Verigene^® Warfarin Metabolism Nucleic Acid Test (IVD)

Background

Warfarin is the most widely prescribed oral anticoagulant for thromboembolic therapy in North America and Europe^1,2. Warfarin therapy-associated hemorrhage is one of the leading causes of drug-related adverse events, including death, in many Western countries^3,4,5.

Both individualistic and genetic factors influence a given patient’s response to warfarin. In terms of individualistic factors, a patient’s age, body weight or body surface area, diet, concurrent medications, and other factors are all known to affect dose requirements. In terms of genetic factors, there are both pharmacokinetic and pharmacodynamic effects on warfarin treatment.

Warfarin consists of two racemic isomers -- an S-isomer and an R-isomer; the S-isomer is 3-5 times more potent than the R-isomer. The main metabolizing enzymes for warfarin are members of the cytochrome P450 family, with the S-isomer of warfarin specifically metabolized by cytochrome P450, subfamily IIc, polypeptide 9 protein (CYP2C9, hereafter). Genetic variations of CYP2C9 are responsible for the pharmacokinetic effect on warfarin metabolism. The molecular target of warfarin in vivo is the protein product of the Vitamin K Epoxide Reductase Complex, Subunit 1gene (VKORC1, hereafter), which is inhibited by warfarin. Genetic variations of VKORC1 are responsible for the pharmacodynamic affect on warfarin. In one study, combining the CYP2C9*2, CYP2C9*3, and VKORC1 1173C>T genotype results, as much as 56% of the inter-individual variability of the warfarin pharmacodynamic response was accounted for⁶.

CYP2C9
The CYP2C9 gene, located on the long arm of human chromosome 10 (10q24), plays a key role in the metabolism of the S-isomer of warfarin. Many allelic variants of CYP2C9 have been described in the literature, most of which encode dysfunctional or nonfunctional proteins. The two most common alleles of CYP2C9 that affect warfarin metabolism are CYP2C9*2 (also known as R144C) and CYP2C9*3 (also known as I359L). Polymorphisms at these alleles produce defective CYP2C9 protein that exhibits reduced activity for metabolizing warfarin. Individuals who are heterozygous or homozygous variant for either CYP2C9*2 or CYP2C9*3 are generally more sensitive to standard doses of warfarin than similar individuals who lack the variant⁷.

VKORC1
The molecular target of warfarin in vivo was recently characterized as the protein product of the VKORC1 gene^6,8. The VKORC1 gene, located on the short arm of human chromosome 16 (16p11.2), encodes the VKORC1 protein which plays an essential role in gamma-carboxylation of Vitamin K-dependent blood clotting factors. During carboxylation, reduced Vitamin K is oxidized to Vitamin K 2,3 epoxide, from which reduced Vitamin K is regenerated by VKORC1 to participate in another round of clot-promoting catalysis. Warfarin inhibits VKORC1, reducing clotting efficiency and creating the anticoagulation effect of the drug. Many polymorphisms have been described in the VKORC1 gene, which tend to occur in haplotype blocks with particular combinations of polymorphisms exhibiting strong linkage disequilibrium such that they are rarely observed in the absence of one another⁹. As a result, identification of one member of a haplotype block is often predictive for the overall haplotype. The VKORC1 polymorphism 1173C>T has been shown to be in strong linkage disequilibrium with another frequently assayed polymorphism, -1639G>A. Several large studies have genotyped subjects at both of these positions and confirmed the strong linkage disequilibrium between these two polymorphisms, suggesting that either may be used as a marker to identify a single phenotypic group^7,10.

Table 1 shows the allele frequency across various ethnic groups for the CYP2C9*2, CYP2C9*3, and VKORC1 mutations.

 

  

  

  

  

  

Intended Use

The Verigene^® Warfarin Metabolism Nucleic Acid Test is an in vitro diagnostic for the detection and genotyping of the *2 and *3 alleles of the CYP2C9 gene and a singlepoint polymorphism (C to T at position 1173) of the VKORC1 gene, from EDTA-anticoagulated whole blood samples, as an aid in the identification of patients at risk for increased warfarin sensitivity. The test is intended to be used on the Verigene^® System.

Clinical Study Highlights

Accuracy of the Verigene^® Warfarin Metabolism Nucleic Acid Test was assessed using two hundred thirty eight (n=238) clinical samples in comparison to bi-directional DNA sequence analysis of the same samples. The percent agreement between the two methods was 100%. In a three-site study designed to assess reproducibility of the Verigene^® Warfarin Metabolism Nucleic Acid Test, five genomic DNA samples, covering all possible genotypes for all three alleles, were each tested in triplicate on a daily basis by the same operators for three days. One site performed the reproducibility testing twice each day, using two different operators. The qualitative reproducibility between all sites, lots, and operators showed 100% agreement between the calls made and expected results.

Literature Cited

1. Scordo MG et al. Clin Pharmacol Ther 2002; 72: 702-710.
2. Coumadin® (Warfarin Sodium Tablets, USP) website: http://www.coumadin.com/coumadin/home/consumer_index.jsp?BV_UseBVCookie=Yes
   Please see Full Prescribing Information.
3. Landefeld C & Beyth R. Am J Med 1993; 95: 315-318.
4. Levine M et al. Chest 1998; 114: 511S-523S.
5. Pirmohamed M et al. BMJ 2004; 329: 15-19.
6. Wadelius M et al. Pharmacogenetics J 2005; 5: 262-270.
7. Rieder MJ et al. NEJM 2005; 352: 2285-2293.
8. Rost S et al. Nature 2004; 427: 537-541.
9. Li T et al. Nature 2004; 427: 541-544.
10. McCain MR et al. A rapid ACCE review of CYP2C9 and VKORC1 allele testing to inform warfarin dosing in adults at elevated risk for thrombotic events to avoid serious bleeding. 2007.
11. Schelleman H et al. Clin Pharmacol Ther 81(5):742:747.
12. Lee CR et al. Pharmacogenetics 2002; 12: 251-263.

 

 

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