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

Abstract

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.

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

“…CO-reduced difference spectroscopy is one of the most effective approaches for determining the structural stability of P450 holoproteins (Omura and Sato, 1964). The height of the absorption maximum at 450 nm tends to decrease holoprotein stability (Kumondai et al, 2018;Watanabe et al, 2018;Gutiérrez et al, 2020;Kumondai et al, 2021aKumondai et al, , 2021bKumondai et al, , 2021c. The Ser454Ter substitution did not encode a heme-binding region; thus, neither holoprotein content nor enzyme activity could be detected.…”

“…The study was approved by the Tohoku University Graduate School of Pharmaceutical Sciences ethics committee (permission number 14-08) and the Tohoku Medical Megabank Organization ethics committee (permission number 2017-4-26, 2017-4-58, and 2017-4-090), and written informed consent was obtained from all subjects prior to sample collection. Polymerase chain reaction (PCR) amplification of genomic DNA extracted from leucocytes was conducted using a Gentra Puregene Blood Kit (Qiagen, Hilden, Germany) as previously described (Kumondai et al, 2021a(Kumondai et al, , 2021b(Kumondai et al, , 2021c. At least 10 ng genomic DNA, 2× AmpliTaq Gold 360 Master Mix (Applied Biosystems, Foster City, CA, USA), and 0.5 μM of each primer (Supplementary Table S2), in a total volume of 20 μL, were used for PCR amplification.…”

“…4.1.0 (ProteinSimple) software to evaluate CYP4F2 protein expression levels. As previously described (Kumondai et al, 2021b(Kumondai et al, , 2021c, each well was loaded with 100 ng of microsomal protein containing CYP4F2 wild-type and variant proteins. CYP4F2 was detected using a polyclonal anti-human CYP4F2 antibody (diluted 1:100) and HRP-conjugated goat anti-rabbit IgG.…”

Advances in Personalized Medicines along with Functional Genomics and Pharmacogenomics

“…This Special Issue, Functional Genomics and Pharmacogenomics in Human Disease, covers 15 publications conducted by various groups, including nine original research papers, five review papers, and one case report. Original research papers consist of genetic discovery and functional characterization of CYP1A2 and CYP2C9 [ 1 , 2 ], genetic studies of oral cancer [ 3 , 4 , 5 ], chronic obstruction pulmonary disease (COPD) [ 6 ], asthma [ 7 ], diabetic kidney disease [ 8 ], and web-based pharmacogenomics tool development [ 9 ]. Review papers include the thiopurine treatments with NUDT15 polymorphisms [ 10 ], lipid lowering therapies in familial hypercholesterolemia [ 11 ], incorporation of genetic addiction risk severity (GARS) to overcome the addiction pandemic [ 12 ], and the latest update on genetic variants of UGT [ 13 ] and SULT genes [ 14 ].…”

“…Functional studies of the protein variants discovered in the CYP1A2 [ 1 ] and CYP2C9 [ 2 ] genes were conducted, and the studies identified multiple genetic variants with significantly reduced enzyme activity. This is expected to make a great contribution to drug metabolism, environmental substance metabolism, and toxicology research with regard to the CYP1A1 and CYP2C9 substrates.…”