Identification and Functional Characterization of a New CYP2C9 Variant (CYP2C9*51) Expressed among African Americans
- Leslie J. Dickmann1,
- Allan E. Rettie1,
- M. Byron Kneller1,
- Richard B. Kim2,
- Alastair J. J. Wood2,
- C. Michael Stein2,
- Grant R. Wilkinson2 and
- Ute I. Schwarz2
- 1Department of Medicinal Chemistry, University of Washington, Seattle, Washington (L.J.D., A.E.R., M.B.K.); and 2Division of Clinical Pharmacology, Departments of Medicine and Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee (R.B.K., A.J.J.W., C.M.S., G.R.W., U.I.S.)
- Richard B. Kim, 572 MRB-1, Vanderbilt University School of Medicine, Nashville, TN 37232-6602. E-mail: richard.kim{at}mcmail.vanderbilt.edu
Abstract
CYP2C9 is a polymorphic gene for which there are four known allelic variants; CYP2C9*1,CYP2C9*2, CYP2C9*3, andCYP2C9*4. In the present study, DNA from 140 European Americans and 120 African Americans was examined by single-strand conformational polymorphism and restriction fragment length polymorphism analyses, resulting in the identification of a new CYP2C9 variant, CYP2C9*5. This variant is derived from a C1080G transversion in exon 7 of CYP2C9 that leads to an Asp360Glu substitution in the encoded protein. TheCYP2C9*5 variant was found to be expressed only in African Americans, such that approximately 3% of this population carries the CYP2C9*5 allele. The variant was expressed in, and purified from, insect cells infected with a recombinant baculovirus. Comparative kinetic studies using the purified wild-type protein CYP2C9*1; the Ile359Leu variant, CYP2C9*3; and the Asp360Glu variant, CYP2C9*5 were carried out using (S)-warfarin, diclofenac, and lauric acid as substrates. The major effect of the Asp360Glu mutation was to increase the K mvalue relative to that of CYP2C9*1 for all three substrates: 12-fold higher for (S)-warfarin 7-hydroxylation, 5-fold higher for the 4′-hydroxylation of diclofenac, and 3-fold higher for the ω-1 hydroxylation of lauric acid. V max values differed less than K m values between the CYP2C9*1 and CYP2C9*5 proteins. In vitro intrinsic clearances for CYP2C9*5, calculated as the ratio ofV max/K m, ranged from 8 to 18% of CYP2C9*1 values. The corresponding ratio for CYP2C9*3 was 4 to 13%. Accordingly, the in vitro data suggest that carriers of the CYP2C9*5 allele would eliminate CYP2C9 substrates at slower rates relative to persons expressing the wild-type protein.
Footnotes
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↵1 CYP2C9 allele submitted to and number designated by the Human Cytochrome P450 Allele Nomenclature Committee.
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This work was supported in part by United States Public Health Service Grants GM31304 and GM32165. The Molecular Biomarkers Laboratory in the Center for Ecogenetics and Environmental Health at the University of Washington is supported by National Institute of Environmental Health Sciences Grant ES07033.
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Portions of this work have been reported in abstract form: Schwarz UI, Choo EF, Dresser GK, Stein CM, Wood AJJ, Roden DM, Wilkinson GR and Kim RB (2000) Identification of a new CYP2C9 variant in African-Americans. Clin Pharmacol Ther 67:169.
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↵2 CYP2C9 variant proteins (CYP2C9*1, CYP2C9*2, etc.) are named according to the recommendations made by the Human Cytochrome P450 Allele Nomenclature Committee (http://www.imm.ki.se/CYPalleles).
- Abbreviations:
- P450
- cytochrome P450
- SNP
- single nucleotide polymorphism
- PCR-RFLP
- polymerase chain reaction-restriction fragment length polymorphism
- SSCP
- single-strand conformational polymorphism
- TBE
- Tris/borate/EDTA
- MALDI-TOF
- matrix-assisted laser desorption ionization time-of-flight
- HPLC
- high-performance liquid chromatography
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- Received February 20, 2001.
- Accepted May 14, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
