Warfarin Response (CYP2C9 and VKORC1)

Clinical Usage

Background Information

Warfarin acts as an anticoagulant by binding to and inhibiting VKORC which is a clotting factor. CYP2C9 is an enzyme that metabolizes Warfarin for removal from circulation.  Warfarin occurs in two enantiomeric forms, the R- and the S-enantiomers. The S- form is 3-5 times more active than the R-enantiomer.  Polymorphisms in CYP2C9 and VKORC1 genes have been reported to affect Warfarin blood levels and efficacy in blood. Identifying inherited variants involved with warfarin metabolism or efficacy can be used to optimize dosing and reduce the time needed to achieve therapeutic International Normalized Ratio (INR). 

There are three phenotypic groups for Warfarin sensitivity associated with polymorphisms on CYP2C9 and VKORC1 genes: Normal, Moderate, and High. Normal phenotype individuals may be administered Warfarin following standard dosing guidelines. A Moderate phenotype is associated with increased Warfarin sensitivity. These individuals may be at increased risk of an adverse drug reaction and may require a reduced dose. Individuals with High Warfarin sensitivity require a lower Warfarin dose to achieve desired effects. 

Warfarin acts in the vitamin K cycle by interfering with the regeneration of 
reduced vitamin K from oxidized vitamin K in the VKOR (Vitamin K 
OxidoReductase) complex; the major subunit of VKOR is VKORC1.  Mutations in the VKORC1 gene cause decreased VKORC1 enzyme activity and result in changes in Warfarin sensitivity.  The VKORC1 G allele has normal enzyme activity and the A allele has decreased enzyme activity. 

The more active  S-form of Warfarin is metabolized by the cytochrome P450 enzyme CYP2C9. CYP2C9*1 is the normal, or wild type (wt) allele causing normal CYP2C9 enzyme activity and a normal or Extensive Metabolizer (EM) phenotype.  Most other CYP2C9 variant alleles cause a decrease in activity leading to an Intermediate Metabolizer (IM) or Poor Metabolizer (PM) phenotype, with moderately decreased or greatly decreased ability to metabolize Warfarin, respectively.  The decreased enzyme activity can cause an increase in warfarin sensitivity.

Other factors such as weight, body mass index, age, diet and concurrent medication are also known to affect the Warfarin dose requirement.

Gene Information

The CYP2C9 gene is located on chromosome 10, on the long arm (q) at location q24. More precisely, the CYP2C9 gene is located from base pair (bp) 96,698,349 to base pair 96,749,485 on chromosome 10.  The gene consists of 9 exons.  The *2 variant is found on exon 3 of the gene and is a substitution of a T for a C at bp position 430 of CYP2C9.  The *2 variant causes an amino acid change of an arginine to a cysteine at position 144 of the cytochrome P450 2C9.  The *3, *5, and *11 variants are found on exon 7 of the CYP2C9 gene.  The *3 allele is a substitution of a C for an A at bp position 1075.  The *3 variant causes an amino acid change from isoleucine to leucine at position 359 in the CYP2C9 protein.  The *5 allele is a substitution of a G for a C at bp 1080.  The *5 variant causes a change of aspartic acid to a glutamic acid at position 360 of the CYP2C9 protein.  The *11 variant causes a mutation of arginine to tryptophan at amino acid position 335 of the CYP2C9 protein.

The VKORC1 gene is located on the short (p) arm of chromosome 16 from base pair 31,102,174 to base pair 31,106,698.  The G1639A variant is a change of a G to an A in the promoter region of the gene which is the -1639 bp.  This variant may be called the *2 allele of VKORC1.

Population Information

This information was adapted from Cavallari et al, 2011, and from Cavallari and Perera, 2012.

Test Method

These assays were developed using CLSI guidelines.  Control DNA samples of known genotype are tested together with each patient sample to ensure correct results.  Genomic DNA is extracted from the submitted buccal swab sample and subjected to polymerase chain reaction (PCR).  The following CYP2C9 alleles were tested: *2, *3, *5,*11.  The wild type or normal allele (*1) was assigned by default if none of the variant alleles were detected.  The G1639A variant of VKORC1 was tested, and was labeled an A allele.  The normal VKORC1 allele was called the G allele.

Specimen

Collection

• Buccal swab
• Whole blood,  2-5 mL, into:
i. EDTA-containing tube (purple or lavender top), or
ii. ACD-containing tube (yellow top), or
iii. Citrate-containing tube (blue top).
• Store at 2-8°C.  Ship by overnight carrier at ambient temperature.

Rejection Criteria

• Buccal swab:
i. Physical damage
ii. Specimen appears to have microbial contamination or other visible  contamination
iii. The name on the tube does not match the name on the paperwork.
iv. It is older than 10 days.
• Blood specimen:
i. It is collected in a heparin-containing tube because heparin can inhibit  the PCR reaction.
ii. It leaked in the shipping container.
iii. The name on the tube does not match the name on the paperwork.

iv. It is older than 10 days.

Interpretation

CPT code

81355

Test Limitations

The detection of genetic variants does not replace the need for therapeutic drug monitoring or other appropriate clinical monitoring by the health care provider.  Additional mutations for the tested genes that are not described in the methodology section will not be detected.  CYP2C9 metabolism is also influenced by concomitant medications, inhibitors, inducers, diet and various disease states.  These tests were developed and the performance characteristics were determined by MDL.  The CYP2C9 and VKORC1 tests have not been cleared or approved by the US Food and Drug Administration.  The FDA has determined that such approval is not necessary.

This test is approved for use on New York state residents.

References

1.    Cavallari LH et al.  Role of Pharmacogenomics in the Management of Traditional and Novel Oral Anticoagulants. Pharmacotherapy  31(12): 1192-1207.  2011

2.    Cavallari LH and Perrera MA.  The future of warfarin pharmacogenetics in under-represented minority groups.  Future Cardiol.  8(4): 563-576.  2012

3.    Flockhart DA, et al.  Pharmacogenetic Testing of CYP2C9 and VKORC1 Alleles for Warfarin.  American College of Medical Genetics Policy Statement.  Genet Med  10(2): 139-150.  2008
doi: 10.1097/GIM.0b013e318163c35f

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5.Johnson JA, et al.  Clinical Pharmacogenetics Implementation Consortium Guidelines for CYP2C9 and VKORC1 Genotypes and Warfarin Dosing.  Clinical Pharmacology & Therapeutics.  2011 doi:10.1038/clpt.2011.185    

6.Jorgensen AL, et al.  Influence of CYP2C9 and VKORC1 on Patient Response to Warfarin:  A Systematic Review and Meta-Analysis.  PLOS ONE  7(8): 1-20.  2012 e44064.  doi:10.1371/journal.pone.0044064

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8.Klein TE, et al.  The International Warfarin Pharmacogenetics Consortium.  Estimation of the WWararin Dose with Clinical and Pharmacogenetic Data.  The New England Journal of Medicine.  360(8):753-764.  2009

9.Langley MR, Booker JK, Evans JP, McLeod HL, Weck KE.  Validation of Clinical Testing for Warfarin Sensitivity.  Comparison of CYP2C9-VKORC1 Genotyping Assays and Warfarin-Dosing Algorithms.  Journal of Molecular Diagnostics.  11(3): 216-225.  2009 DOI: 10.2353/jmoldx.2009.080123

10. Limdi NA, et al.  Influence of CYP2C9 and VKORC1 on Warfarin Response During Initiation of Therapy.  Blood Cells Mol Dis.  43(1): 119-128.  2009
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11. Limdi, Nita A., et al.  Warfarin Pharmacogenetics: a single VKORC1 polymorphism is predictive of dose across 3 racial groups. Blood Journal 115: 3827-3834.  2010 doi: 10.1182/blood-2009-12-255992.

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13. Reider, Mark J., et al.  Effect of VKORC1 Haplotypes on Transcriptional Regulation and Warfarin Dose. The New England Journal of Medicine 352(22): 2285-93.  2005

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17. Yuan HY, Chen JJ, Lee MT, Wung JC, Chen YF, Charng MJ, Lu MJ, Hung CR, Wei CY, Chen CH, Wu JY, Chen YT.  A novel functional VKORC1 promoter polymorphism is associated with inter-individual and inter-ethnic differences in warfarin sensitivity. Hum Mol Genet. 14(13):1745-51. 2005