CYP2C9基因多态性研究及与格列本脲代谢的关系
摘要
【目的】
研究健康正常人与糖尿病患者CYP2C9基因多态性情况;分析人体内CYP2C9酶突变等位基因对格列本脲药物代谢的影响。从个体遗传学角度阐明临床治疗中药物反应的个体差异性问题,为指导格列本脲等糖尿病药物的临床合理应用提供依据,实现糖尿病的个体化医疗,为药物遗传学研究打下基础。
【方法】
采用聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)及测序鉴定等方法,对150例健康正常人和100例Ⅱ型糖尿病患者以及7例长期服用格列本脲治疗的Ⅱ型糖尿病患者CYP2C9基因启动子区、第2外显子、第3外显子、第4外显子、第6外显子及第7外显子突变位点及突变频率进行多态性研究;并采用HPLC等技术对7例长期服用格列本脲治疗的Ⅱ型糖尿病患者进行服药后1、2.5、4、8 h不同时间点药物代谢水平研究,利用3P97药动学程序计算药动学参数并采用配对t检验进行比较。
【结果】
1.检测的150例健康正常人中,CYP2C9基因启动子区-1188C/T位点、第2外显子269T>C位点、第3外显子430C>T位点、第6外显子895A>G位点及第7外显子1075A>C位点分别有53.9%、1.3%、0%、2%、7%的杂合携带者;100例Ⅱ型糖尿病患者在这些位点分别有60.4%、0%、1%、1%、7%的杂合携带者。第4外显子中未发现多态性位点。
2.统计分析显示健康正常人与糖尿病患者在CYP2C9基因启动子区-1188C/T位点、第2外显子269T>C位点及第3外显子430C>T位点差异较大,但无统计学意义。本课题研究结果与中国其他地区人群、亚洲地区日本韩国人群、欧美人群CYP2C9等位基因频率比较结果发现:第3外显子430位点与欧美人群存在显著性差异(Χ2p=65.3154,P<0.05);第7外显子1075位点与亚洲地区日本韩国人群(Χ2p=5.6488,P<0.05)及欧美人群(Χ2p=11.6795,P<0.05)均存在显著性差异,其他位点比较结果差异不显著(P>0.05)。
3.在格列本脲的药物代谢实验中发现了一例弱代谢者(PM),基因测序表明,该受试PM的CYP2C9基因为第6外显子895A>G位点为CYP2C9*1/*16基因型杂合子。该受试PM的格列本脲血浆半衰期为51.87h,而其他受试糖尿病个体(基因型CYP2C9*1/*1)平均半衰期为5.775h,是CYP2C9*1/*1基因型者的约9倍。
【结论】
1.多态性研究表明,CYP2C9基因启动子区-1188C/T位点、第2外显子269T>C位点、第3外显子430C>T位点、第6外显子895A>G位点及第7外显子1075A>C位点均存在多态位点,150例健康正常人及100例Ⅱ型糖尿病患者共250例样品中这些位点分别有56.5%、0.8%、0.4%、1.6%、7.0%的杂合携带者,第4外显子中未发现多态位点。
2.本课题研究结果与中国其他地区人群、亚洲地区日本韩国人群、欧美人群CYP2C9等位基因频率比较结果发现:第3外显子430位点与欧美人群存在显著性差异,第7外显子1075位点与亚洲地区日本韩国人群及欧美人群均存在显著性差异(P<0.05)。
3.对糖尿病患者CYP2C9酶等位基因CYP2C9*16(第6外显子895A>G位点)研究表明该位点突变对格列本脲的药代动力学有显著性影响。本课题研究结果表明,CYP2C9基因分型对指导临床合理用药和个体化医疗具有重要意义。
【Objective】
Study of the CYP2C9 gene polymorphism in healthy controls and diabetics; Analysis the effect of the human body CYP2C9 enzyme mutant allele on glibenclamide drug metabolism. Elucidate the clinical treatment of drug reaction individual differences from the perspective of individual genetics, to provide a evidence for guide glibenclamide and other diabetes drugs clinical rational application , to achieve diabetes individuation medical ,and to lay the foundation for the Pharmacogenetics study.
【Methods】
Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequencing methods were used to study the CYP2C9 gene polymorphism on promoter region、exon 2、exon 3、exon 4、exon 6 and exon 7 mutational site and frequency analysis in 150 healthy controls、100 typeⅡdiabetics and 7 cases of long-term use of glibenclamide treatment of typeⅡdiabetics;HPLC techniques were used to study the drug metabolism for 7 cases of long-term use of glibenclamide treatment of typeⅡdiabetics after 1、2.5、4、8h at different time points. 3P97 pharmacokinetic software was used to calculate pharmacokinetic parameters and paired t-test to comparison.
【Results】
1. In the detection of 150 healthy controls, CYP2C9 gene promoter region -1188C/T sites、exon 2 269T>C sites、exon 3 430C>T sites、exon 6 895A>G sites and exon 7 1075A>C sites were 53.9% 1.3%,0%,2%,7% heterozygous carriers; 100 diabetics in these sites were 60.4%,0%,1%,1%,7% heterozygous carriers . Exon 4 found no polymorphism loci.
2. Statistical analysis showed that CYP2C9 gene promoter region -1188C/T、exon 2 269T>C and exon 3 430C>T mutational site exist quite different, but no statistical significance . The present study results with the other parts of China in total population, the Asian population( Japanese and Korean), Europe and the United States population CYP2C9 allele frequency comparisons found : exon 3 430C>T sites,with the population Europe and the United States, there was significant difference (Χ2p =65.3154, P<0.05); exon 7 1075A>C sites with the Asian population ( Japanese and Korean) (Χ2p =5.6488. P <0.05) and Europe and the United States population (Χ2p =11.6795. P <0.05) were significantly different, Other results of the comparison sites no significant difference (P>0.05).
3. In glibenclamide drug metabolism experiment found one poor metabolizer(PM). CYP2C9 gene sequencing showed that the subject of PM is a exon 6 895A>G site CYP2C9*1/*16 heterozygous genotype. The subject of PM glibenclamide plasma half-life of 51.87h. and other diabetics subjects (CYP2C9*1/*1) on average half-life of the individual 5.775h. It is about 9 times than CYP2C9*1/*1 genotype.
【Conclusion】
1. Polymorphism study showed, CYP2C9 gene promoter -1188C/T sites. Exon 2 269T>C sites, exon 3 430C>T sites, Exon 6 895A>G sites and exon 7 1075A>C sites there are polymorphic loci. Including 150 healthy controls and 100 typeⅡdiabetics these sites were 56.5%、0.8%、0.4%、1.6%、7.0% heterozygous carriers,and in exon 4, we found no polymorphism loci.
2. The present study results with the other parts of China population, the Asian population( Japanese and Korean), Europe and the United States population CYP2C9 allele frequency comparisons found: exon 3 430C>T sites, there was significant difference with the population Europe and the United States;exon 7 1075A>C sites,there was significant difference with the Asian population ( Japanese and Korean)、 Europe and the United States population(P<0.05).
3. Diabetics with glibenclamide metabolism study shows that CYP2C9 enzyme mutant allele CYP2C9*16(Exon 6 895A>G sites)to glibenclamide Pharmacokinetics significant affected, The research show that according to CYP2C9 gene typing of clinical specimens and Individual medical is of great guiding significance.
研究健康正常人与糖尿病患者CYP2C9基因多态性情况;分析人体内CYP2C9酶突变等位基因对格列本脲药物代谢的影响。从个体遗传学角度阐明临床治疗中药物反应的个体差异性问题,为指导格列本脲等糖尿病药物的临床合理应用提供依据,实现糖尿病的个体化医疗,为药物遗传学研究打下基础。
【方法】
采用聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)及测序鉴定等方法,对150例健康正常人和100例Ⅱ型糖尿病患者以及7例长期服用格列本脲治疗的Ⅱ型糖尿病患者CYP2C9基因启动子区、第2外显子、第3外显子、第4外显子、第6外显子及第7外显子突变位点及突变频率进行多态性研究;并采用HPLC等技术对7例长期服用格列本脲治疗的Ⅱ型糖尿病患者进行服药后1、2.5、4、8 h不同时间点药物代谢水平研究,利用3P97药动学程序计算药动学参数并采用配对t检验进行比较。
【结果】
1.检测的150例健康正常人中,CYP2C9基因启动子区-1188C/T位点、第2外显子269T>C位点、第3外显子430C>T位点、第6外显子895A>G位点及第7外显子1075A>C位点分别有53.9%、1.3%、0%、2%、7%的杂合携带者;100例Ⅱ型糖尿病患者在这些位点分别有60.4%、0%、1%、1%、7%的杂合携带者。第4外显子中未发现多态性位点。
2.统计分析显示健康正常人与糖尿病患者在CYP2C9基因启动子区-1188C/T位点、第2外显子269T>C位点及第3外显子430C>T位点差异较大,但无统计学意义。本课题研究结果与中国其他地区人群、亚洲地区日本韩国人群、欧美人群CYP2C9等位基因频率比较结果发现:第3外显子430位点与欧美人群存在显著性差异(Χ2p=65.3154,P<0.05);第7外显子1075位点与亚洲地区日本韩国人群(Χ2p=5.6488,P<0.05)及欧美人群(Χ2p=11.6795,P<0.05)均存在显著性差异,其他位点比较结果差异不显著(P>0.05)。
3.在格列本脲的药物代谢实验中发现了一例弱代谢者(PM),基因测序表明,该受试PM的CYP2C9基因为第6外显子895A>G位点为CYP2C9*1/*16基因型杂合子。该受试PM的格列本脲血浆半衰期为51.87h,而其他受试糖尿病个体(基因型CYP2C9*1/*1)平均半衰期为5.775h,是CYP2C9*1/*1基因型者的约9倍。
【结论】
1.多态性研究表明,CYP2C9基因启动子区-1188C/T位点、第2外显子269T>C位点、第3外显子430C>T位点、第6外显子895A>G位点及第7外显子1075A>C位点均存在多态位点,150例健康正常人及100例Ⅱ型糖尿病患者共250例样品中这些位点分别有56.5%、0.8%、0.4%、1.6%、7.0%的杂合携带者,第4外显子中未发现多态位点。
2.本课题研究结果与中国其他地区人群、亚洲地区日本韩国人群、欧美人群CYP2C9等位基因频率比较结果发现:第3外显子430位点与欧美人群存在显著性差异,第7外显子1075位点与亚洲地区日本韩国人群及欧美人群均存在显著性差异(P<0.05)。
3.对糖尿病患者CYP2C9酶等位基因CYP2C9*16(第6外显子895A>G位点)研究表明该位点突变对格列本脲的药代动力学有显著性影响。本课题研究结果表明,CYP2C9基因分型对指导临床合理用药和个体化医疗具有重要意义。
【Objective】
Study of the CYP2C9 gene polymorphism in healthy controls and diabetics; Analysis the effect of the human body CYP2C9 enzyme mutant allele on glibenclamide drug metabolism. Elucidate the clinical treatment of drug reaction individual differences from the perspective of individual genetics, to provide a evidence for guide glibenclamide and other diabetes drugs clinical rational application , to achieve diabetes individuation medical ,and to lay the foundation for the Pharmacogenetics study.
【Methods】
Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequencing methods were used to study the CYP2C9 gene polymorphism on promoter region、exon 2、exon 3、exon 4、exon 6 and exon 7 mutational site and frequency analysis in 150 healthy controls、100 typeⅡdiabetics and 7 cases of long-term use of glibenclamide treatment of typeⅡdiabetics;HPLC techniques were used to study the drug metabolism for 7 cases of long-term use of glibenclamide treatment of typeⅡdiabetics after 1、2.5、4、8h at different time points. 3P97 pharmacokinetic software was used to calculate pharmacokinetic parameters and paired t-test to comparison.
【Results】
1. In the detection of 150 healthy controls, CYP2C9 gene promoter region -1188C/T sites、exon 2 269T>C sites、exon 3 430C>T sites、exon 6 895A>G sites and exon 7 1075A>C sites were 53.9% 1.3%,0%,2%,7% heterozygous carriers; 100 diabetics in these sites were 60.4%,0%,1%,1%,7% heterozygous carriers . Exon 4 found no polymorphism loci.
2. Statistical analysis showed that CYP2C9 gene promoter region -1188C/T、exon 2 269T>C and exon 3 430C>T mutational site exist quite different, but no statistical significance . The present study results with the other parts of China in total population, the Asian population( Japanese and Korean), Europe and the United States population CYP2C9 allele frequency comparisons found : exon 3 430C>T sites,with the population Europe and the United States, there was significant difference (Χ2p =65.3154, P<0.05); exon 7 1075A>C sites with the Asian population ( Japanese and Korean) (Χ2p =5.6488. P <0.05) and Europe and the United States population (Χ2p =11.6795. P <0.05) were significantly different, Other results of the comparison sites no significant difference (P>0.05).
3. In glibenclamide drug metabolism experiment found one poor metabolizer(PM). CYP2C9 gene sequencing showed that the subject of PM is a exon 6 895A>G site CYP2C9*1/*16 heterozygous genotype. The subject of PM glibenclamide plasma half-life of 51.87h. and other diabetics subjects (CYP2C9*1/*1) on average half-life of the individual 5.775h. It is about 9 times than CYP2C9*1/*1 genotype.
【Conclusion】
1. Polymorphism study showed, CYP2C9 gene promoter -1188C/T sites. Exon 2 269T>C sites, exon 3 430C>T sites, Exon 6 895A>G sites and exon 7 1075A>C sites there are polymorphic loci. Including 150 healthy controls and 100 typeⅡdiabetics these sites were 56.5%、0.8%、0.4%、1.6%、7.0% heterozygous carriers,and in exon 4, we found no polymorphism loci.
2. The present study results with the other parts of China population, the Asian population( Japanese and Korean), Europe and the United States population CYP2C9 allele frequency comparisons found: exon 3 430C>T sites, there was significant difference with the population Europe and the United States;exon 7 1075A>C sites,there was significant difference with the Asian population ( Japanese and Korean)、 Europe and the United States population(P<0.05).
3. Diabetics with glibenclamide metabolism study shows that CYP2C9 enzyme mutant allele CYP2C9*16(Exon 6 895A>G sites)to glibenclamide Pharmacokinetics significant affected, The research show that according to CYP2C9 gene typing of clinical specimens and Individual medical is of great guiding significance.
引文
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