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Rapid melting curve analysis for genetic variants that underlie inter-individual variability in stable warfarin dosing

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Abstract

Warfarin anticoagulation therapy is complicated by its narrow therapeutic index and by wide inter-individual differences in dosing requirements arising, in part, from genetic factors. The present report describes the development, validation and feasibility testing of a rapid genotyping assay that concurrently detects the CYP2C9*2 and *3 variants along with the VKORC1 C1173T polymorphism. The study employed melting curve analysis using labeled probes and compared two detection instruments (the HR-1 and the R.A.P.I.D. LT) to two previously validated methods, 5′ nuclease allelic discrimination (Taqman®) assay and cycle sequencing. The HR-1 detected 189 true negatives and 113 true positives; 1 wild-type sample was mistyped as a heterozygote by both instruments. Sequencing of that sample confirmed it to be a CC homozygote; however, a rare C > T polymorphism was discovered 1 base 5′ from the *2 polymorphic site, presumably causing the mistaken genotype by melting curve. Both methods had sensitivity = 1.00 and specificity > 0.99. Combined with a method for rapid buccal swab DNA extraction, genotyping results were obtained in a median of 59 min. These methods should facilitate genotype-driven warfarin dosing in “real-time” clinical practice.

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Acknowledgements

This work was supported in part by a grant from the Intermountain Healthcare Deseret Foundation, Salt Lake City, UT, USA (JFC) and a grant from the Critical-Path Institute, Tucson, AZ and Bethesda, MD, USA (JFC and JLA).

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Authors and Affiliations

  1. Department of Internal Medicine, Division of Cardiology, University of Utah School of Medicine, Salt Lake City, UT, USA

    John F. Carlquist, Joseph B. Muhlestein & Jeffrey L. Anderson

  2. Cardiovascular Department, LDS Hospital, Salt Lake City, UT, 84143, USA

    John F. Carlquist, Zachary P. Nicholas, Jessica L. Clark, Samera F. Kahn, Benjamin D. Horne, Joseph B. Muhlestein, Heidi T. May & Jeffrey L. Anderson

  3. Idaho Technology, Salt Lake City, UT, USA

    Jason T. McKinney

  4. Department of Medical Informatics, Division of Genetic Epidemiology, University of Utah School of Medicine, Salt Lake City, UT, USA

    Benjamin D. Horne

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  1. John F. Carlquist
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  2. Jason T. McKinney
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  3. Zachary P. Nicholas
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  4. Jessica L. Clark
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  5. Samera F. Kahn
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  6. Benjamin D. Horne
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  7. Joseph B. Muhlestein
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  8. Heidi T. May
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  9. Jeffrey L. Anderson
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Correspondence to John F. Carlquist.

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Carlquist, J.F., McKinney, J.T., Nicholas, Z.P. et al. Rapid melting curve analysis for genetic variants that underlie inter-individual variability in stable warfarin dosing. J Thromb Thrombolysis 26, 1–7 (2008). https://doi.org/10.1007/s11239-007-0077-x

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  • DOI: https://doi.org/10.1007/s11239-007-0077-x

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