Evaluation of the eSensor® XT-8 System for Determining CYP2C9 and VKORC1 Genotypes to Aid in Correct Warfarin Dosing

Abstract: 

Warfarin is a commonly prescribed anticoagulant used to treat a variety of disorders; however it has a narrow therapeutic window and response varies widely among individuals. Addition of genetic information, especially for genes CYP2C9 and VKORC1, could potentially improve dosing algorithms. The eSensor® XT-8 is novel technology capable of detecting polymorphisms in these genes. CYP2C9 is the gene that codes for the enzyme that metabolizes warfarin into its inactive metabolites, and mutant *2 and *3 alleles decrease warfarin clearance. VKORC1 codes for an enzyme the recycles reduced vitamin K making it available for use by vitamin K-dependent coagulation factors, and the mutant A allele decreases action. The anticoagulant activity of warfarin is due to inhibition of the VKORC1 enzyme. The results of this study revealed that 47% of the 98 participants had the mutant A allele in the VKORC1 gene and 34% had the mutant *2 or *3 allele for CYP2C9. As a result, these individuals are likely more sensitive to the action of warfarin and/or less effective at clearing the drug. Genetic information could be helpful in determining warfarin dosage but is only one of many factors in warfarin dosing, including age, weight, height, and ethnicity. Validation of the eSensor® Warfarin Sensitivity Test is ongoing at Scott and White Memorial Hospital.

Table of Contents: 

    Introduction

    The drug warfarin, also known as Coumadin®, is one of the most commonly prescribed anticoagulants worldwide, with more than 21 million prescriptions in 2003 in the United States alone (Caraco, Blotnick, and Muszkat 2008). It is also the only available oral anticoagulant. Warfarin is used to treat a variety of thromboembolic disorders such as superficial and deep vein thrombosis and pulmonary embolism. It is also prescribed prophylactically to prevent thrombosis in patients at risk of stroke or who have suffered trauma, surgery, or myocardial infarction (Rodak et al. 2007). The drug exerts its anticoagulant effect by inhibiting the vitamin K epoxide reductase (VKORC1) enzyme that recycles reduced vitamin K. Several coagulation factors, including factors II, VII, IX, and X are dependent on vitamin K, and its absence results in noncarboxylated, nonfunctional factors. As a result, therapeutic warfarin dosage decreases the active form of each factor by approximately 30% to 50% (Bristol-Myers Squibb n.d.; Rodak et al. 2007; Wang et al. 2008).

    Unfortunately, warfarin has a rather narrow therapeutic window and must maintain the prothromin time (PT) expressed as the international normalized ratio (INR) between 2.0 and 3.0 (McMahan et al. 1998; Sconce et al 2005). Any deviations below 2.0, the patient is at risk of recurring thromboembolism, and an INR above 3.0 increases the risk of hemorrhage and uncontrolled bleeding. This risk is increased especially if the patient has a history of alcohol abuse, chronic renal insufficiency, or previous gastrointestinal bleeds (McMahan et al. 1998). In addition, individuals’ response to warfarin can vary greatly, leading to the need for a more individualized dosing regimen.

    It has been previously reported that, in addition to such factors as age and body weight, a person’s genotype also plays a role in achieving a stable warfarin dose. Two of the most important genes in warfarin metabolism and efficacy are CYP2C9 and VKORC1. Warfarin is administered as a racemic mixture of R and S enantiomers and most of the anticoagulant activity (66%) is attributed to the S form. However, it also has a more rapid clearance. CYP2C9 encodes for the polymorphic enzyme that metabolizes warfarin into its inactive metabolites and two variant alleles, CYP2C9*2 and CYP2C9*3, are known to cause decreased S-warfarin clearance. Thus, individuals who carry one or more of these alleles require lower doses of warfarin and are more at risk of overdosage and bleeding episodes (Bristol-Myers Squib n.d.; Caraco, Blotnick, and Muszkat 2008; Linder 2009). Variants of the VKORC1 gene can also affect warfarin dosage required to achieve a therapeutic INR of 2.0 to 3.0. Polymorphisms in the promoter SNP-1639G>A account for a large amount of the variability in VKORC1 activity. The -1639 A allele is associated with reduced dosage requirements because VKORC1 activity can be as low as half that of other haplotypes for individuals carrying this allele (Briston-Myers Squib, Sconce et al. 2005; Wang et al. 2008).

    As a result, knowing an individual’s genotype at these two loci could lead to a much faster and safer determination of optimal warfarin dosage and decrease the incidence of bleeding and/or subtherapeutic dosing. This might also decrease many health care providers’ apprehension towards prescribing the medication to patients who could really benefit from its use. The purpose of this study is to evaluate the use of the eSensor® XT-8 system for determining CYP2C9 and VKORC1 genotypes at Scott & White Memorial Hospital to potentially help determine appropriate warfarin dosing regimens.

    Materials and Methods

    eSensor® XT-8 System

    The eSensor® XT-8 System, manufactured by Osmetech Molecular Diagnostics, is “an in vitro diagnostic device intended for genotyping multiple mutations or polymorphisms in an amplified DNA sample utilizing electrochemical detection technology (Osmetech Molecular Diagnostics n.d., 1)” The system requires single-stranded DNA that has been amplified by multiplex PCR and digested by exonuclease. The specimen combines with signal buffer that contains allele-specific oligonucleotide probes for the tested polymorphisms, and these probes are labeled with a genotype-specific ferrocene derivative. This mixture is loaded into the cartridge that contains single-stranded capture probes bound to gold plated electrodes. After being loaded into the XT-8 System, the targets hybridize to the complementary sequences of both probes and the genotype of the polymorphism is determined by voltammetry (Osmetech Molecular Diagnostics n.d.).

    Sample Collection

    The samples that were genotyped were selected at random by the Scott & White Pharmacy from patients that were currently undergoing Coumadin therapy, and the sample was whole blood in a blue top tube containing the anticoagulant sodium citrate.

    Materials

    The materials required for the extraction, amplification, exonuclease digestion, and genotyping of the sample include: PCR grade water, vortex mixer, 2 mL tubes, microfuge tubes, heat block, pipettes, thermal cycler, and micro-centrifuge. Extraction requires nucleic acid isolation reagent cartridges. In addition, the following are required and provided by Osmetech Molecular Diagnostics, including: Warfarin Sensitivity Test Cartridge, Warfarin Sensitivity Test PCR mix, Taq polymerase, exonuclease, Warfarin Sensitivity Test Signal Buffer, XT Buffer-1, and XT Buffer-2. The procedure is as follows, with additional information contained in the Single-channel eSensor® Warfarin Sensitivity Test Worksheet.

    Procedure

    Extraction

    The extraction was carried out using the MagNA Pure Compact Nucleic Acid Isolation Kit I manufactured by Roche. The required sample was 200 µL of anticoagulated whole blood. The extraction process consisted of 4 stages: lysing, binding, washing, and eluting.

    Amplification

    The amplification of the sample used PCR methods. The PCR Master Mix was made according to the “PCR Master Mix Set-Up” located on the Single-channel eSensor®Warfarin Sensitivity Test Worksheet and contained 28 µL of PCR Mix and 2 µL Taq polymerase for a total of 30 µL per sample. The PCR Master Mix was dispensed into labeled PCR reaction tubes, followed by the addition of 5 µL of genomic DNA (2-200 ng/µL). A negative control containing 5 µL of water was also set up. All tubes were mixed and placed into a thermal cycler to amplify according to the “Thermal Cycling Protocol-PCR” located on the Single-channel eSensor® Warfarin Sensitivity Test Worksheet.

    Exonuclease Digestion

    After amplification was completed, the tubes were removed from the thermal cycler. Five µL of exonuclease was then added to each PCR tube. The tubes were mixed and placed back into the thermal cycler according to the “Thermal Cycling Protocol-Exonuclease” located on the Single-channel eSensor® Warfarin Sensitivity Test Worksheet.

    Hybridization and Genotyping

    The Hybridization Master Mix was made according to the “Hybridization Solution Set-Up” located on the Single-channel eSensor® Warfarin Sensitivity Test Worksheet and included 70 µL of Warfarin Sensitivity Test Signal Buffer, 10 µL XT Buffer-1, and 20 µL XT Buffer-2. The XT Buffer-1 was added before the XT Buffer-2. 100 µL of Master Mix was added to each PCR tube containing exonuclease-digested amplicon. The PCR tubes were mixed, and then 125 µL of Hybridization-sample mix were pipetted into labeled test cartridges. After closing the cartridge and entering appropriate sample and reagent information into the XT-8 System, the cartridge was placed into the instrument and locked into place. The XT-8 then performed genotyping analysis.

    Results

    A total of 98 patients were genotyped for both the CYP2C9 and VKORC1 genes with the following results. The patient genotypes for VKORC1, located in Table 1, showed that the majority (53%) of individuals have the normal GG allele combination, with 35% and 12% of individuals having the GA and AA combinations respectively. However, if the data is separated by ethnicity, as in Table 4, one can see that the variant alleles are found at a much higher frequency in Caucasian individuals than in either African American or Hispanic individuals. The patient genotypes for CYP2C9, located in Table 2, again showed that the majority of those tested (65%) have the normal genotype, *1/*1. Of the remainder, 21% have the *1/*2 allele combination, 11% have *1/*3, and *2/*2 and *2/*3 are found in one individual each. The variant alleles at this locus were also seen more in Caucasian individuals than in African Americans and Hispanics.

    Discussion

    The eSensor® XT-8 System is a novel instrument that uses a solid-phase electrochemical method to determine the genotype for several different polymorphisms, including the CYP2C9 and VKORC1 genes. The results show that the majority of individuals carry the normal *1/*1, GG genotypes and can be expected to show normal warfarin metabolism and sensitivity, based solely on genetics and disregarding other factors. The *2 allele and *3 allele, caused by the 430C>T and 1075A>C polymorphisms in the CYP2C9 gene respectively, occur at a lower frequency. However, patients with this genotype do not metabolize warfarin as well as those without the polymorphism and are at risk of over-dosage. The data also shows that the VKORC1 polymorphic A allele is much more common in Caucasian individuals than in African American and Hispanic individuals; however, the small number of these individuals sampled could play a role. These numbers are supported by the literature, which states that distribution of the polymorphism is highly population-dependent and can be found in approximately 40% of Caucasian individuals and up to 90% of Asian individuals (Yuan et al. 2005). Individuals with a GA or AA genotype have an increased sensitivity to the actions of warfarin and are also at risk of over-dosage.

    The eSensor® XT-8 system is currently undergoing validation for the Single-channel Warfarin Sensitivity Test Kit, but there are some who question the usefulness of genetic information in warfarin dosing regardless of the validity. Most modern warfarin dosing algorithms include multiple factors when calculating an appropriate dose, and some say the high cost of molecular testing is not economical for the information gained. For example, one individual in the study who had the genotype *1/*2 A/A was given 1 mg of warfarin on day 1 of the dosing regimen, and the subsequent INR was 3.0, already almost supra-therapeutic. Another individual in the study with the same genotype was given 2.5 mg of warfarin and had a sub-therapeutic INR of 1.5 on day 1 and 1.8 on day 3. This shows the variability in dosing requirements because, in addition to genotype, an individual’s age, weight, height, ethnicity, overall health, and current medications can affect the warfarin dose needed. It has been shown previously that required dosage falls with age and that warfarin dosage is positively correlated with weight and height. Liver health also affects required dosage, as this is the site of action for both enzymes and where coagulation factors are produced. Alcohol use, through its effects on the liver, also affects dosage. Also such varied medications as statins and antifungals can affect warfarin metabolism. This can lead to markedly different responses to the same dosage.

    Additional studies are needed to confirm the usefulness of genetic information in warfarin dosing regimens, and validation of the eSensor® XT-8 system at Scott & White is ongoing. Future studies should use a larger sample population and include more individuals from minority ethnicities. Also, if one could isolate all of the variables in warfarin dosing, the information might be more helpful. That being said, molecular testing could soon provide doctors one more piece of information in determining safe and effective doses of warfarin for patients who need the drug.

    References

    • Bristol-Myers Squibb Company, comp. Coumadin Tablets (Warfarin Sodium Tablets, USP) Crystalline. Bristol-Myers Squibb.
    • Caraco, Y., S. Blotnick, and M. Muszkat. 2008. CYP2C9 Genotype-guided Warfarin prescribing enhances the efficacy and safety of anticoagulation: A prospective randomized controlled study. Clinical Pharmacology & Therapeutics 83: 460-70.
    • Linder, Mark. 2009. Rationale for CYP2C9/VKORC1 genotyping of patients prescribed Warfarin.” Critical Values 2: 14-17.
    • McMahan, Deborah, David Smith, Mark Carey, and Xiao Zhou. 1998. Risk of major hemorrhage for outpatients treated with Warfarin. Journal of General Internal Medicine 13: 311-16.
    • Rodak, Bernadette F., George A. Fritsma, and Kathryn Doig. 2007. Hematology Clinical Principles and Applications, 3rd ed. Philadelphia: Saunders.
    • Sconce, Elizabeth, Tayyaba Khan, Hilary Wynne, Peter Avery, and Louise Monkhouse. 2005. The impact of CYP2C9 and VKORC1 genetic polymorphism and patient characteristics upon Warfarin dose requirements: Proposal for a new dosing regimen.” Blood 106: 2329-333.
    • Wang, Danxin, Huizi Chen, Kathryn Momary, Larisa Cavallari, Julie Johnson, and Wolfgang Sadee. 2008. Regulatory polymorphism in Vitamin K Epoxide Reductase Complex Subunit 1 (VKORC1) Affects gene expression and Warfarin dose requirement. Blood 112: 1013-021.
    • Yuan, HY, J Chen, M Lee, J Wung, and Y Chen. 2005. A novel functional VKORC1 promoter polymorphism is associated with inter-individual and inter-ethnic differences in Warfarin sensitivity.” Human Molecular Genetics 14: 1745-1751.
    • Osmetech Molecular Diagnostics. “Single-channel eSensor® Warfarin Sensitivity Test Kit Product Insert.” Product Insert.

    Table 1: VKORC1 Genotypes

    VKORC1
    Genotype

    Number

    Percent

    GG

    52

    53%

    GA

    34

    35%

    AA

    12

    12%

    Total #

    98

     

    GG represents the normal genotype, with the other two possibilities containing the mutant A allele at the SNP-1639 location of the VKORC1 gene.

    Table 2: CYP2C9 Genotypes

    CYP2C9

    Genotype

    Number

    Percent

    *1/*1

    64

    65%

    *1/*2

    21

    21%

    *1/*3

    11

    11%

    *2/*2

    1

    1%

    *2/*3

    1

    1%

    Total #

    98

     

    The *1/*1 allelic combination represents a normal genotype, the other combinations contain one or more of the mutant *2 and/or *3 alleles.

    Table 3: Polymorphisms in the eSensor® Warfarin Sensitivity Test Panel

    Polymorphism

    Allele

    CYP450 2C9 430 C>T

    *2

    CYP450 2C9 1075 A>C

    *3

    VKORC1 -1639G>A

    GG, GA, or AA

    Source: Osmetech Single-channel eSensorÒ Warafarin Sensitivity Test Kit Product Insert.

    Table 4: Genotype by Ethnicity

    Caucasian

    VKORC1

    Genotype

    Number

    Percent

    CYP2C9

    Genotype

    Number

    Percent

    GG

    33

    45%

    *1/*1

    47

    64%

    GA

    30

    40%

    *1/*2

    18

    24%

    AA

    11

    15%

    *1/*3

    9

    12%

    Total #

    74

     

     

    74

     

     

    African American

    GG

    12

    100%

    *1/*1

    11

    92%

    GA

    0

     

    *1/*2

    0

     

    AA

    0

     

    *1/*3

    1

    8%

    Total #

    12

     

     

    12

     

     

    Hispanic

    GG

    5

    71%

    *1/*1

    5

    71%

    GA

    1

    14%

    *1/*2

    2

    29%

    AA

    1

    14%

    *1/*3

    0

     

    Total #

    7

     

     

    7

     

    Note that two white individuals, one with the genotype *2/*2, GA and one with the genotype *2/*3, GA, as well as 3 individuals with unknown ethnicity, are not listed on this table.