Gene Polymorphisms Of CYP2D6~*10 In Chinese

Objective:β-receptor blocker, especially metoprolol, is one of the most imported cardiovascular drugs. There is an obvious individual difference in clinic. It appeared that the drug reaction is more sensitive in the westerns than the orients. The different metabolism and reaction of drugs in the individuals and different race is a normal phenomenon in clinical medication. Drug metabolism enzyme plays an important role in it. The cytochrome P450s (CYP450s) mainly distributed in human liver are a multi-gene family of constitutive and inducible heme-containing monooxygenases, playing a big role in the metabolism of many endogenous and exogenous substrates and the oxidative metabolism of a wide range of environmental chemicals including carcinogens and toxins, as well as the therapeutic drugs. Cytochrome P450 2D6 (CYP2D6) is the most polymorphic member of the CYP450 gene superfamily. Up to now, there are more than 70 kinds of gene mutations being detected. About 20%-30% of the commonly prescribed drugs includingβ-receptor blocker, antiarrhythmics, antipertensive, antidepressants, antipsychotics, analgesics and cough suppressants are metabolized by CYP2D6. CYP2D6 polymorphism determinates different metabolism degree of drug and influences a different choice of clinical medication and clinical dosage. Most of lipophilic beta blockers including Metoprolol and Propranolol are metabolized by CYP2D6 enzyme, while water-solubility beta blockers including Atenolol and Nadolol are almost excreted by the renal as what it is be. Metoprolol is aβ1-selective adrenoceptor antagonist, clinically used in the treatment of hypertension, angina pectoris and arrhythmia. It is extensively metabolized in liver through the following two main pathways, O-demethylation andα-hydroxylation, O-demethylation is catalized partly by CYP2D6,α-hydroxylation is completely mediated by CYP2D6 and formedα-hydroxylation Metoprolol. About 70%-80% of oral administration dose are metabolized by CYP2D6, Enzyme activity of CYP2D6 is a main influence factor for metoprolol metabolism and the disposition ofβ–adrenoceptor antagonist propranolol and Metoprolol are further examples of some of the clinical consequences observed in Asians that were due to the presence of the CYP2D6*10 allele. Because of the different drug efficacy and variance of adverse reaction in individuals, we cannot make a forecast of the clinical efficacy that leads to an excess therapy, serious adverse reaction and so on. So it is very necessary to study the relationship between medicine reaction and CYP2D6 polymorphism, and then get more information of CYP2D6 gene mutation in different race. To investigate the polymorphism of CYP2D6 and to determine the activity of the enzyme can be of critical significance. Many clinical trials suggested that genotype can have a prominent effect on the pharmacokinetics and pharmacodynamics of its substrates, namely there was a gene-dose-effect. The main mutation style of CYP2D6 is a deletion or substitution of single nucleotide causing frameshift mutation or a deletion of large DNA fragment. Gene mutation can cause different enzyme activity and different enzyme quantities which result in notable individual difference in drug reaction aspect in human. Population can be divided into ultrarapid metabolizer (UM), extensive metabolizer (EM), intermediate metabolizer (IM) and poor metabolizer (PM). In that, Gene with two normal active alleles (CYP2D6*1 or CYP2D6*2) are called UM. Gene with one normal active allele is called EM, Gene with two decreased active alleles (CYP2D6*10) are called IM, Gene with two incompetence alleles (CYP2D6*3 or CYP2D6*4) or a gene deletion (CYP2D6*5) are called PM. CYP2D6*1 is the wild allele. CYP2D6 mutation can cause loss, decrease and even increase in the activity of the corresponding enzyme. The stronger metabolic capability is, the shorter drug resistant time in the body is. Because CYP2D6 activity plays a significant role in the kinetics and dynamics of many routinely used drugs, estimation of its invivo activity would be of tremendous importance. Phenotype of CYP2D6 is indicated by probe drug, there are three major probe drugs having been used so far, namely debrisoquine, dextromethorphan and metoprolol. Phenotype can reflect the metabolism rate directly but cannot detect the genotype. Sometimes, the detection of phenotype is limited because of the expensive cost, drug adverse reaction, ethics and so on. At the moment, the peripheral lymphocyte in blood is collected to extract DNA and polymerase chain reaction is adopted to get gene fragment to know the genotype. Among the Asians, the most frequently distributed allele is CYP2D6*10, having two point mutations of C188T in exon 1and G4268C in exon 9, C188T is an more important mutation, causing a Pro34Ser amino acid substitution and G4268C causes a Ser486Thr amino acid substitution. It is speculated that a substitution in this region might increase the degradation of the enzyme, and correspondingly the enzyme activity. That is, CYP2D6*10 causes a reduction in enzyme activity, instead of a complete loss and many studies about the relationship of C188T and drug reaction can be seen. Gene mutant can be divided into three types according to genotype: with two enzymatic normal active alleles at point 188 in exon 1 is called wild type homozygote C/C (CYP2D6*1/*1); with two decreased enzymatic active alleles is called mutant homozygote T/T (CYP2D6*10/*10); with one normal enzymatic active allele is called heterozygosis C/T (CYP2D6*1/*10). Lot us of studies prove that the gene mutation frequencies of CYP2D6*10 between westerners and oriental have an obviously racial difference. There is a information of small sample size, different research method, deviated conclusion occurred or a poor repetition at present in many studies, while a small sample size will affect the results, so we need to have a well detective method.The frequencies of CYP2D6*10 allele in Chinese are different in different reports. This article is to investigate the gene polymorphisms of CYP2D6*10, especially about the point mutation of C188T in Chinese people. The analysis of genotypes was conducted by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method in a large sample size. To get to know the genotypic frequencies and genic mutation frequencies at point C188T in order to guide clinical medication.Methods:CYP2D6*10 gene polymorphisms were detected in 491 Chinese people by PCR-RFLP method.1 Blood collectedMore than 3ml upper limb fresh venous blood of 491 Chinese people was drawn in EDTA anticoagulated tubes.2 Genomic DNA extractedGenomic DNA was extracted from blood samples with Genomic DNA Purification Kit according to the instruction of the Kit.3 Analyze the gene polymorphisms of CYP2D6*10 by PCR-RFLP method(1) Amplified fragment was 272bp DNA fragments(2) RFLP analysisAnalyze genotypes by RFLP method and electrophoresis. The amplified 272bp DNA fragments contained a HphI cleavage site. Thus the cleavage of the 272bp fragments were produced into fragments of 213bp and 59bp which were confirmed to be the presence of C/C188 allele; cleavage of the 272bp fragments were produced into fragments of 112bp, 101bp and 59bp which were confirmed to be the presence of T/T188 allele; Also, 213bp, 112bp, 101bp and 59bp were confirmed to be the presence of C/T188.Direct counting method was adopted to know genotypic frequency and gene mutation frequency. Hardy-Weinberg equilibrium was tested using the chi-square goodness of fit test. Chi-square criterion was adopted to analyze whether there was sex difference in the three genotypes and whether there were ethnic differences in gene mutation frequency among this study and the other ethnics.Results: The observed frequencies of the C/C, C/T, T/T genotypes were 16.7%, 43.4%, and 39.9% respectively; which met Hardy-Weinberg equilibrium(P>0.05), the sample had a representation of population. The frequencies of two alleles were as follows: C allele 38.4 %, T allele 61.6%. Compare sex differences through the three gen0types, there were no significant differences among the three genotypes (P>0.05). The result of Chi-square criterion showed there was a significant difference between the western and the oriental in gene mutation frequency, but there was no difference in oriental and Chinese.Conclusion: The genotype frequencies of C/C, C/T, T/T were 16.7%, 43.4%, and 39.9% respectively; which met Hardy-Weinberg equilibrium indicating this was a representative sample. The gene mutation frequency was 61.6% and there were no sex differences among the three genotypes. That is, there was no sex-dependent metabolism of drugs. There was a significant difference between the western and the oriental in gene mutation frequency, but there were no significant differences in the oriental and Chinese.