Study On CYP2C9 Gene Polymorphism In Chinese Han Population Based On Multiplex PCR Targeted Capture Sequencing Technolog

ObjectiveTo explore the genetic polymorphism distribution of cytochrome P450 2C9(CYP2C9)in Chinese Han population,and develop a new genotyping system of CYP2C9 gene based on MPCR and next-generation sequencing technology.Furthermore,a complete in vitro expression system was constructed to explore the biological function and drug metabolism of the newly discovered CYP2C9 variants.MethodsIn this study,1163 Chinese Han population were recruited for blood sample collection,and the genomic DNA of each sample was extracted by magnetic bead method for subsequent genetic screening.At the same time,a new genotyping system based on Multiplex PCR and next-generation sequencing technology has been successfully developed,which more efficiently realized the establishment of CYP2C9 genetic information database.In vitro expression system of yeast cells,CYP2C9 variants and CYPOR were co-expressed in S.cerevisiae microsomes,and the expression of target protein was verified by Western Blot and quantitative analysis.The metabolic activity analysis of 10 novel CYP2C9 variants against two typical probe drugs losartan and glimepiride was determined in vitro using a drug metabolism research platform.Results1.Except for wild type CYP2C9*1,a total of 40 allele variants of CYP2C9 gene were detected in this study.Including 17 previously reported non-synonymous variants(CYP2C9*2,*3,*8,*13,*16,*29,*31,*34,*36,*37,*39,*45,*48,*53,*56,*60,*75),13 synonymous variants and 10 novel non-synonymous variants not registered in PharmVar’s website(L71R,P163S,T301M,E326K,C372R,I389V,H396Y,N398H,G431R,I488F).2.The results of this study show that CYP2C9*3 is the most common allele in Chinese Han population,and its allele frequency is 3.998%,among which 7.57%of research objects are heterozygotes of*1/*3.Most of the other non-synonymous alleles were heterozygous with the wild type,and the total genotype frequency was less than 4.5%,indicating that these alleles were rare in Chinese Han population.3.Western Blot and quantitative analysis showed that the protein expression levels of Pro163Ser,Thr301Met,Glu326Lys,Gly431Arg and Ile488Phe in yeast cells were significantly lower than those of the wild type(p<0.05).The expression levels of the remaining newly discovered CYP2C9 variants were similar to those of the wild type.4.Two typical CYP2C9 probe drugs,losartan and glimepiride,were used to evaluate the metabolic activity of the novel variants.Results showed that Thr301Met,Glu326Lys and Gly431Arg almost lost their catalytic activity,while most of the other variants significantly increased their drug metabolic activity in vitro.Conclusions1.This study has successfully developed a new genotyping system for CYP2C9 gene,realizing the establishment of CYP2C9 genetic information database efficiently,accurately and at a low cost.2.The distribution pattern of CYP2C9 genetic polymorphism in Chinese Han population is characterized by*3 as the dominant variant type,and a variety of rare variants with very low frequency,which are mostly heterozygous with wild type,and the total genotype frequency is less than 4.5%.3.Ten novel non-synonymous variants were identified in this study,seven of which have not yet been registered in public databases and can be regarded as international first reports.4.In vitro biological function studies showed that among the 10 novel nonsynonymous variants,except Pro163Ser,Thr301Met,Glu326Lys,Gly431Arg and Ile488Phe,the protein expression levels of the other variants were similar to those of the wild type.Besides,Except Thr301Met,Glu326Lys and Gly431Arg,most of the other variants were belonged to the rapid metabolizers.