CYP2D6 Gene Polymorphism Frequency And Metabolic Activity In Vitro Analysis Of The Differences In Uygur

Objective:High blood pressure and cardiovascular disease is one of the main cause of death in the world, especially in our country Uygur populations in the frequency, serious threat to human life and health. Metoprolol and debrisoquine as a classic drugs in the treatment of hypertension and cardiovascular are widely used in clinical treatment, but the curative effect of metoprolol and debrisoquine shows differences between individuals, clinical adverse drug reaction more easily. Clinical studies have shown that drug reactions individual differences closely related to pharmacogenomics. Drug metabolism enzymes, drug receptors and their downstream protein gene genetic variation are the main factor causing drug reaction individual differences. Debrisoquine and metoprolol metabolism in human body is mainly composed of CYP2D6. CYP2D6is one of the important members of cytochrome oxidase P450enzymes, participate in metabolism of20%-25%of the clinical prescription drugs. CYP2D6is so far found the most genetic polymorphisms of metabolic enzyme, and gene polymorphism with racial differences. China’s regional ethnic composition is complex, differences in genetic background between all nationalities. Drug adverse reactions of ethnic minority population is urgency. This study set up genomic DNA sample library by using the method of molecular biology, analysis CYP2D6gene polymorphism frequency and metabolic phenotype differences in Uighur, aimed to explore the mechanism of drug adverse reactions based on racial differences, in order to optimize the treatment dose, reduce the adverse drug reaction, also contribute to protect and utilizate the genetic resources of ethnic minorities in China.Methods:1. Continue to expand Chinese ethnic minorities genomic DNA sample library.Freshmen in the university as the object, through questionnaire survey, screening minority healthy adults had no history of marriage with other nation within three generations, with the principles of informed consent, collecting blood in accordance with the standards for clinical principal, and extract blood genomic DNA by un-centrifugal column method. At present, we have established genetic samples2264cases.2. CYP2D6gene polymorphism differences in vitro analysis.Using CYP2D6CDS as template, got2444-2444,2444g> A,4124g> C three mutant gene through point-mutations PCR method, and using Double Digestion, connection and transformation method constructed eukaryotic expression plasmid of three mutations. Western blot testing protein expression; fluorescein kit P450TM-Glo assay kit testing enzyma activity; protein incubate with metoprolol and Debrisoquine, LCMS-IT-TOF testing enzyma metabolic efficiency.Results:1. Expanded the Chinese minority DNA sample library.Collected blood samples2264cases, include ethnic Han526cases, the Hui nationality249cases, Uygur233cases, Mongolian215cases, Korean154cases, Tibetan169cases, kazak161cases,other minorities557cases.2. The CYP2D6gene polymorphism in vitro metabolic phenotype variance analysisthe amount of protein expression levels of2459g> A is99.68%of CYP2D6CDS’ s, the amount of protein expression levels of4124g> C is98.27%of CYP2D6CDS’ s, both have no significant difference between CYP2D6CDS, otherwise2444-2445insG has no protein expression. Compared with wild type CYP2D6enzyme activity,4124g> C enzyme activity is slightly decreased, activity of2459g> A decreased obviously, the activity of2444-2445insG lowest.Conclusion:4124g> C SNP have effects on the expression of genes on purpose, but can still guarantee purpose protein expression and most active, the expressed protein still have similar metabolic activity to those of wild type;2459g> A SNP has no apparent impact on the number of purpose gene protein expression, but has greater influence on the structure and activity of proteins, expressed protein loss of most of the activity.2444-2445insG SNP directly lead to protein inactivation.