CYP4F2基因变异与华法林严重出血并发症的关联性及功能机制研究

发布时间：2018-06-06 03:21

本文选题：CYP4F2 + 基因　；参考：《中国人民解放军医学院》2017年硕士论文

【摘要】：背景:尽管新型口服抗凝药已经问世,华法林作为一线口服抗凝药仍然在临床广泛应用;由于应用华法林抗凝治疗存在严重出血并发症的风险,因此,如何安全有效的个体化应用华法林抗凝治疗仍然是临床亟待解决的难题。华法林药物基因组学研究发现,除临床环境因素外,华法林代谢和作用通路上的基因遗传变异很大程度上影响了华法林的疗效和副作用。细胞色素P450家族成员CYP4F2具有氧化水解维生素K的功能,因此,编码基因CYP4F2的基因变异可能通过改变维生素K循环直接参与凝血因子的活化,进而影响华法林的抗凝效果和出血并发症发生。然而,目前尚缺乏关于CYP4F2变异型与华法林出血并发症相关联及其功能机制的报道。第一部分:CYP4F2候选基因变异与华法林严重出血并发症的关联性和生物信息功能预测分析:队列研究目的:筛查并验证与华法林主要出血并发症相关联的CYP4F2候选基因变异型。方法:前瞻性分阶段连续募集解放军总医院首次接受华法林抗凝治疗的患者,并进行为期至少3个月的随访。利用Hapmap和NCBIdsSNP数据库以及Haploview软件筛查CYP4F2的候选标签SNPs,通过Snapshot方法对所有入选患者的外周血DNA进行基因分型,利用logistic回归筛查并验证与华法林主要出血事件相关联的CYP4F2基因变异型。利用生物信息学分析寻找具有潜在生物功能的CYP4F2出血关联变异型。结果:本课题组于2008年1月至2011年9月期间和2014年1月至2015年12月期间,分别连续募集了符合入选标准并完成随访的312例和241例华法林抗凝治疗患者,前期收集病例作为筛查组,后期收集病例作为验证组。所有入选患者中包括:房颤410例(74.14%),瓣膜置换术后106例(18.63%);肺栓塞12例(2.17%),静脉血栓18例(3.25%),其他患者9例(1.63 %)。筛选出并成功行基因分型检测的CYP4F2标签SNPs总计8个,经logistic回归分析发现,筛查组中CYP4F2rs3093168 CC基因型携带者主要出血事件的发生风险明显高于CT+TT 基因型携带者(7.79% vs.2.13%, OR:5.39,95% CI:1.28-23.12,p=0.02)。验证组中,上述相关性依然存在(6.45% vs. 1.68%,OR:7.93,95%CI:1.03-61.06,p=0.04)。在所有病例中,CYP4F2rs3093168CC 基因型携带者主要出血事件的发生风险同样明显高于CT和TT基因型携带者者7.19%vs.1.93%, OR:5.23,95%CI:1.78-15.39,p=0.003 )。通过连锁不平衡分析发现,CYP4F2启动子上游第44,244碱基处存在与rs3093168相连锁的SNP位点rs2079288。利用 SNPalyze V4.0 软件分析发现,rs2079288 处于 linc RNA(NONHSAGO25013.2 )编码区,其功能机制可能与lincRNA通过cis调控CYP4F2基因表达有关。结论:CYP4F2rs3093168 CC基因型与华法林严重出血并发症密切相关,生物信息分析发现其功能机制可能与影响CYP4F2基因表达调控有关。第二部分:CYP4F2目标基因再测序和生物信息功能分析筛查与华法林严重出血并发症相关联的基因变异型:病例对照研究目的:通过CYP4F2目标基因再测序和生物信息功能分析筛查与华法林严重出血并发症相关联的基因变异型。方法:在前瞻性连续募集的解放军总医院首次接受华法林抗凝治疗的患者中,选取华法林主要出血病例及与之相匹配的未发生出血病例,对CYP4F2基因进行再测序。对所有基因变异型进行最小等位基因频率计算及Hardy-Weiberg平衡检验。利用logistic回归分析筛查出的CYP4F2基因变异型与主要出血事件的相关性,并利用生物信息学分析寻找具有潜在生物功能的CYP4F2出血关联变异型。结果:再测序分析总计发现了 64个CYP4F2基因变异型,其中12个为首次发现的新的基因变异,以上基因变异型均符合Hardy-Weiberg平衡。新发现的一处位于CYP4F2第3内含子区的插入缺失变异CYP4F2-1705 ins AGAT -1733 del CAGA仅见于严重出血病例中,发生率为22.22%。Mutation Taster软件分析该插入缺失变异位于第3个内含子和外显子剪接位点下游,并可能通过影响mRNA的剪接从而影响基因表达。结论:CYP4F2-1705 ins AGAT-1733 del CAGA与华法林严重出血并发症密切相关,生物信息分析发现其功能机制可能与影响CYP4F2基因转录有关。第三部分:华法林严重出血并发症相关联的CYP4F2基因变异型的体外生物功能分析目的:对华法林主要出血相关联CYP4F2基因变异型进行体外生物功能分析。方法:募集健康男性志愿者,提取外周全血DNA,对严重出血相关联基因变异(rs3093168 及 CYP4F2-1705 ins AGAT-1733 del CAGA )进行基因分型,筛选人口统计学和其他遗传背景相匹配的主要出血相关基因型携带者和非携带者。提取白细胞mRNA,利用qRT-PCR分析基因变异对CYP4F2基因转录的影响;提取血浆,利用CYP4F2 Elisa试剂盒分析基因变异对CYP4F2蛋白表达影响,利用FIXaAssay试剂盒分析基因变异对FIXa活化的影响。利用pIRES-FIX质粒构建pIRES-FIX-CYP4F2共转染质粒并酶切鉴定。将pIRES-FIX质粒,pIRES-FIX-CYP4F2质粒以及空白对照质粒分别转染L02细胞,western-blot检测CYP4F2及FIX的蛋白表达。利用FIXa检测试剂盒检测CYP4F2对FIX羧化功能的影响。结果:rs3093168 CC基因型与TT+CT基因型携带者的CYP4F2mRNA表达水平无明显差异,CYP4F2-1705 ins AGAT-1733 del CAGA插入缺失变异基因型携带者较非携带者CYP4F2mRNA表达水平明显升高。rs3093168CC基因型携带者及 CYP4F2-1705 ins AGAT-1733 del CAGA rs3093168 基因型携带者 CYP4F2蛋白表达水平较对照组均明显升高。FIXa活性检测发现,rs3093168 CC基因型携带者以及CYP4F2-1705 ins AGAT-1733 del CAGA插入缺失变异携带者的FIXa活性较对照组明显降低。转染相应质粒后,CYP4F2及FIX的蛋白成功过表达。在过表达FIX的L02细胞培养上清和细胞裂解液中,FIXa活性均较对照组显著升高;同时过表达CYP4F2和FIX后,L02细胞培养上清和细胞裂解液中的FIXa活性较单独过表达FIX组明显降低。结论:与华法林主要出血并发症相关的rs3093168 CC基因变异型及CYP4F2插入缺失变异可能通过增加CYP4F2蛋白表达,抑制凝血因子活化,导致变异携带者服用华法林的出血风险增加。
[Abstract]:Background: Although the new oral anticoagulants have come out, Hua Falin is still widely used as a first-line anticoagulant in the clinic. Because of the risk of severe bleeding complications in the application of Hua Falin anticoagulant therapy, how to apply Hua Falin anticoagulant therapy safely and effectively is still a difficult problem to be solved in clinic. Hua Falin drug is a difficult problem. Genomic studies have found that, in addition to the clinical environmental factors, the genetic variation in Hua Falin's metabolic and action pathways greatly affects the efficacy and side effects of Hua Falin. The cytochrome P450 family member CYP4F2 has the function of oxidative hydrolysis of vitamin K, so the gene mutation of the CYP4F2 gene may be altered by the change of vitamin K Circulation directly participates in the activation of coagulation factors and further affects the anticoagulant effect and bleeding complications of Hua Falin. However, there is still a lack of reports on the association of the CYP4F2 variant with the complications of Hua Falin bleeding and its functional mechanism. Part 1: the association and biology of the variant of the CYP4F2 candidate gene and the complications of Hua Falin's severe bleeding. Information functional prediction analysis: cohort study objective: to screen and verify the CYP4F2 candidate gene variant associated with the major bleeding complications of warfarin. Methods: a prospective, continuous recruitment of warfarin anticoagulants for the first time in the General Hospital of the Liberation Army, and a follow-up of at least 3 months. Use of Hapmap and NCBIdsSNP data. The library and the Haploview software screened the candidate label SNPs for CYP4F2. The Snapshot method was used to genotyping the peripheral blood DNA of all selected patients. The logistic regression was used to screen and verify the CYP4F2 gene variant associated with the major bleeding events of the warfarin. The bioinformatics analysis was used to find the CYP4F2 out of the potential biological function. Results: during the period from January 2008 to September 2011 and January 2014 to December 2015, we collected 312 cases and 241 cases of warfarin anticoagulant treatment, respectively, which met the criteria of admission and completed the follow-up. The early collection cases were selected as the screening group and the latter period was collected as the verification group. There were 410 cases of atrial fibrillation (74.14%), 106 cases (18.63%) after valve replacement, 12 cases of pulmonary embolism (2.17%), 18 cases of venous thrombosis (3.25%) and 9 cases (1.63%) of other patients (1.63%). A total of 8 CYP4F2 tags were selected and successfully detected by genotyping, and the main bleeding events of the CYP4F2rs3093168 CC genotype carriers in the screening group were found to be the main bleeding events in the screening group. The risk of occurrence was significantly higher than that of CT+TT genotype carriers (7.79% vs.2.13%, OR:5.39,95% CI:1.28-23.12, p=0.02). In the verification group, the above correlation still existed (6.45% vs. 1.68%, OR:7.93,95%CI:1.03-61.06, p=0.04). In all cases, the risk of major bleeding events in the CYP4F2rs3093168CC genotype carriers was also significantly higher than that of C. T and TT genotype carriers 7.19%vs.1.93%, OR:5.23,95%CI:1.78-15.39, p=0.003). Through linkage disequilibrium analysis, it was found that the SNP site linked to the rs3093168 phase in the 44244th base of the CYP4F2 promoter was found by the SNPalyze V4.0 software analysis of the rs3093168 phase. The mechanism may be related to the lincRNA regulation of CYP4F2 gene expression through CIS. Conclusion: the CYP4F2rs3093168 CC genotype is closely related to the severe bleeding complications of warfarin. Bioinformatics analysis found that its functional mechanism may be related to the regulation of CYP4F2 gene expression. The second part: CYP4F2 target gene re sequencing and biological information functional analysis. Screening and genetic variants associated with severe warfarin bleeding complications: a case-control study: screening of the gene variants associated with the complications of warfarin bleeding through CYP4F2 target gene sequencing and bioinformatic functional analysis. Methods: warfarin anticoagulants were first accepted in the PLA General Hospital of prospective continuous recruitment. The CYP4F2 gene was re sequenced in the main bleeding cases of warfarin and the non bleeding cases matched with them. The minimum allele frequency and Hardy-Weiberg balance test of all gene variants were performed. The CYP4F2 gene variant was screened by logistic regression and the major bleeding events were screened by logistic regression analysis. Correlation, and using bioinformatics analysis to find the associated variant of CYP4F2 bleeding associated with potential biological function. Results: 64 CYP4F2 gene variants were found by re sequencing analysis, of which 12 were new genetic variants found for the first time, and all of the above variants were in line with Hardy-Weiberg balance. A new site was found in CYP4F2 third. The insertion deletion mutation CYP4F2-1705 ins AGAT -1733 del CAGA in the intron is only in severe bleeding cases, the occurrence rate is 22.22%.Mutation Taster software analysis that the insertion deletion mutation is located downstream of the third introns and exon splicing sites, and may affect the gene expression by affecting the shear splicing of mRNA. Conclusion: CYP4F2-1705 in S AGAT-1733 del CAGA is closely related to the severe bleeding complications of warfarin. Bioinformatics analysis found that its functional mechanism may be related to the impact of CYP4F2 gene transcription. The third part: in vitro biofunctional analysis of the CYP4F2 gene variant associated with the severe hemorrhage complications of warfarin: related to the main bleeding phase of the warfarin CYP4F2 base Methods: in vitro biological function analysis of the variant. Methods: the healthy male volunteers were collected, the peripheral blood DNA was extracted, the gene mutation (rs3093168 and CYP4F2-1705 ins AGAT-1733 del CAGA) of the severe hemorrhage phase was genotyped, and the major haemorrhagic related genotype carriers, which matched the demographic and other genetic scenes, were screened. Non carrier. Extract leukocyte mRNA, use qRT-PCR to analyze the effect of gene mutation on CYP4F2 gene transcription; extract plasma, use CYP4F2 Elisa kit to analyze the effect of gene mutation on the expression of CYP4F2 protein, analyze the effect of gene mutation on FIXa activation by FIXaAssay kit. Use pIRES-FIX plasmid to construct pIRES-FIX-CYP4F2 co transfection. Plasmid and enzyme digestion were identified. PIRES-FIX plasmid, pIRES-FIX-CYP4F2 plasmid and blank control plasmid were transfected into L02 cells respectively. The protein expression of CYP4F2 and FIX was detected by Western-blot. The effect of CYP4F2 on FIX carboxylic function was detected by FIXa detection kit. Results: the expression level of rs3093168 CC genotype and TT+CT genotype carrier. There was no significant difference between the CYP4F2-1705 ins AGAT-1733 del CAGA insertion mutant genotype carriers and the CYP4F2mRNA expression level of the non carriers significantly higher.Rs3093168CC genotype and CYP4F2-1705 ins AGAT-1733. It was found that the rs3093168 CC genotype carriers and the FIXa activity of the CYP4F2-1705 ins AGAT-1733 del CAGA insertion deletion carriers were significantly lower than those of the control group. The proteins of CYP4F2 and FIX were overexpressed successfully after the transfection of the corresponding plasmid. At the same time, after overexpression of CYP4F2 and FIX, the activity of FIXa in L02 cell culture supernatant and cell lysate was significantly lower than that in FIX group. Conclusion: the rs3093168 CC gene variant and CYP4F2 insertion deletion mutation associated with the major bleeding complications of warfarin may be induced by increasing the expression of CYP4F2 protein and inhibiting the activation of coagulation factor. Carriers with warfarin had a higher risk of bleeding.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R973.2

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