12):3683.Link 30.Flockhart DA, O’Kane D, Williams MS, Watson MS, Gage B, Gandolfi R, et al. Pharmacogenetic testing of CYP2C9 and VKORC1 alleles for warfarin. Genetics in Medicine. 2008;10(2):139.Pubmed 31.Schwarz UI, Ritchie MD, Bradford Y, Li C, Dudek SM, Frye-Anderson A, et al. Genetic determinants of response to warfarin during initial anticoagulation. N Engl J Med.2008;358(10):999-1008.Pubmed 32.Loebstein R, Yonath H, Peleg D , Almog S, Rotenberg M , Lubetsky A , et al. Interindividual variability in sensitivity to warfarin—nature or nurture? Clin Pharmacol Ther. 2001;70:159–64.Pubmed 33.Gladstone DJ, Bui E, Fang J, Laupacis A, Lindsay MP, Tu JV. et al. Potentially preventable strokes in high-risk patients with atrial fibrillation who are not adequately anticoagulated. Stroke. 2009 Jan;40(1):235-40.Pubmed 34.Geisen C, Watzka M, Sittinger K, Steffens M, Daugela L, Seifried E,et al. VKORC1 haplotypes and their impact on the inter-individual and inter-ethnical variability of oral anticoagulation. Thromb Haemost. 2005;94(4):773-9.Pubmed 35. Higashi MK, Veenstra DL, Kondo LM, Wittkowsky AK, Srinouanprachanh SL, Farin FM, Rettie AE. Association between CYP2C9 genetic variants and anticoagulation-related outcomes during warfarin therapy. JAMA. 2002 Apr 3;287(13):1690-8.Pubmed 36.Vecsler M LR, Almog S, Kurnik D, Goldman B, Halkin H, Gak E. Combined genetic profiles of components and regulators of the vitamin K dependent gammacarboxylation system affect individual sensitivity to warfarin. Thromb Haemost. 2006;95:205-11.Pubmed 37.Takahashi H, Wilkinson G.R, Caraco Y, Muszkat M, Kim R.B., Kashima T, et al. Population differences in S-warfarin metabolism between CYP2C9 genotypematched Caucasian and Japanese patients. Clin Pharmacol Ther. 2003;73:253– 63.Pubmed 38.Hill CE, Duncan A, Wirth D, Nolte FS. Detection and identification of cytochrome P-450 2C9 alleles *1, *2, and *3 by high-resolution melting curve. Am J Clin Pathol. 2006;125:584-91.Pubmed 39.Gage BF, Eby C, Milligan PE, Banet GA, Duncan JR, McLeod HL. Use of pharmacogenetic and clinical factors to predict the therapeutic dose of warfarin. Clin Pharmacol Ther.2008;84(3):326-31.Pubmed 40.Hamdy SI, Hiratsuka M, Narahara K, Endo N, ElEnany M, Moursi N, et al. Genotyping of four genetic polymorphisms in the CYP1A2 gene in the Egyptian population. British journal of clinical pharmacology. 2003;55(3):321-4.Pubmed 41.Scott SA, Khasawneh R, Peter I, Kornreich R, Desnick RJ. Combined CYP2C9, VKORC1 and CYP4F2 frequencies among racial and ethnic groups. Pharmacogenomics. 2010;1(6):781-91.Pubmed 42.Scordo MG, Pengo V, Spina E, Dahl ML, Gusella M, Padrini R. Influence of CYP2C9 and CYP2C19 genetic polymorphisms on warfarin maintenance dose and metabolic clearance&ast. Clinical Pharmacology & Therapeutics. 2002;72(6):702-10.Pubmed 43.Takahashi H, Kashima T, Nomizo Y, Muramoto N, Shimizu T, Nasu K, et al. Metabolism of warfarin enantiomers in Japanese patients with heart disease having different CYP2C9 and CYP2C19 genotypes&ast. Clinical Pharmacology & Therapeutics. 1998;63(5):519-28. 44.Taube J, Halsall D, Baglin T. Influence of cytochrome P-450 CYP2C9 polymorphisms on warfarin sensitivity and risk of over-anticoagulation in patients on long-term treatment. Blood. 2000;96(5):1816-9.Pubmed 45. Limdi N, McGwin G, Goldstein J, Beasley T, Arnett D, Adler B, et al. Influence of CYP2C9 and VKORC1 1173C/T genotype on the risk of hemorrhagic complications in African-American and European-American patients on warfarin. Clinical Pharmacology & Therapeutics. 2007;83(2):312-21.Pubmed 46. Rosemary J, Adithan C. The pharmacogenetics of CYP2C9 and CYP2C19: ethnic variation and clinical significance. Current clinical pharmacology. 2007;2(1):93109.Pubmed 47.Borgiani P, Ciccacci C, Forte V, Romano S, Federici G, Novelli G. Allelic variants in the CYP2C9 and VKORC1 loci and interindividual variability in the anticoagulant dose effect of warfarin in Italians. Pharmacogenomics. 2007;8(11):1545-50.Pubmed 48.Rosemary J, Adithan C. The pharmacogenetics of CYP2C9 and CYP2C19: ethnic variation and clinical significance. Current clinical pharmacology. 2007;2(1):93109.Pubmed 49. Obayashi K, Nakamura K, Kawana J, Ogata H, Hanada K, Kurabayashi M, et al. VKORC1 gene variations are the major contributors of variation in warfarin dose in Japanese patients&ast. Clinical Pharmacology & Therapeutics. 2006;80(2):16978.Pubmed

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50.Takahashi H, Echizen H. Pharmacogenetics of CYP2C9 and interindividual variability in anticoagulant response to warfarin. The pharmacogenomics journal. 2003;3(4):202-14.Pubmed 51.Moyer TP, O'Kane DJ, Baudhuin LM, Wiley CL, Fortini A, Fisher PK, et al. Warfarin sensitivity genotyping: a review of the literature and summary of patient experience Mayo Clin Proc. 2009 Dec;84(12):1079-94. doi: 10.4065/mcp.2009.0278.Pubmed 52.Yuan HY, Chen JJ, Lee MTM, Wung JC, Chen YF, Charng MJ, et al. A novel functional VKORC1 promoter polymorphism is associated with interindividual and inter-ethnic differences in warfarin sensitivity. Human molecular genetics. 2005;14(13):1745-51.Pubmed 53.Xie HG, Prasad H.C, Kim R.B. and Stein C.M. CYP2C9 allelic variants: ethnic distribution and functional significance. Adv Drug Delivery Rev.54:1257– 70.Pubmed 54.Elizabeth A. Sconce, Tayyaba I. Khan, Hilary A. Wynne, Peter Avery, et al. The impact of CYP2C9 and VKORC1 genetic polymorphism and patient characteristics upon warfarin dose requirement: proposal for a new dosing regimen. Blood. 2005;106:2329–33.Pubmed 55.Sanderson S , Emery j, Higgins J . CYP29 gene variants , drug dose ,and bleeding risk in warfarin treated patients: HuGent ™ systematic review and metaanalysis . Genetics in Medicine .2005; 792):97.Pubmed 56. Mark J. Rieder, Alexander P. Reiner, Brian F. Gage, Deborah A, et al. Effect of VKORC1 haplotypes on transcriptional regulation and warfarin dose. N Engl J Med. 2005;352:2285–93.Pubmed 57. Rost S, Fregin A, Ivaskevicius V, Conzelmann E , Hortnagel K, Pelz H.J , et al. Mutations in VKORC1 cause warfarin resistance and multiple coagulation factor deficiency type 2. Nature. 2004;427:537– 41.Pubmed 58. Kamali F, Khan TI, King BP, Frearson R, Kesteven P, Wood P, et al. Contribution of age, body size, and CYP2C9 genotype to anticoagulant response to warfarin&ast. Clinical Pharmacology & Therapeutics. 2004;75(3):204-12.Pubmed 59.Higashi MK, Veenstra DL, Kondo LM, Wittkowsky AK, Srinouanprachanh SL, Farin FM, et al. Association between CYP2C9 genetic variants and anticoagulation-related outcomes during warfarin therapy. JAMA: The Journal of the American Medical Association. 2002;287(13):1690-8.Pubmed 60.Meckley LM, Wittkowsky AK, Rieder MJ, Rettie AE, Veenestra DL. An analysis of the relative effects of VKORC1 and CYP2C9 variants on. ThrombHaemost. 2008;100:220–39.Pubmed


Dalal Nasser Eldin ElKaffash,Aymen Abdel Hay, Nermine Hossam Zakaria & Mounir Elhag 61.Rieder MJ, Reiner AP, Gage BF, Nickerson DA, Eby CS, McLeod HL, et al. Effect of VKORC1 haplotypes on transcriptional regulation and warfarin dose. New England Journal of Medicine. 2005;352(22):2285-93.Pubmed 62. Tatarūnas V, Lesauskaitė V, Veikutienė A, Jakuška P, Benetis R. The Influence of CYP2C9 and VKORC1 Gene Polymorphisms on Optimal Warfarin Doses After Heart Valve Replacement. Medicina (Kaunas). 2011;47(1):25-30.Pubmed 63.Gan GG, Phipps ME, Lee MMT, Lu LS, Subramaniam RY, Bee PC, et al. Contribution of VKORC1 and CYP2C9 polymorphisms in the interethnic variability of warfarin dose in Malaysian populations. Annals of Hematology. 2011;90(6):635-41.Pubmed 64.H Echizen H Ta. Pharmacogenetics of CYP2C9 and interindividual variability in anticoagulant response to warfarin. Pharmacogenomics.2003;3:202–14.Pubmed 65.Limdi NA, Wadelius M, Cavallari L, Eriksson N, Crawford DC, Lee MT, et al. Warfarin pharmacogenetics: a single VKORC1 polymorphism is predictive of dose across 3 racial groups. Blood. 2010 May 6;115(18):3827-34.Pubmed 66.Ensom MHH, T. K. H. Chang , and P. Patel. Pharmacogenetics: the therapy drug monitoring of the future? Clin Pharmacokinet. 2001;40:783–802.Pubmed

67.Dorothy M. Adcock CK, Domnita Crisan and Frederick L. Kiechle. Effect of Polymorphisms in the Cytochrome P450 CYP2C9 Gene on Warfarin Anticoagulation. Archives of Pathology & Laboratory Medicine. 2004 December;128(12):1360-3.Pubmed 68.Mannuci PM. Genetic control of anticoagulation. Lancet. 1999;353:688– 9.Pubmed 69.Poller I A .Mckernan, J M Thomson and M Elstein . Fixed mini dose warfarin:a new approach to prophylaxis against venous thromboembolism after major surgery .Br Med J. 1987; 295:1309-12.Pubmed

Conflict of Interest: - Authors has not declared any conflict of interest. Source of funding: - None

Copyright © 2013 Dalal Nasser Eldin ElKaffash,Aymen Abde l Hay, Nermine Hossam Zakaria & Mounir Elhag. This is an op en access article d istributed u nder the C reative Comm ons Attribution License, which permits unrestricted use, distribution, and reproduction in any m edium, provid ed the original work is properly cited .

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