Warfarin Sensitivity Genotype by Sequence Analysis, Blood
Clinical Information Discusses physiology, pathophysiology, and general clinical aspects, as they relate to a laboratory test
Warfarin is a coumarin-based drug commonly utilized in anticoagulation therapy to prevent thrombosis due to inherited and acquired hemostatic disorders. The drug is also used in a number of other medical conditions and treatments including atrial fibrillation and hip replacement surgery. Warfarin acts by interfacing with the metabolism of vitamin K, which is necessary for production of key coagulation factors. Warfarin inhibits vitamin K recycling by blocking its metabolism at the vitamin K-epoxide intermediate thereby decreasing the amount of available vitamin K. Warfarin has a narrow therapeutic window; under medicating increases the risk for thrombosis and overmedicating increases the risk for cerebrovascular accidents. Warfarin therapy has one of the highest rates of severe adverse drug reactions.
Warfarin is dosed using non genetic factors including gender, weight, and age, and is monitored by coagulation testing in order to maintain the international normalized ratio (INR) within specific limits. However, warfarin metabolism is highly variable and dependent upon genetic factors. Polymorphisms within 2 genes are known to affect the metabolism of warfarin and the dose needed to maintain the correct serum drug level and degree of anticoagulation, as measured by the INR.
The cytochrome P450 2C9 gene (CYP2C9) encodes an enzyme that metabolizes the more active isomer of warfarin (S-warfarin) to inactive products. Polymorphisms in this gene decrease the activity of the enzyme and may cause increases in serum warfarin and overmedicating, driving INR above the therapeutic target level.
The second gene (VKORC1) encodes vitamin K epoxide reductase complex subunit-1 (VKORC1), a small transmembrane protein of the endoplasmic reticulum that is part of the vitamin K cycle and the target of warfarin therapy.(1) VKORC1 is primarily transcribed in the liver, although it is present in smaller amounts in the heart and pancreas. Vitamin K epoxide, a by-product of the carboxylation of blood coagulation factors, is reduced to vitamin K by VKORC1. A polymorphism within the promoter of VKORC1 decreases expression of the gene, decreasing the availability of vitamin K. This may cause increases in serum warfarin and overmedicating, driving INR above the therapeutic target level.
Thus, in both situations (polymorphisms in either CYP2C9 or VKORC1), a reduced warfarin dose is needed to compensate for the effects of the polymorphism in order to maintain the target INR.
CYP2C9 metabolizes a wide variety of drugs including warfarin and many nonsteroidal anti-inflammatory drugs. It is also partially responsible for metabolizing other drugs such as fluoxetine, fluvastatin, phenytoin, and oral hypoglycemic drugs.
A number of specific polymorphisms have been found in the CYP2C9 gene that results in enzymatic deficiencies. The following information outlines the relationship between the polymorphisms detected in this assay and the effect on the activity of the enzyme encoded by that allele:
Effect on Enzyme Metabolism
None (wild type)
Extensive metabolizer (normal)
Dosing of warfarin, which is metabolized through CYP2C9, may require adjustment for the individual patient. Patients who are poor metabolizers (reduced activity) may benefit by dose reductions or by being switched to other comparable drugs that are not metabolized primarily by CYP2C9. The following is a partial listing of drugs known to affect CYP2C9 activity as of the date of this report. A more complete listing is presented in the drug label, available from URL:
Drugs that undergo metabolism by CYP2C9:
-Angiotensin II blockers: irbesartan, losartan
-Anticoagulants: warfarin (more active S-isomer)
-Antidepressants: amitriptyline (minor), fluoxetine
-Nonsteroidal anti-inflammatory drugs (NSAIDS): celecoxib, diclofenac, ibuprofen, naproxen, piroxicam, suprofen
-Oral hypoglycemic agents: glipizide, glimepiride, glyburide/glibenclamide, nateglinide, tolbutamide
-Miscellaneous drugs: fluvastatin, phenytoin, sulfamethoxazole, tamoxifen, torsemide
-Coadministration of these drugs may decrease the rate of elimination of other drugs metabolized by CYP2C9
Drugs known to increase CYP2C9 activity:
-Phenobarbital, rifampin, secobarbital
-Coadministration of these drugs increase the synthesis of CYP2C9, resulting in increased CYP2C9 activity and metabolism of warfarin. A dose increase may be needed to maintain the INR in the target range
Drugs known to decrease CYP2C9 activity:
-Amiodarone, fenofibrate, fluconazole, fluvastatin, isoniazid, lovastatin, phenylbutazone, sertraline, sulfamethoxazole, sulfaphenazole, teniposide, ticlopidine, voriconazole, zafirlukast
-Coadministration of these drugs may decrease the rate of metabolism of CYP2C9-metabolized drugs, including warfarin, increasing the possibility of toxicity
The -1639 promoter polymorphism is located in the second nucleotide of an E-Box (CANNTG) and its presence disrupts the consensus sequence, reducing promoter activity. In vitro experiments show a 44% higher transcription level of the G versus the A allele.(1) The -1639 G->A nucleotide change results in decreased gene expression and reduced enzyme activity.
Identifying patients who may require warfarin dosing adjustments(2,3) including:
-Patients who have previously been prescribed warfarin and have required multiple dosing adjustments to maintain the international normalized ratio in the target range
-Patients with a history of thrombosis or bleeding when previously taking warfarin
-Patients being started on a first prescription for warfarin
An interpretive report will be provided.
The normal genotype (wild-type) for CYP2C9 is termed CYP2C9*1. Other genotypes that lead to inactive or reduced activity alleles include CYP2C9*2, CYP2C9*3, CYP2C9*4, CYP2C9*5, and CYP2C9*6. An individual who has homozygous wild-type, CYP2C9*1/CYP2C9*1, is considered an extensive metabolizer.
The normal genotype for VKORC1 is -1639G. A polymorphism at -1639A reduces VKORC1 expression. The VKORC1 GA or AA genotype leads to a significant decrease in mRNA expression in the liver compared with individuals with the GG genotype.
Individuals who have polymorphisms in both the VKORC1 promoter (GA or AA) and also in CYP2C9 should receive a reduced dose of warfarin to maintain the international normalized ratio in the target range; dosing adjustments are required when polymorphisms in both genes are present.
Drug-drug interactions and drug-metabolite inhibition must be considered when dealing with heterozygous individuals. Drug-metabolite inhibition can occur, resulting in inhibition of residual functional CYP2C9 or VKORC1 catalytic activity. A clinical pharmacologist should be consulted for assessing the potential for drug interactions.
Patients may also develop toxicity problems if liver and kidney function are impaired.
Cautions Discusses conditions that may cause diagnostic confusion, including improper specimen collection and handling, inappropriate test selection, and interfering substances
This assay should be ordered on patients who require decreased warfarin dosing to maintain the international normalized ratio in the therapeutic range.
This test detects only the specified polymorphisms. Additional findings, such as small insertions and deletions or novel mutations, will be reported if found. Other polymorphisms in the primer binding regions can affect the testing, and, ultimately, the genotyping assessments made.
Warfarin metabolism may be inhibited through drug-drug interactions, including amiodarone and statins.
Genotyping patients using DNA obtained from leukocytes may not provide useful information in patients who have had a bone marrow or liver transplant or a recent transfusion. To obtain an accurate genotype on a bone marrow transplant recipient, buccal cells should be provided. To obtain an accurate genotype for a patient who has received a donor liver, testing must be done on donor cells. If the patient has been transfused, wait 4 to 6 weeks until transfused cells have left the circulation.
Reference Values Describes reference intervals and additional information for interpretation of test results. May include intervals based on age and sex when appropriate. Intervals are Mayo-derived, unless otherwise designated. If an interpretive report is provided, the reference value field will state this.
An interpretive report will be provided.
Clinical References Provides recommendations for further in-depth reading of a clinical nature
1. Oldenburg J, Bevens C, Muller C, Watzka M: Vitamin K epoxide reductase complex subunit I (VKORC1): the key protein of the vitamin K cycle. Antioxid Redox Signal 2006;8(3-4):347-353
2. Yuan H, Chen J, Lee M, et al: A novel functional VKORC1 promoter polymorphism is associated with inter-individual and inter-ethnic differences in warfarin sensitivity. Hum Mol Genet 2005;14:1745-1751
3. Sconce E, Khan T, Wynne H, 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;106:2329-2333