CYP2C19 基因多态性与冠心病危险因素对氯吡格雷抵抗的影响

目的:观察细胞色素P450系统药物代谢酶CYP2C19基因多态性以及相关临床因素对氯吡格雷抵抗的影响。方法:选择2010年11月至2011年5月我科拟行PCI术治疗的冠心病患者共145例,均给予氯吡格雷300mg负荷剂量,75mg维持剂量。①通过流式细胞仪检测血管舒张因子刺激酸磷蛋白血小板反应性指数VASP PRI(以VASP PRI≥50%,定义为氯吡格雷抵抗)分为氯吡格雷抵抗组和氯吡格雷反应组。②检测入选患者的药物代谢酶CYP2C19的基因型;根据不同等位基因功能缺失,分为快代谢基因型(*1/*1)、中间代谢基因型(*1/*2、*1/*3)和慢代谢基因型(*2/*2、*2/*3、*3/*3)。③观察CYP2C19基因型及相关临床危险因素对氯吡格雷反应性的影响,④观察氯吡格雷抵抗与临床不良终点事件主要临床不良终点事件[心源性死亡、再发心肌梗死、靶病变再次血运重建术(TLR)]和次要临床终点事件(支架内血栓形成、脑血管意外、大出血)之间的相关性。结果:检测出氯吡格雷抵抗的患者31例,其发生率为20.67%;检测出CYP2C19慢代谢基因型携带患者19例,所占比例为12.67%。慢代谢基因型患者与(快代谢基因型+中间代谢基因型患者)之间VASP PRI比为(49.20±8.45)%VS(44.17±5.41)%,P<0.05,氯吡格雷抵抗发生率之比为35.49%(n=11)VS16.81%(n=20),P<0.05。多元回归分析提示CYP2C19慢代谢基因型(OR:4.43;95%CI:3.28-8.37,P<0.05)和2型糖尿病(OR:2.76;95%CI:2.13-6.14;P<0.05)是氯吡格雷抵抗的两种危险因素。临床随访结果显示氯吡格雷抵抗组与氯吡格雷反应组主要临床不良终点事件的发生率比为6.45%(n=2)vs2.63%(n=3),P<0.05。结论:携带CPY2C19慢代谢基因型和患有2型糖尿病是导致氯吡格雷抵抗的两种重要的危险因素,氯吡格雷抵抗的发生增加了临床不良终点事件的风险。     

CYP2C19遗传多态性的研究进展

S-美芬妥英羟化代谢多态性不仅存在个体差异,而且存在种族差异。CYP2C19基因是决定S-美芬妥英羟化代谢的决定基因,该基因的突变是导致S-美芬妥英羟化代谢多态性的分子机制。近年来对CYN50s基因型和表型相关性的研究越来越受到重视,人们希望利用基因型分析来了解个体中该药物代谢酶的活性,期望既能提高药物治疗水平同时又降低不良反应的发生。有关CYP2C19的研究在此方面已树立了一个成功的典范。     

ABCB1、CES1 和 CYP2C19 基因多态性与 氯吡格雷抵抗的相关性研究进展

氯吡格雷是目前全球临床使用最为广泛的血小板受体抑制剂,但其抗血小板效应存在明显个体差异,部分病人服用常规剂量氯吡格 雷后存在抵抗现象,甚至发生不良临床事件。多项研究表明,ABCB1、CES1 和 CYP2C19 基因多态性对氯吡格雷抵抗的产生发挥重要作用。 简介氯吡格雷体内吸收与代谢机制和氯吡格雷抵抗的定义,综述 ABCB1、CES1 和 CYP2C19 基因多态性对氯吡格雷抵抗的影响。     

应用HRM技术对CYP2C19*2和CYP2C19*3进行双重SNP分型

氯吡格雷是一种广泛用于预防静脉血栓形成的抗血小板药物。研究表明, 携带有CYP2C19基因功能缺失型等位基因CYP2C19*2、CYP2C19*3的病人, 其体内代谢氯吡格雷成为其活性形式的能力降低, 导致氯吡格雷抑制血小板聚集功能减弱。文章旨在建立一种利用高分辨率熔解曲线分析(High-resolution melting curve analysis,HRM)技术在闭合单管中同时对CYP2C19*2、CYP2C19*3两个多态性位点进行简便、准确分型的方法。本实验针对两个SNP位点分别设计特异性的HRM引物, 并在两个位点引物的5′端分别加上富含AT和GC的序列, 保证两个位点的扩增产物熔解峰无重叠。利用HRM技术, 快速、灵敏地对64例随机DNA样本的CYP2C19*2 、CYP2C19*3两个多态性位点进行了基因分型, 且HRM方法的分型结果与测序验证结果完全一致。因此, 利用HRM技术可以实现在闭合单管中简便、准确地对CYP2C19*2 、CYP2C19*3两个多态性位点同时进行基因分型。该方法有望应用于临床, 指导氯吡格雷的个体化用药。     

CYP2C9 1061 A／C与VKORC1—1639 G／A基因多态性对华法林应用剂量的影响

目的探讨维生素K环氧化物还原酶复合物1基因（VKORC1）-1639G／A与细胞色素P450酶2C9基因CYP2C9 1061 A／C多态性对中国汉族人华法林应用剂量的影响。方法应用PCR—RFLP方法检测129例长期口服华法林的患者VKORC1—1639G／A及CYP2C9 1061 A／C多态性,并按基因型分组,分别比较VKORC1和CYP2C9不同基因型间平均华法林剂量。结果病例组检出VKORC1—1639AA、AG、GG型分别有96例（74．4％）、30例（23．3％）、3例（2．3％）,等位基因A和G频率分别为86％、14％,CYP2C91061;AA、AC、CC型分别有117例（90．7％）、11例（8．5％）、1例（0．8％）,等位基因A和c频率jj别为95％、5％;病例组与正常人组VKORC1—1639、CYP2C9 1061各基因型分布差异无统计学意义;VKORC1—1639不同基因型间患者所需华法林平均剂量AA型低于AG型,后者又要低于GG型;携带CYP2C9 1061 C等位基因的患者（AC和CC型）华法林平均剂量要低于AA型。结论华法林应用剂量偏低可能与VKORC1-1639G→A和CYP2C9 1061A→C转变有关,VKORC1-1639AA型占多数可能是中国汉族人群所需华法林剂量普遍较低的重要原因。     

用均匀设计优化apo E基因的PCR扩增方案

由于PCR影响因素多,获得理想扩增结果比较困难,有必要寻找一种简单有效的方法建立最佳扩增体系.针对Mg²⁺浓度、二甲基亚砜(DMSO)浓度、变性时间、延伸时间、循环次数等因素进行4因素6水平和6因素10水平均匀设计实验优化apo E基因244 bp片段的PCR扩增条件.采用纯化模板和简易模板,只需6～10次实验即可获得特异性、高产率扩增结果.研究表明,将均匀设计用于PCR条件优化以及有关的样品处理、试剂选择等研究方面可以避免盲目性,迅速获得满意结果.     

ACE、CYP11B2基因多态性与慢性心力衰竭患者醛固酮脱逸的关系

目的:研究CYP11B2-344C/T(醛固酮合成酶)及ACEI/D(血管紧张素转化酶)基因多态性与慢性心力衰竭(CHF)患者实施ACEI治疗后出现醛固酮脱逸表现的关系。方法:回顾分析2008年10月至2012年10月我科收治的252例CHF患者,全部患者应用ACEI治疗3月,醛固酮在基线以上为醛固酮脱逸,依据此标准将患者分为研究组(脱逸组,n=86)与对照组(非脱逸组,n=166),依据PCR(聚合酶链反应)及RFLP(片段长度限制多态性)等方法分别检测两组CYP11B2及ACE基因型,比较两组基因型频率的分布。结果:252例患者中,共86例出现醛固酮脱逸,发生率为34.1%。全部受试患者CYP11B2基因型及ACE基因型频率与Weinberg-Hardy平衡均相符(P均0.05)。研究组ACE I/D三种基因型的组间分布与对照组相较,无统计学差异(P0.05);CYP11B2基因TT型的频率与对照组相较,呈明显统计学差异(P0.05),等位基因C/T频率的组间分布同对照组相较,亦呈明显差异(P0.05)。研究组ACEI/D的基因多态性及CYP11B2-344C/T的多态性中,基因型联合组间分布与对照组相较,无统计学差异(P0.05)。结论:ACE基因多态性与CHF患者ACEI治疗后出现醛固酮脱逸无关,CYP11B2基因T等位基因及TT基因型多态性可能是CHF患者ACEI治疗后发生醛固酮脱逸的高危因素。醛固酮脱逸时,ACE、CYP11B2基因不具有协同效果。     

杨梅RAPD-PCR体系的正交优化研究

以杨梅DNA为模板,对影响杨梅RAPD-PCR扩增的重要参数进行了优化试验,以期建立杨梅RAPD反应的最佳体系。通过采用正交试验设计的方法,对杨梅RAPD-PCR条件进行了优化,结果表明最佳的杨梅RAPD-PCR的反应体系(20μl)中含有1×buffer,1.0U TaqDNA聚合酶,3.0mmol/L MgCl2,0.30mmol/L dNTPs,1.5μmol/L引物和模板DNA 30-40ng。适宜的扩增条件为94℃预变性3min,再进入38个PCR循环(94℃变性30s,38℃退火30s,72℃延伸90s),72℃延伸7min,4℃保存。     

中国汉族人群CYP4F3基因多态性的研究

目的:研究CYP4F3基因单核苷酸多态性(SNP)在中国汉族人群中的分布,为进一步研究该基因群体遗传学特征及与疾病易感性的相关性提供更为详实的数据。方法:对CYP4F3基因进行重测序,构建连锁不平衡模式,选择标签SNP在192例北京和424例广州汉族个体中进行基因分型。结果:CYP4F3基因重测序共检出30个SNP,连锁不平衡分析显示广州和北京地区人群的连锁不平衡模式不同,但选择的8个标签SNP的等位基因和基因型频率分布在2个人群中的差异无统计学意义。结论:中国北京地区汉族与广州地区汉族人群CYP4F3基因多态性无显著差异,但不同种族间存在差异。     

CYP19基因表达与芳香化酶活性调控因子的研究进展

芳香化酶是雌激素合成中的关键酶,催化睾酮和雄烯二酮转化为雌激素。本文在对芳香化酶的蛋白结构、基因特征和分布进行阐述的基础上,重点对编码该蛋白的CYP19基因的调控因子以及芳香化酶活性的调节进行探讨。CYP19基因的调控因子包括cAMP反应元件(CRE)、类固醇生成因子1/肾上腺4结合蛋白(SF-1/Ad4BP)、雌激素受体(ER)等顺式作用因子和TATA结合蛋白(TBP)、生长因子等反式作用因子。主要通过cAMP依赖性蛋白激酶信号通路在转录水平对其进行调节。而芳香化酶表达及其酶活性的调控因子主要集中于性类固醇激素和促性腺激素,此外还受到温度等外部因子等因素的调节。芳香化酶的调节对维持雌雄激素间作用的平衡、保证机体的正常生理功能具有重要意义。     

Role of CYP2C9 and CYP2C19 polymorphisms in patients with atherosclerosis

The arachidonic acid metabolizing CYP enzymes with prominent roles in vascular regulation are epoxygenases of the two gene family which generate epoxyeicosatrienoic acids. Carriers of CYP2C9 mutant alleles exhibit a diminished CYP2C9 metabolic capacity leading to decreased endothelium-derived hyperpolarizing factors (EDHF) synthesis and an increased risk for atherosclerosis. We investigated whether the polymorphisms of CYP2C9/19 are related with atherosclerosis. We examined 108 patients having angioraphically > or =70 coronary artery narrowing and 90 healthy controls. CYPC2C9/19*2 and CYP2C9/19*3 alleles were investigated in both patients and controls by a real time PCR instrument. There was no significant difference in the distribution of the CYP2C9*2/*3 alleles between cases and the controls. We found that smoker patients having CYP2C9*2 heterozygote genotype have 3.7-fold risk of developing atherosclerosis. CYP2C19*3 heterozygote alleles are more frequent in patients than in controls (10.2%, 5.6% respectively) and it is related with a three-fold risk of atherosclerosis (odds ratio (OR) = 3.75, confidence interval (CI) = 0.75-18.65). It becomes clear that cigarette smoking can cause almost all major diseases prevalent today, such as cancer or heart disease. This inter-subject variability in cigarette-induced pathologies is partly mediated by genetic variants of genes that may participate in detoxification processes, e.g., cytochrome P450 (CYP), cellular susceptibility to toxins, such as p53, or disease development such as atherosclerosis.     

Lipophilicity Relationships in Inhibitors of CYP2C9 and CYP2C19 Enzymes

Quantitative structure-activity relationships (QSARs) within a series of cytochrome P450 2C9 (CYP2C9) and cytochrome P450 2C19 (CYP2C19) inhibitors are reported. In particular, it is noted that compound lipophilicity, in the form of log P values (where P is the octanol/water partition coefficient), is an important factor in explaining the variation in inhibitory potency within these series of compounds, many of which also act as substrates for the respective enzymes. In addition, there is a role for hydrogen bonding and π-π stacking interactions within the P450 active site which represent secondary factors in the binding processes of these compounds.     

Rabeprazole Can Overcome the Impact of CYP2C19 Polymorphism on Quadruple Therapy

Objectives: The prospective study was designed to clarify the impact of CYP2C19 on quadruple therapies and survey the efficacies of rabeprazole‐based quadruple therapy for Helicobacter pylori infection after failure of standard triple therapies. Patients and Methods: From January 2007 to March 2009, 1055 H. pylori‐infected patients received standard triple regimens (proton‐pump inhibitor (PPI), clarithromycin, and amoxicillin). Helicobacter pylori eradication was achieved in 865 (81.9%) subjects. One hundred ninety eradication‐failure patients were enrolled and randomly assigned to receive a 7‐day eradication therapy. Ninety‐six patients were treated with esomeprazole‐based quadruple rescue therapies (EB), while 94 patients were treated with rabeprazole‐based quadruple rescue therapies (RB). Follow‐up endoscopy was done 16 weeks later to assess the treatment response. Patients’ responses, CYP2C19 genotypes, and antibiotics resistances were also examined. Results: Intention‐to‐treat analysis revealed that RB had better eradication rates than EB (EB: 72.9%; 95% CI: 64.9–80.9% and RB: 78.7%; 95% CI 72.5–84.9%) (p value = .543). Per‐protocol results were EB = 75.3%; 95% CI: 70.3–80.3% and RB = 85.1%; 95% CI: 80.6–89.6% (p value = .0401). Both regimens had similar compliance (p value = 0.155) and adverse events (p value = 0.219). We also surveyed those patients without resistance of any antibiotics. RB still showed better outcome than EB. Our data showed that esomeprazole‐based regimen and CYP2C19 Hom EM genotype were important predictors for eradication failure. Conclusions: In quadruple therapy, rabeprazole‐based regimens had better efficacy than esomeprazole‐based regimens. CYP2C19 polymorphism also played an important role in quadruple therapy. It seems advisable to change PPI to rabeprazole in second‐line quadruple therapy.     

Active-site characteristics of CYP2C19 and CYP2C9 probed with hydantoin and barbiturate inhibitors

Three series of N-3 alkyl substituted phenytoin, nirvanol, and barbiturate derivatives were synthesized and their inhibitor potencies were tested against recombinant CYP2C19 and CYP2C9 to probe the interaction of these ligands with the active sites of these enzymes. All compounds were found to be competitive inhibitors of both enzymes, although the degree of inhibitory potency was generally much greater towards CYP2C19. Inhibitor stereochemistry did not markedly influence K(i) towards CYP2C9, and log P adequately predicted inhibitor potency for this enzyme. In contrast, stereochemistry was an important factor in determining inhibitor potency towards CYP2C19. (S)-(+)-N-3-Benzylnirvanol and (R)-(-)-N-3-benzylphenobarbital emerged as the most potent and selective CYP2C19 inhibitors, with K(i) values of < 250nM--at least two orders of magnitude greater inhibitor potency than towards CYP2C9. Both inhibitors were metabolized preferentially at their C-5 phenyl substituents, indicating that CYP2C19 prefers to orient the N-3 substituents away from the active oxygen species. These features were incorporated into expanded CoMFA models for CYP2C9, and a new, validated CoMFA model for CYP2C19.     

Stereoselective determination of the CYP2C19 probe drug mephenytoin in human urine by gas chromatography-mass spectrometry

A sensitive, specific and reproducible gas chromatographic assay utilizing mass-selective detection has been developed for the stereoselective determination of mephenytoin (MP) in human urine. Following extraction of urine samples using methyl tert.-butyl ether, separation of R- and S-MP was achieved with a chiral capillary column; detection and quantitation were accomplished by mass spectrometry in the single ion monitoring mode (m/z 104 and 189). Excellent linearity was observed for both enantiomers over the concentration range of 5-1000 ng/ml with corresponding correlation coefficients (r)>0.99. The intra- and inter-day precision and accuracy were within +/-5%. This method employs a simplified processing procedure, demonstrates improved extraction recovery, and provides at least 5-fold greater sensitivity than previously reported assays. This method is well suited for the phenotypic evaluation of CYP2C19 activity using mephenytoin.     

中国苗族人群中细胞色素P450 2C19基因多态性的研究

为研究细胞色素P45 0 2C19在中国苗族人群中的基因型 ,采用聚合酶链式反应与限制性内切核酸酶片段长度多态性技术分析了 98例无血缘关系苗族人群的基因型。结果显示 5 0人为CYP2C19野生型纯合子 (wt/wt) ;39人为CYP2C19m1杂合子 (wt/m1) ;9人为CYP2C19m1突变型纯合子 (m1/m1)。与国内外相关报道比较 ,本实验得出的CYP2C19m1突变频率与文献报道相符