Pharmacogenomic Next-Generation DNA Sequencing: Lessons from the Identification and Functional Characterization of Variants of Unknown Significance in CYP2C9 and CYP2C19 .

Autor:	Devarajan S; Departments of Molecular Pharmacology and Experimental Therapeutics (S.D., I.M., M.-F.H., L.W., R.M.W., J.M.R.) and Health Sciences Research (N.B.L., S.J.B.), Personalized Genomics Laboratory, Department of Laboratory Medicine and Pathology (A.M.M., J.L.B.), and Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic Graduate School of Biomedical Sciences (D.R.N.), Mayo Clinic, Rochester, Minnesota; and Human Genome Sequencing Center, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas (S.E.S.)., Moon I; Departments of Molecular Pharmacology and Experimental Therapeutics (S.D., I.M., M.-F.H., L.W., R.M.W., J.M.R.) and Health Sciences Research (N.B.L., S.J.B.), Personalized Genomics Laboratory, Department of Laboratory Medicine and Pathology (A.M.M., J.L.B.), and Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic Graduate School of Biomedical Sciences (D.R.N.), Mayo Clinic, Rochester, Minnesota; and Human Genome Sequencing Center, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas (S.E.S.)., Ho MF; Departments of Molecular Pharmacology and Experimental Therapeutics (S.D., I.M., M.-F.H., L.W., R.M.W., J.M.R.) and Health Sciences Research (N.B.L., S.J.B.), Personalized Genomics Laboratory, Department of Laboratory Medicine and Pathology (A.M.M., J.L.B.), and Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic Graduate School of Biomedical Sciences (D.R.N.), Mayo Clinic, Rochester, Minnesota; and Human Genome Sequencing Center, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas (S.E.S.)., Larson NB; Departments of Molecular Pharmacology and Experimental Therapeutics (S.D., I.M., M.-F.H., L.W., R.M.W., J.M.R.) and Health Sciences Research (N.B.L., S.J.B.), Personalized Genomics Laboratory, Department of Laboratory Medicine and Pathology (A.M.M., J.L.B.), and Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic Graduate School of Biomedical Sciences (D.R.N.), Mayo Clinic, Rochester, Minnesota; and Human Genome Sequencing Center, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas (S.E.S.)., Neavin DR; Departments of Molecular Pharmacology and Experimental Therapeutics (S.D., I.M., M.-F.H., L.W., R.M.W., J.M.R.) and Health Sciences Research (N.B.L., S.J.B.), Personalized Genomics Laboratory, Department of Laboratory Medicine and Pathology (A.M.M., J.L.B.), and Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic Graduate School of Biomedical Sciences (D.R.N.), Mayo Clinic, Rochester, Minnesota; and Human Genome Sequencing Center, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas (S.E.S.)., Moyer AM; Departments of Molecular Pharmacology and Experimental Therapeutics (S.D., I.M., M.-F.H., L.W., R.M.W., J.M.R.) and Health Sciences Research (N.B.L., S.J.B.), Personalized Genomics Laboratory, Department of Laboratory Medicine and Pathology (A.M.M., J.L.B.), and Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic Graduate School of Biomedical Sciences (D.R.N.), Mayo Clinic, Rochester, Minnesota; and Human Genome Sequencing Center, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas (S.E.S.)., Black JL; Departments of Molecular Pharmacology and Experimental Therapeutics (S.D., I.M., M.-F.H., L.W., R.M.W., J.M.R.) and Health Sciences Research (N.B.L., S.J.B.), Personalized Genomics Laboratory, Department of Laboratory Medicine and Pathology (A.M.M., J.L.B.), and Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic Graduate School of Biomedical Sciences (D.R.N.), Mayo Clinic, Rochester, Minnesota; and Human Genome Sequencing Center, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas (S.E.S.)., Bielinski SJ; Departments of Molecular Pharmacology and Experimental Therapeutics (S.D., I.M., M.-F.H., L.W., R.M.W., J.M.R.) and Health Sciences Research (N.B.L., S.J.B.), Personalized Genomics Laboratory, Department of Laboratory Medicine and Pathology (A.M.M., J.L.B.), and Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic Graduate School of Biomedical Sciences (D.R.N.), Mayo Clinic, Rochester, Minnesota; and Human Genome Sequencing Center, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas (S.E.S.)., Scherer SE; Departments of Molecular Pharmacology and Experimental Therapeutics (S.D., I.M., M.-F.H., L.W., R.M.W., J.M.R.) and Health Sciences Research (N.B.L., S.J.B.), Personalized Genomics Laboratory, Department of Laboratory Medicine and Pathology (A.M.M., J.L.B.), and Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic Graduate School of Biomedical Sciences (D.R.N.), Mayo Clinic, Rochester, Minnesota; and Human Genome Sequencing Center, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas (S.E.S.)., Wang L; Departments of Molecular Pharmacology and Experimental Therapeutics (S.D., I.M., M.-F.H., L.W., R.M.W., J.M.R.) and Health Sciences Research (N.B.L., S.J.B.), Personalized Genomics Laboratory, Department of Laboratory Medicine and Pathology (A.M.M., J.L.B.), and Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic Graduate School of Biomedical Sciences (D.R.N.), Mayo Clinic, Rochester, Minnesota; and Human Genome Sequencing Center, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas (S.E.S.)., Weinshilboum RM; Departments of Molecular Pharmacology and Experimental Therapeutics (S.D., I.M., M.-F.H., L.W., R.M.W., J.M.R.) and Health Sciences Research (N.B.L., S.J.B.), Personalized Genomics Laboratory, Department of Laboratory Medicine and Pathology (A.M.M., J.L.B.), and Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic Graduate School of Biomedical Sciences (D.R.N.), Mayo Clinic, Rochester, Minnesota; and Human Genome Sequencing Center, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas (S.E.S.)., Reid JM; Departments of Molecular Pharmacology and Experimental Therapeutics (S.D., I.M., M.-F.H., L.W., R.M.W., J.M.R.) and Health Sciences Research (N.B.L., S.J.B.), Personalized Genomics Laboratory, Department of Laboratory Medicine and Pathology (A.M.M., J.L.B.), and Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic Graduate School of Biomedical Sciences (D.R.N.), Mayo Clinic, Rochester, Minnesota; and Human Genome Sequencing Center, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas (S.E.S.) reid@mayo.edu.
Jazyk:	angličtina
Zdroj:	Drug metabolism and disposition: the biological fate of chemicals [Drug Metab Dispos] 2019 Apr; Vol. 47 (4), pp. 425-435. Date of Electronic Publication: 2019 Feb 11.
DOI:	10.1124/dmd.118.084269
Abstrakt:	CYP2C9 and CYP2C19 are highly polymorphic pharmacogenes; however, clinically actionable genetic variability in drug metabolism due to these genes has been limited to a few common alleles. The identification and functional characterization of less-common open reading frame sequence variation might help to individualize therapy with drugs that are substrates for the enzymes encoded by these genes. The present study identified seven uncharacterized variants each in CYP2C9 and CYP2C19 using next-generation sequence data for 1013 subjects, and functionally characterized the encoded proteins. Constructs were created and transiently expressed in COS-1 cells for the assay of protein concentration and enzyme activities using fluorometric substrates and liquid chromatography- tandem mass spectrometry with tolbutamide ( CYP2C9 ) and (S)-mephenytoin ( CYP2C19 ) as prototypic substrates. The results were compared with the SIFT, Polyphen, and Provean functional prediction software programs. Cytochrome P450 oxidoreductase (CPR) activities were also determined. Positive correlations were observed between protein content and fluorometric enzyme activity for variants of CYP2C9 ( P < 0.05) and CYP2C19 ( P < 0.0005). However, CYP2C9 709G>C and CYP2C19 65A>G activities were much lower than predicted based on protein content. Substrate intrinsic clearance values for CYP2C9 218C>T, 343A>C, and CYP2C19 337G>A, 518C>T, 556C>T, and 557G>A were less than 25% of wild-type allozymes. CPR activity levels were similar for all variants. In summary, sequencing of CYP2C9 and CYP2C19 in 1013 subjects identified low-frequency variants that had not previously been functionally characterized. In silico predictions were not always consistent with functional assay results. These observations emphasize the need for high-throughput methods for pharmacogene variant mutagenesis and functional characterization. (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)
Databáze:	MEDLINE
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