Pharmacogenomic Next‐Generation DNA Sequencing: Identification and Functional Characterization of Variants of Unknown Significance in CYP2C9 and CYP2C19
Authors: Devarajan Sandhya , Moon Irene , Black John Logan , Bielinski Suzette , Larson Nicholas , Neavin Drew , Wang Liewei , Weinshilboum Richard , Reid Joel 
Published: November 2023

ObjectiveCYP2C9 and CYP2C19 are highly polymorphic pharmacogenes that encodes enzymes that metabolize a large number of clinically relevant drugs. While the majority of variability in drug metabolism can be explained by a few common alleles, identification of gene sequence variation that alters enzyme activity and incorporation of that information into pharmacogenomic rules for clinical decision support might lead to improved therapy. Using sequence data derived from the Right Drug, Right Dose, Right Time: Using Genomic Data to Individualize Treatment (RIGHT Protocol), we sought to identify variants of unknown significance (VUSs) for CYP2C9 and CYP2C19, and to characterize the function of the encoded enzymes.MethodsTargeted next‐generation sequencing for 84 pharmacogenes using the PGRN‐Seq v1.0 capture reagent was performed with DNA samples from 1013 RIGHT Protocol participants. Plasmids containing VUSs were created using site directed mutagenesis and were transiently expressed in COS‐1 cells. Initial determination of enzyme activity was performed using fluorogenic substrates. Enzyme kinetic parameters (Km and Vmax) for each of the variant allozymes were determined using liquid chromatography mass spectrometry with tolbutamide (CYP2C9) and mephenytoin (CYP2C19) as prototypic substrates. We also compared our results with in silico functional predictions by SIFT and Polyphen.ResultsSeven VUSs were identified for each gene, none of which had been functionally characterized. There were significant positive correlations between protein content and fluorometric enzyme activity for all variants for both CYP2C9 (P<0.05) and CYP2C19 (P<0.005). Notably, CYP2C9 V237L and CYP2C19 A173V activity was much lower than predicted based on protein content. The substrate intrinsic clearance of CYP2C9 variants P73L, S115R, V237L [6.23E‐05±1.08E‐05; 2.55E‐05±4.1E‐06; 7.28E‐05±2.4E‐05 (uL/mg protein/min)], and CYP2C19 variants V113I, A173V, R186C and R186H [2.49E‐04±2.78E‐05; 1.80E‐05±2.19E‐06; 2.33E‐05±9.2E‐06;1.45E‐04±1.2E‐05 (uL/mg protein/min)] were less than 25% of their respective WT enzyme.ConclusionSequencing of CYP2C9 and CYP2C19 from 1013 subjects identified low frequency variants that had not been functionally characterized. In silico predictions which showed variable results, can be helpful but are not always accurate, hence functional validation is necessary for VUSs requiring protein expression and determination of kinetic parameters. Efforts to identify variation in diverse populations is resulting in a very large number of VUSs, so there is a need for the application of high throughput mutagenesis approaches and functional characterization methods to help pharmacogenomics contribute to precision medicine.Support or Funding InformationNIH RO1 GM28157 and the Mayo Center for Individualized Medicine Pharmacogenomics Program.