RT Journal Article
SR Electronic
T1 Pharmacogenomic Next-Generation DNA Sequencing: Lessons from the Identification and Functional Characterization of Variants of Unknown Significance in CYP2C9 and CYP2C19
JF Drug Metabolism and Disposition
JO Drug Metab Dispos
FD American Society for Pharmacology and Experimental Therapeutics
SP dmd.118.084269
DO 10.1124/dmd.118.084269
A1 Sandhya Devarajan
A1 Irene Moon
A1 MingFen Ho
A1 Nicholas B Larson
A1 Drew R Neavin
A1 Ann M Moyer
A1 John L Black
A1 Suzette J Bielinski
A1 Steven E Scherer
A1 Liewei Wang
A1 Richard M Weinshilboum
A1 Joel M Reid
YR 2019
UL http://dmd.aspetjournals.org/content/early/2019/02/11/dmd.118.084269.abstract
AB CYP2C9 and CYP2C19 are highly polymorphic pharmacogenes, but clinically actionable genetic variability in drug metabolism due to these genes has primarily 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 were transiently expressed in COS-1 cells for the assay of protein concentration and enzyme activities using fluorometric substrates and LC-MS/MS with tolbutamide (CYP2C9) and mephenytoin (CYP2C19) as prototypic substrates. Results were compared with SIFT, Polyphen, and Provean functional prediction software. Cytochrome P450 oxidoreductase CPR activities were also determined. Positive correlations were observed between protein content and fluorometric enzyme activity for variants for 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 (WT) 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.