Molecular Diagnostics


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Development of Warfarin Sensitivity Test based on Genetic Variability in P-450 Enzymes: A Pharmacogenomic Approach

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  • 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>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.
  • Molecular Diagnostics

    1. 1. Princeton Inc 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
    2. 2. Princeton Inc 2 Overview • Clinical Challenges of Warfarin Safety • Warfarin Dosing & Inter-Individual Variation • Case Study • Warfarin Dosing Algorithms • Why Test? • Princeton Solutions
    3. 3. Princeton Inc 3 Clinical Challenge: Warfarin Safety • 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 reaction (ADR) – 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
    4. 4. Princeton Inc 4 Risks of Warfarin ADR Strongly Depend on INR Value • INR below 2 = high risk of stroke • INR above 4 = high risk of hemorrhage
    5. 5. Princeton Inc 5 Warfarin Dosing Inter-individual Variation •Narrow therapeutic index & non- linear response. •Patients within target INR only 32% to 56% of time. •Dose influenced by age, ethnicity, drugs, environmental and genetic factors.
    6. 6. Princeton Inc 6 Current Methods for Warfarin Dosing • Initial dose can be modified by age, gender, body mass, co-morbidities. • This will predict only 17-21% of the inter-individual variation. • Subsequent dosing based on INR.
    7. 7. Princeton Inc 7 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
    8. 8. Princeton Inc 8 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 INR. Warfarin Dose & VKORC1
    9. 9. Princeton Inc 9 Cytochrome P450 2C9 (CYP2C9) • Variations explain approximately 15% of patient variability in Warfarin dose response. • 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 therapeutic INR. Warfarin Dose & CYP2C9
    10. 10. Princeton Inc 10 Case Study • 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 existing algorithms.
    11. 11. Princeton Inc 1111 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 INR
    12. 12. Princeton Inc Pharmacogenetics: Warfarin • CYP450 2C9 Gene – Warfarin Metabolism – 2C9 430C>T (*2) Allele – 2C9 1075A>C (*3) Allele • VKCOR Gene – Warfarin Sensitivity – VKCORC1 -1639G>A Allele
    13. 13. Princeton Inc Warfarin Metabolism 2C9 Genotype/Phenotype Allele Genotype Result Summary Result Phenotype Description 430C>T (*2) C/C 2C9 *1/*1 Extensive (normal) warfarin metabolism anticipated 1075A>C (*3) A/A 430C>T (*2) C/T 2C9 *1/*2 Intermediate warfarin metabolism anticipated 1075A>C (*3) A/A 430C>T (*2) C/C 2C9 *1/*3 Slow warfarin metabolism 1075A>C (*3) A/C 430C>T (*2) T/T 2C9 *2/*2 1075A>C (*3) A/A 430C>T (*2) C./T 2C9 *2/*3 1075A>C (*3) A/C 430C>T (*2) C/C 2C9 *3/*3 Extremely slow warfarin metabolism anticipated 1075A>C (*3) C/C
    14. 14. Princeton Inc Warfarin Sensitivity VKORC1 Genotype/Phenotype Allele Genotype Result Summary Result Phenotype Description -1639G>A G/G -1639 G/G Low warfarin sensitivity anticipated G/A -1639 G/A Intermediate warfarin sensitivity anticipated A/A -1639 A/A High warfarin sensitivity anticipated
    15. 15. Princeton Inc Signal Probe Ferrocene Label Target DNA Capture Probe Hybridization of Target at Working Electrode Surface eSensor® DNA Detection Technology
    16. 16. Princeton Inc eSensor® DNA Detection Technology Insulator Electrode CaptureProbe TargetMolecule CaptureProbeWT Signal ProbeLabel 1 WT TargetMolecule Mut Signal ProbeLabel 2 Mut
    17. 17. Princeton Inc Signal Processing -0.2 0.0 0.2 0.4 0.6 100 110 120 130 140 150 160 170 180 nA/mm2 Potential (V) eSensor® DNA Detection Technology
    18. 18. Princeton Inc -0.2 0.0 0.2 0.4 0.6 100 110 120 130 140 150 160 170 180 nA/mm2 Potential (V) • Mutant (MUT) • Wild Type (WT) • Heterozygous (HET) eSensor® DNA Detection Technology
    19. 19. Princeton Inc Assay Workflow Exonuclease 5 uL dsDNA Amplicon Sample EXONUCLEASE DIGESTION EXONUCLEASE ADDITION Signal Buffer 70 uL XT-Buffer 1 10 uL XT-Buffer 2 20 uL Hybridization Solution 100 uL ssDNA Amplicon Sample GENOTYPING SETUP Genomic DNA 5 uL PCR PCR Reaction Mix 35 uL PCR SET-UP PCR Master Mix 30 uL PCR MIx 28 uL Taq Polymerase 2 uL 125 uL eSensor® XT-8 Cartridge HYBRIDIZATION / DETECTION
    20. 20. Princeton Inc Acknowledgements Molecular Cellular & Development Biology/Chemistry University of Colorado at Boulder, CO • NIH, NSF, DOE Chemistry, Eastern New Mexico University Portales, NM • NIH NCRR P20-61480