Functional Characterization of 29 Cytochrome P450 4F2 Variants Identified in a Population of 8380 Japanese Subjects and Assessment of Arachidonic Acid ω-Hydroxylation.

Cytochrome P450 4F2 (CYP4F2) is an enzyme that is involved in the metabolism of arachidonic acid (AA), vitamin E and K, and xenobiotics including drugs. CYP4F2*3 polymorphism (rs2108622; c.1297G>A; p.Val433Met) has been associated with hypertension, ischemic stroke, and variation in the effectiveness of the anticoagulant drug warfarin. In this study, we characterized wild-type CYP4F2 and 28 CYP4F2 variants, including a Val433Met substitution, detected in 8380 Japanese subjects. The CYP4F2 variants were heterologously expressed in 293FT cells to measure the concentrations of CYP4F2 variant holoenzymes using carbon monoxide-reduced difference spectroscopy, where the wild type and 18 holoenzyme variants showed a peak at 450 nm. Kinetic parameters [Vmax , substrate concentration producing half of Vmax (S50 ), and intrinsic clearance (CL int ) as Vmax /S50 ] of AA ω-hydroxylation were determined for the wild type and 21 variants with enzyme activity. Compared with the wild type, two variants showed significantly decreased CL int values for AA ω-hydroxylation. The values for seven variants could not be determined because no enzymatic activity was detected at the highest substrate concentration used. Three-dimensional structural modeling was performed to determine the reason for reduced enzymatic activity of the CYP4F2 variants. Our findings contribute to a better understanding of CYP4F2 variant-associated diseases and possible future therapeutic strategies. SIGNIFICANCE STATEMENT: CYP4F2 is involved in the metabolism of arachidonic acid and vitamin K, and CYP4F2*3 polymorphisms have been associated with hypertension and variation in the effectiveness of the anticoagulant drug warfarin. This study presents a functional analysis of 28 CYP4F2 variants identified in Japanese subjects, demonstrating that seven gene polymorphisms cause loss of CYP4F2 function, and proposes structural changes that lead to altered function.

Identification and functional validation of novel pharmacogenomic variants using a next-generation sequencing-based approach for clinical pharmacogenomics.

Inter-individual variability in pharmacokinetics and drug response is heavily influenced by single-nucleotide variants (SNVs) and copy-number variations (CNVs) in genes with importance for drug disposition. Nowadays, a plethora of studies implement next generation sequencing to capture rare and novel pharmacogenomic (PGx) variants that influence drug response. To address these issues, we present a comprehensive end-to-end analysis workflow, beginning from targeted PGx panel re-sequencing to in silico analysis pipelines and in vitro validation assays. Specifically, we show that novel pharmacogenetic missense variants that are predicted or putatively predicted to be functionally deleterious, significantly alter protein activity levels of CYP2D6 and CYP2C19 proteins. We further demonstrate that variant priorization pipelines tailored with functional in vitro validation assays provide supporting evidence for the deleterious effect of novel PGx variants. The proposed workflow could provide the basis for integrating next-generation sequencing for PGx testing into routine clinical practice.

Functional Characterization of 21 Rare Allelic CYP1A2 Variants Identified in a Population of 4773 Japanese Individuals by Assessing Phenacetin O-Deethylation.

Cytochrome P450 1A2 (CYP1A2), which accounts for approximately 13% of the total hepatic cytochrome content, catalyzes the metabolic reactions of approximately 9% of frequently used drugs, including theophylline and olanzapine. Substantial inter-individual differences in enzymatic activity have been observed among patients, which could be caused by genetic polymorphisms. Therefore, we functionally characterized 21 novel CYP1A2 variants identified in 4773 Japanese individuals by determining the kinetic parameters of phenacetin O-deethylation. Our results showed that most of the evaluated variants exhibited decreased or no enzymatic activity, which may be attributed to potential structural alterations. Notably, the Leu98Gln, Gly233Arg, Ser380del Gly454Asp, and Arg457Trp variants did not exhibit quantifiable enzymatic activity. Additionally, three-dimensional (3D) docking analyses were performed to further understand the underlying mechanisms behind variant pharmacokinetics. Our data further suggest that despite mutations occurring on the protein surface, accumulating interactions could result in the impairment of protein function through the destabilization of binding regions and changes in protein folding. Therefore, our findings provide additional information regarding rare CYP1A2 genetic variants and how their underlying effects could clarify discrepancies noted in previous phenotypical studies. This would allow the improvement of personalized therapeutics and highlight the importance of identifying and characterizing rare variants.