The test genotypes three polymorphisms in two genes that correlate with warfarin dose and allow individualization of therapy based on genotype. The more active S-enantiomer of warfarin is metabolized to inactive forms by the liver enzyme cytochrome P450 (CYP450) 2C9. Two polymorphisms in the gene for CYP450 2C9 (*2, *3) reduce enzyme activity and are correlated with reduced warfarin dosage and an increased incidence of adverse side effects. The vitamin K epoxide reductase (VKOR) enzyme participates in the pathway of reactions leading to activation of clotting factors and is the target of warfarin action. A warfarin-sensitive haplotype has been identified, and the promoter polymorphism –1639G&gt;A can identify patients with the warfarin-sensitive genotype (–1639AA).
Genotyping of mutations or polymorphisms uses allele-specific signal probes containing ferrocene labels with distinguishable redox potentials. The signal probe matching the wild-type sequence contains a ferrocene label of one electrochemical potential, and a second signal probe matching the mutant sequence contains a second, distinguishable ferrocene label. Both the wild-type and mutant targets bind to the capture probe at a site adjacent to the mutation. Capture probes covalently bound to the electrode hybridize equally to target DNA with both wild-type (WT) and mutant (MUT) sequences. Signal probes complementary to the WT and MUT target sequences are present in the hybridization buffer, and contain ferrocene labels with different redox potentials. The signal probes compete for binding to the region of the target DNA containing the mutation site, and binding of the perfect-match signal probe (i.e., WT signal probe to WT target) predominates. The genotype is determined by measuring the ratio of electrochemical signals from the WT and MUT signal probes
Oxidation of the ferrocene labels and transmission of current through the monolayer depend on proximity of the label to the monolayer surface. As a result, an unbound signal probe is not detected, and washing steps are not required to remove unbound reagents prior to the ACV measurement, even when a large number of signal probes representing multiple target sequences are present. The assay process is simplified, which allows hybridization and detection to be done in a small-footprint instrument without fluid handling or waste containers.
So, for the wildtype, the signal will look like this. For a mutant type, the signal will be offset to indicate that it is not the same signal as a wildtype. For a heterozygote, which carries both a wildtype and mutant form of the gene, the signal will fall under the areas of the wildtype signal and of the mutant signal, with a lower value.
Development of Warfarin Sensitivity Test based on Genetic
Variability in P-450 Enzymes: A Pharmacogenomic Approach
Suneel A. Chhatre*, Erum F. Naqvi¹
*Chemistry & Cell Biology, Medical University of Americas
(MUA), Nevis, WI
¹Centre for Molecular Diagnostics, Princeton Biomedical
Laboratories, PA, USA
MUA Research Day Spring 2010
• Clinical Challenges of Warfarin Safety
• Warfarin Dosing & Inter-Individual Variation
• Case Study
• Warfarin Dosing Algorithms
• Why Test?
• Princeton Solutions
• Widely prescribed dangerous drug.
– 2 million on warfarin, 30 million Rx a year.
– 43,000 ER visits a year, 2nd to Insulin for ER adverse drug
– 87,000 major bleeding events a year.
– 17,000 strokes a year.
– 10,000 deaths a year.
Source: FDA, AEI-Brookings Joint Center, and The Joint Commission
Risks of Warfarin ADR
Strongly Depend on INR Value
• INR below 2 = high risk of stroke
• INR above 4 = high risk of hemorrhage
•Narrow therapeutic index & non-
•Patients within target INR only
32% to 56% of time.
•Dose influenced by age, ethnicity,
drugs, environmental and genetic
Current Methods for Warfarin Dosing
• Initial dose can be modified
by age, gender, body mass,
• This will predict only 17-21% of
the inter-individual variation.
• Subsequent dosing based on
Genetics & Warfarin Dosing
• Single base pair changes in DNA
sequence lead to reduced activities
in two genes.
• These two genes play a key role in
the patient’s response to Warfarin.
− Response – VKORC1 (1386)
− Metabolism – CYP2C9 *2 & *3
Vitamin K Epoxide Reductase 1 (VKORC1)
• Variations explain up to 25% of patient
variability in Warfarin dose response.
• Approximately 37% of Caucasians, 14%
of African-Americans, and 89% of
Asians carry at least one variant copy.
• Patients with certain VKORC1
variations have an increased risk for
anticoagulant overdose, and may
require lower doses of Warfarin to
achieve and maintain therapeutic
Warfarin Dose & VKORC1
Cytochrome P450 2C9 (CYP2C9)
• Variations explain approximately 15%
of patient variability in Warfarin dose
• Approximately 20% of Caucasians, 5%
of African-Americans, and 2% of Asians
carry at least one variant copy.
• Patients with CYP2C9 gene variations
require more time to achieve stable
INR, are at an increased risk of
bleeding, and may require lower doses
of Warfarin to achieve and maintain
Warfarin Dose & CYP2C9
• Patient profile:
− 65 years old
− White male
− 260 LBS, 5’9” tall
− Taking Lipitor®
− Diagnosis: Deep Vein Thrombosis
• Therapeutic dose:
5.6 mg/day using available clinical data and
Case Study Cont’d
• Now we genotype this patient:
– CYP2C9 *3/*3
– VKORC1 A/A
• Resulting optimal warfarin dose:
− Loading dose: 4.1 mg
− Therapeutic dose: 1.8 mg/day
Without genotype data, this
patient would have INR value
>4, with potential hemorrhage,
and slow return to therapeutic
PCR Reaction Mix
PCR Master Mix
HYBRIDIZATION / DETECTION
Molecular Cellular & Development
University of Colorado at Boulder,
• NIH, NSF, DOE
Chemistry, Eastern New Mexico
• NIH NCRR P20-61480