Identification and in vitro functional assessment of 10 CYP2C9 variants found in Chinese Han subjects

Abstract

Cytochrome P450 2C9 (CYP2C9) participates in about 15% of clinical drug metabolism, and its polymorphism is associated with individual drug metabolism differences, which may lead to the adverse drug reactions (ADRs). In this study, 1163 Chinese Han individuals were recruited to investigate their distribution pattern of CYP2C9 gene and find out the variants that may affect their drug metabolic activities. We successfully developed a multiplex PCR amplicon sequencing method and used it for the genetic screening of CYP2C9 in a large scale. Besides the wild type CYP2C9*1, totally 26 allelic variants of CYP2C9 were detected, which included 16 previously reported alleles and 10 new non-synonymous variants that had not been listed on the PharmVar website. The characteristics of these newly detected CYP2C9 variants were then evaluated after co-expressing them with CYPOR in S. cerevisiae microsomes. Immunoblot analysis revealed that except for Pro163Ser, Glu326Lys, Gly431Arg and Ile488Phe, most of newly detected variants showed comparable protein expression levels to wild type in yeast cells. Two typical CYP2C9 probe drugs, losartan and glimepiride, were then used for the evaluation of metabolic activities of variants. As a result, 3 variants Thr301Met, Glu326Lys, and Gly431Arg almost lost their catalytic activities and most of other variants exhibited significantly elevated activities for drug metabolism. Our data not only enriches the knowledge of naturally occurring CYP2C9 variants in the Chinese Han population, but also provides the fundamental evidence for its potential clinical usage for personalized medicine in the clinic.

EnzyACT: A Novel Deep Learning Method to Predict the Impacts of Single and Multiple Mutations on Enzyme Activity

“…There are six key substrate regions in the P450 enzyme (Figure 2B) which are located at residues 96−117, 198−205, 233−240, 286−304, 359−369, and 470− 477, all of them were highlighted by the dashed black boxes. 30 Figure 2A is the heatmap of the DMS experimental score and our predicted score, respectively, which show a clear similar pattern of activity changes upon single-point mutations, especially in these key areas. Ordinarily, substitution close to the substrate most likely changes the enzyme activity.…”

“…We comprehensively analyzed the saturation mutation results of P450 to demonstrate the capability of our method to design enzyme activity. There are six key substrate regions in the P450 enzyme (Figure B) which are located at residues 96–117, 198–205, 233–240, 286–304, 359–369, and 470–477, all of them were highlighted by the dashed black boxes Figure A is the heatmap of the DMS experimental score and our predicted score, respectively, which show a clear similar pattern of activity changes upon single-point mutations, especially in these key areas.…”

Establishment and Application of a Novel Genetic Detection Panel for SNPs in Mongolian Gerbils

“…In comparison with single-SNP amplification and genotyping methods, the multiple-target region capture technology (MultipSeq Primer for the genotyping of SNP loci) based on next-generation sequencing technology is more cost-effective and has higher efficiency, enabling the simultaneous acquisition of a greater number of loci and genotyping results [ 32 , 36 , 37 ]. In the present study, by testing the 219 selected SNPs in 120 animals, it was found that three loci had a low amplification quality, seven loci presented the same genotype in all animals, and three loci were completely linked with other loci.…”

Identification and Functional Assessment of EightCYP3A4Allelic Variants*39–*46Detected in the Chinese Han Population

“…With the similar genotyping approach we previously established for the CYP2C9 gene (Zhang et al, 2023), one multiplex PCR amplicon sequencing and genotyping method for the CYP3A4 gene was developed in this study. The process started with an initial PCR amplification using the primers in Table 1, which enabled us to amplify multiple regions simultaneously, including the promoter, all 13 exons of the CYP3A4 gene, and the exon-intron junction areas.…”