Massively parallel characterization of CYP2C9 variant enzyme activity and abundance

• Published in: American journal of human genetics Vol. 108; no. 9; pp. 1735 - 1751
• Main Authors: Amorosi, Clara J., Chiasson, Melissa A., McDonald, Matthew G., Wong, Lai Hong, Sitko, Katherine A., Boyle, Gabriel, Kowalski, John P., Rettie, Allan E., Fowler, Douglas M., Dunham, Maitreya J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 02.09.2021; Elsevier
• Subjects: Binding Sites; CYP2C9; Cytochrome P-450 CYP2C9 - chemistry; Cytochrome P-450 CYP2C9 - genetics; Cytochrome P-450 CYP2C9 - metabolism; deep mutational scanning; Enzyme Assays; Gene Library; High-Throughput Screening Assays; humanized yeast; Humans; Models, Molecular; Mutagenesis, Site-Directed; Mutation, Missense; pharmacogenomics; Phenytoin - chemistry; Polymorphism, Genetic; Prescription Drugs - chemistry; Prescription Drugs - metabolism; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Transgenes; warfarin; Warfarin - chemistry; Warfarin - metabolism; Xenobiotics - chemistry; Xenobiotics - metabolism; CYP2C9; pharmacogenomics; deep mutational scanning; humanized yeast; warfarin
• ISSN: 0002-9297; 1537-6605
• DOI: 10.1016/j.ajhg.2021.07.001

CYP2C9 encodes a cytochrome P450 enzyme responsible for metabolizing up to 15% of small molecule drugs, and CYP2C9 variants can alter the safety and efficacy of these therapeutics. In particular, the anti-coagulant warfarin is prescribed to over 15 million people annually and polymorphisms in CYP2C9 can affect individual drug response and lead to an increased risk of hemorrhage. We developed click-seq, a pooled yeast-based activity assay, to test thousands of variants. Using click-seq, we measured the activity of 6,142 missense variants in yeast. We also measured the steady-state cellular abundance of 6,370 missense variants in a human cell line by using variant abundance by massively parallel sequencing (VAMP-seq). These data revealed that almost two-thirds of CYP2C9 variants showed decreased activity and that protein abundance accounted for half of the variation in CYP2C9 function. We also measured activity scores for 319 previously unannotated human variants, many of which may have clinical relevance.