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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 16  |  Issue : 4  |  Page : 371-376

The effect of ‘cytochrome P-450 2C9’ and ‘vitamin K epoxide reductase complex 1’ genetic polymorphism upon oral anticoagulation requirements


1 Department of Clinical Pathology, Nasr City Police Hospital, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Al-Azhar University, Cairo, Egypt

Date of Submission30-Aug-2018
Date of Acceptance04-Feb-2019
Date of Web Publication23-Apr-2019

Correspondence Address:
Hossam Y.K Mohammed
Clinical Pathology, Police Hospital, Nasr city
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/AZMJ.AZMJ_91_18

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  Abstract 


Background Polymorphisms in the gene encoding the cytochrome P-450 2C9 enzyme (CYP2C9) are known to contribute to variability in sensitivity to Marevan. CYP2C9 is the enzyme primarily responsible for the metabolic clearance of s-enantiomer of Marevan. The vitamin K epoxide reductase complex 1 (VKORC1) is the target of coumarin anticoagulants, and its common genetic variants result in altered sensitivity to Marevan. VKORC1 polymorphisms are associated with a need for lower doses of Marevan during long-term therapy.
Aim of work This study aimed to assess the allelic frequencies and to investigate the relationship between ‘CYP2C9’ and ‘VKORC1’ genotype and vitamin K antagonist anticoagulation.
Patients and methods This study was conducted on 40 patients. There were 24 females and 16 males, with a male to female ratio of 2 : 3. Their ages ranged from 28 to 72 years. All the studied patients were laboratory investigated with international normalized ratio, complete blood count, and detection of VKORC1 and CYP2C9 by PCR reverse hybridization method using PGX thrombo strip assay (Vienna Lab.).
Results Regarding the distribution of patients according to frequency of deep venous thrombosis (DVT) attacks, 16 (40%) patients showed single attack of DVT and 24 (60%) patients showed recurrent attacks. Patients with single attack of DVT comprised 12 (75%) females and four (25%) males, with male to female ratio of 1 : 3. As for the patients with recurrent attacks of DVT, there were 13 (54.2%) female and 11(45.8%) male patients, with a male to female ratio of 1 : 1.2.
Conclusion Detection of genetic polymorphisms in CYP2C9 and VKORC1 genes before onset of warfarin therapy greatly influenced response to warfarin and shortened the time required to reach target international normalized ratio, and hence reduced the risk of recurrence of DVT.

Keywords: cytochrome P-450 2C9 enzyme, PCR, vitamin K epoxide reductase complex 1, warfarin (Marevan) oral anticoagulant


How to cite this article:
Mohammed HY, Al-Zohairy YZ, Hashish ME. The effect of ‘cytochrome P-450 2C9’ and ‘vitamin K epoxide reductase complex 1’ genetic polymorphism upon oral anticoagulation requirements. Al-Azhar Assiut Med J 2018;16:371-6

How to cite this URL:
Mohammed HY, Al-Zohairy YZ, Hashish ME. The effect of ‘cytochrome P-450 2C9’ and ‘vitamin K epoxide reductase complex 1’ genetic polymorphism upon oral anticoagulation requirements. Al-Azhar Assiut Med J [serial online] 2018 [cited 2019 Oct 15];16:371-6. Available from: http://www.azmj.eg.net/text.asp?2018/16/4/371/256765




  Introduction Top


Oral anticoagulation with the vitamin K antagonist ‘Marevan’ reduces the rate of thromboembolic events in patients with deep venous thrombosis (DVT) [1]. However, Marevan is one of the most widely used anticoagulants, yet interindividual differences in drug response, a narrow therapeutic range, and a high risk of bleeding or stroke complicate its use. To achieve and maintain an optimal Marevan dose, the prothrombin time and international normalized ratio (INR) are monitored, and doses are adjusted to maintain each patient’s INR within a narrow therapeutic range. An INR of less than 2 is associated with an increased risk of thromboembolism [2], and an INR of 4 or more is associated with an increased risk of bleeding [3].

Polymorphisms in the gene encoding the cytochrome P-450 2C9 enzyme (CYP2C9) are known to contribute to variability in the sensitivity to Marevan [4].

CYP2C9 is the enzyme primarily responsible for the metabolic clearance of s-enantiomer of Marevan [5].

Patients with certain common genetic variants of CYP2C9 require a lower dose of Marevan and a longer time to reach a stable dose. They are also at higher risk for over-anticoagulation and serious bleeding [6].

The vitamin K-dependent blood coagulation proteins are components of the calcium-binding proteins family, which includes prothrombin; factors VII, IX, and X; and proteins C, S, and Z. These seven proteins have an essential role in the initiation and regulation of blood coagulation. Vitamin K epoxide reductase complex 1 (VKORC1) recycles the vitamin K epoxide to the reduced form of vitamin K, an essential cofactor in the formation of the active clotting factors II, VII, IX, and X through γ-glutamyl carboxylation [5].

The VKORC1 is the target of coumarin anticoagulants, and its common genetic variants result in altered sensitivity to Marevan. VKORC1 polymorphisms are associated with a need for lower doses of Marevan during long-term therapy [6] and, in some studies, were found to contribute to the variation in dose requirement more than CYP2C9 variants [7].

Based on these observations, the Food and Drug Administration approved a labeling change for Marevan that describes the reported effects of VKORC1 and CYP2C9 on dose requirements; the package insert as of August 2007 stated that ‘Lower initiation doses should be considered for patients with certain genetic Variations in CYP2C9 and VKORC1 enzymes.’ The Food and Drug Administration also approved chemical tests for these genetic variations [8]. However, there is little information about the relative contribution of VKORC1 and CYP2C9 to the anticoagulation response in patients during the initiation of Marevan therapy [9].

The first months of anticoagulant treatment are particularly problematic, as the safe and effective dose for an individual patient is not known and is determined empirically [10].


  Patients and methods Top


This study was conducted on 40 patients attending Nasr City Police Hospital Laboratory for follow-up of their anticoagulation status. There were 24 females and 16 males, with a male to female ratio of 2 : 3. Their ages ranged from 28 to 72 years old.

Inclusion criteria

Patients with DVT, patients with stable continuous intake of oral anticoagulants, and patients who had records of all the previous INR results since the start of treatment were included. Additionally, other inclusion criteria included the choice of resistant nonresponder patients to 2–3 mg dose of vitamin K antagonist oral anticoagulation, food (especially green vegetables rich in vitamin K), and concomitant medications that may affect the response of patients, and those who had a history of recurrence of DVT.

Exclusion criteria

Patients who are less than 18 years old and patients with active malignancy were excluded.

Thorough history taking was done for all patients, stressing on primary indication for oral anticoagulation, the INR achieved with a stable Marevan dose, the target INR, the use of concomitant medications (grouped according to those that increase and those that decrease the INR), details of diet intake, and any history of bleeding or thrombosis with the medication.

For all the studied patients, the following laboratory investigations were done: (a) INR and complete blood count, (b) detection of CYP2C9 and VKORC1 by PCR using PGX ThromboStrip Assay (Vienna Lab., Wien, Austria), and (c) protein C, protein S, and protein Z for studied patients if needed.

Statistical analysis

PASW statistical software package (version 18.0, 2010; IBM SPSS Inc., Chicago, US) was used for data analysis. Data were expressed as mean±SD for quantitative measures and both number and percentage for categorized data. Comparison between two independent mean groups for parametric data was done using Student t test. χ2 test was done to study the association between each two variables or comparison between two independent groups regarding the categorized data. The probability of error at 0.05 was considered significant, whereas at 0.01 and 0.001 were highly significant.


  Results Top


Regarding the distribution of VKORC1 (1639 G>A) genotypes, we found that 65% of patients showed heterozygous form of the gene, 25% were homozygous, and 10% were wild type. We found that all studied patients showed the wild type of the gene. However, none of the patients showed homozygous CYP2C9 (430C>T) genotype. As for the distribution of patients with CYP2C9 (430C>T) genotype, there were six (15%) heterozygous patients and 34 (85%) patients who showed the wild type of the gene. Interestingly, all patients carrying this mutation were as well mutant for VKORC1 (1639 G>A) gene. Phenotypically, all our patients were resistant to vitamin K antagonist anticoagulation at a dose of 2–3 mg. The distribution of patients with CYP2C9 alleles according to sex showed that there were five (83.333%) females with heterozygous CYP2C9 (430C>T), 19 (55.88%) females showing wild type of the gene, 15 (44.1%) males showing the wild type, and one (16.667%) male showing heterozygous type of the same gene. However, CYP2C9 (1075A>C) allele patients had the distribution of 24 (60%) female and 16 (40%) male patients. We found a significant statistical relation between CYP2C9 gene mutation and the age and insignificant relation with sex. There is insignificant relation between CYP2C9 genotypes and the following parameters: response to vitamin K antagonist oral anticoagulation at a dose of more than or equal to 5 mg, response during duration of therapy of less than or equal to 3 months, and frequency of occurrence of DVT. All the studied patients were nonresponders at dose of vitamin K antagonist oral anticoagulation, of 2–3 mg. Overall, 36/40 (90%) patients were mutant regarding VKORC1 gene [10 (25%) of them were homozygous and 26 (65%) were heterozygous]. Increasing the dose of vitamin K antagonist oral anticoagulation to more than or equal to 5 mg, 27/40 (67.5%) of the patients, achieved appropriate response; of them four (14.8%) were homozygous to VKORC1 gene, 20 (77%) heterozygous, and three (11.5%) were wild type. The remaining 13/40 (35%) were persistently resistant to dose of vitamin K antagonist oral anticoagulation of more than or equal to 5 mg; of which six (46.1%) were homozygous, six (46.1%) were heterozygous, and only one (7.8%) patient was wild type for the VKORC1 gene. Some patients were responders at lower doses (2–3 mg) of vitamin K antagonist oral anticoagulation, whereas other patients required higher doses (≥5 mg) to reach their target INR. Regarding the distribution of CYP2C9 (1075A>C), all studied patients showed the wild type of the gene. As for the distribution of CYP2C9 (430C>T), 6/40 (15%) were heterozygous. The correlation between VKORC1 gene mutation and frequency of occurrence of DVT in the present study was found to be statistically insignificant. The patients with combined VKORC1 and CYP2C9 gene mutation were all female patients with history of recurrence of DVT. Their number was 6. The genotype of 5 of them was combined heterozygous mutation for both VKORC1 and CYP2C9 (430C>T), and only one female patient showed homozygous mutation for VKORC1 and heterozygous gene mutation for CYP2C9 (430C>T). All these patients showed wild type of the gene CYP2C9 (1075A>C). There is a significant relation between INR at more than 5-mg Marevan dose and genotypes of VKORC1 (1639 G>A) and CYP2C9 (430C>T). There were no data available in other studies regarding the relation between VKORC1 (1639 G>A) and CYP2C9 (430C>T) genotypes and INR at high dose of Marevan of more than 5 mg ([Table 1],[Table 2],[Table 3],[Table 4],[Table 5],[Table 6],[Table 7]).
Table 1 Association between vitamin K epoxide reductase complex 1 gene mutation and the studied parameters, age and sex

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Table 2 Association between cytochrome P-450 2C9 enzyme (430C>T) gene mutations and the studied parameters, age and sex

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Table 3 Association between vitamin K epoxide reductase complex 1 (1639 G>A) gene mutation and frequency of recurrence

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Table 4 Association between cytochrome P-450 2C9 enzyme (430C>T) gene mutation and frequency of recurrence

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Table 5 Association between vitamin K epoxide reductase complex 1 (1639 G>A) gene mutation, cytochrome P-450 2C9 enzyme (430C>T) gene mutation, and response to Marevan dose at more than or equal to 5 mg

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Table 6 Association between cytochrome P-450 2C9 enzyme (430C>T) gene mutation, vitamin K epoxide reductase complex 1 (1639 G>A) gene mutation, and response to Marevan during 3 months of therapy

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Table 7 Association between genotypes and the following parameters: white blood cell count, hemoglobin, platelet count, international normalized ratio at 2–3 mg Marevan dose, and international normalized ratio at more than 5 mg Marevan dose

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  Discussion Top


Warfarin is an oral anticoagulant widely used in the treatment and prophylaxis of arterial and venous thromboembolic diseases. The treatment efficacy and safe use of warfarin is followed with prothrombin time, expressed as INR. INR values exceeding the therapeutic levels lead to increase in hemorrhage risk, whereas INR values below the therapeutic levels cause an increase in thromboembolic events [11],[12].

The differences among individuals regarding daily effective maintenance warfarin dose caused difficulty in establishing initial dose and follow-up of therapy. Warfarin dose requirement is affected by many factors such as sex, age, diet, race, concomitant medication, and genetic factors. In recent years, the influence of genetic factors on warfarin dose requirements had come under investigation [13].

Polymorphisms in the genes encoding for VKORC1 and CYP2C9 are known to contribute to variations in the sensitivity to warfarin. Multiple in-vivo studies also showed that several mutant CYP2C9 genotypes were associated with significant reduction in metabolism and daily dose requirements of selected CYP2C9 substrate [14]. Nucleotide variations in VKORC1 are a common cause of pharmacodynamic warfarin resistance [15].

In the present study, regarding the distribution of VKORC1 (1639 G>A) genotypes, we found that 65% of patients showed heterozygous form of the gene, 25% were homozygous, and 10% were wild type. In contrary, Herman et al. [16] reported that regarding VKORC1 genotypes, heterozygous VKORC1 genotype was found in 34.3% of patients, homozygous was found in 2.1% patients, and wild type was found in 63.6%% patients. Moreover, D’Andrea et al. [17] reported that the distribution of patients with VKORC1 (1639 G>A) genotype was 11 (45.8%) patients were heterozygous and 13 (54.2%) patients showed the wild type (1639 AA) of the gene. Budnitz et al. [18] reported that there were 35.7% heterozygous, 29.7% homozygous, and 32.5% wild type, which is different from the previous studies.

These allelic differences could explain in part the ethnic variability of warfarin dose requirements, as warfarin dose requirements vary significantly by race, with higher mean maintenance doses in African–Americans and lower mean doses in Asians compared with doses in white population [4],[19].

CYP2C9 is an important cytochrome P-450 enzyme with a major role in the oxidation of both xenobiotic and endogenous compounds. CYP2C9 makes up approximately 18% of the cytochrome P-450 protein in liver microsomes. Some 100 therapeutic drugs are metabolized by CYP2C9, including drugs with a narrow therapeutic index such as warfarin and phenytoin and other routinely prescribed drugs such as acenocoumarol, tolbutamide, and some NSAIDs. By contrast, the known extrahepatic CYP2C9 often metabolizes important endogenous compound such as arachidonic acid, 5-hydroxytryptamine, and linoleic acids [20].

In the present study, regarding the distribution of patients with CYP2C9 alleles according to sex, we found that they were five (83.333%) female patients with heterozygous CYP2C9 (430C>T), 19 (55.88%) female patients showing wild type of the gene, 15 (44.1%) male patients showing the wild type, and one (16.667%) male patient showing heterozygous type of the same gene. However, patients with CYP2C9 (1075A>C) allele had the distribution of 24 (60%) female and 16 (40%) male patients.

It is evident that ethnic differences in allelic frequencies of VKORC1–1639 G>A SNP and CYP2C9 *2 and *3 do exist. These allelic differences could explain in part the ethnic variability of warfarin dose requirements, as warfarin dose requirements vary significantly by race, with higher mean maintenance doses in African–Americans and lower mean doses in Asians compared with doses in white population [4],[21].

This difference in warfarin dose requirement could be attributed to the fact that CYP2C9*1 metabolizes warfarin normally, CYP2C9*2 reduces warfarin metabolism by 66%, and CYP2C9*3 reduces warfarin metabolism by 95%. Consequently, warfarin given to patients with *2 or *3 variants were metabolized less efficiently, and the drug will remain in circulation longer, so lower warfarin doses were needed to achieve anticoagulation [21].

In the present study, we found a significant statistical relation between CYP2C9 gene mutation and the age and insignificant with sex. On alignment of these observations, we found insignificant relation between CYP2C9 genotypes and the following parameters: response to vitamin K antagonist oral anticoagulation at a dose of more than or equal to 5 mg, response during duration of therapy of less than or equal to 3 months, and frequency of occurrence of DVT. Zhang et al. [22] revealed that the results of the association of each of the CYP2C9 genotypes with the warfarin maintenance dose was significantly related to the genotype.

In the present study, a significant association was found between VKORC1 genotypes and age. This is the same as Rettie et al. [21] who stated that there was a significant association between age and VKORC1.

As for nonresponder patients at Marevan dose of more than or equal to 5 mg, there were 13 (32.5%) patients. Their distribution according to CYP2C9 (430C>T) gene mutation revealed that three (23.1%) were heterozygous and 10 (76.9%) showed the wild type of the gene. Regarding their distribution according to VKORC1 (1639 G>A), six (46.2%) were heterozygous, six (46.2%) were homozygous, and one (7.6%) showed the wild type of the gene.

In the current study, 16 (40%) patients had a single attack of DVT. Of them, 12 (75%) were female and four (25%) were male patients, with a male to female ratio of 1 : 3. As for patients with recurrent attacks of DVT, there were 24 (60%) patients, with 13 (54.2%) females and 11 (45.8%) male patients, with a male to female ratio of 1 : 1.2.

The correlation between VKORC1 gene mutation and frequency of occurrence of DVT in the present study was found to be statistically insignificant.

In the present study, there was a significant relation between INR at more than 5 mg Marevan dose and genotypes of VKORC1 (1639 G>A) and CYP2C9 (430C>T). There were no data available in other studies regarding the relation between VKORC1 (1639 G>A) and CYP2C9 (430C>T) genotypes and INR at high dose of Marevan more than 5 mg.


  Conclusion Top


Detection of genetic polymorphisms in CYP2C9 and VKORC1 genes before the onset of warfarin therapy greatly influences response to warfarin and shortens the time required to reach target INR, and hence reduces the risk of recurrence of DVT.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]



 

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