血栓性疾病中抗凝蛋白的遗传变异及功能研究

发布时间：2018-05-30 07:19

本文选题：抗凝血酶 + 组织因子途径抑制物　；参考：《华中科技大学》2016年博士论文

【摘要】：第一部分抗凝血酶基因变异与静脉血栓的关联研究目的：研究抗凝血酶基因多态性(SERPINC1)与静脉血栓的关联及其致病的潜在的分子遗传学机制。方法和结果：随机选择60例年龄不大于65岁及无蛋白C、蛋白S缺乏的非急性期VTE患者,对其SERPINC1基因启动子区域、编码区及侧翼序列进行重测序,在两位VTE患者基因组DNA中新发现了位于5'非翻译区的同一杂合突变c.-11GA(rs483352843)。对该变异进行大样本(1760例病例 VS 2200例对照)的酶切分型分析显示,c.-11GA突变显著增加携带者患静脉血栓的风险(0R=6.91; 95% CI:1.53-31.22;P=0.0035)。杂合突变携带者的抗凝血酶平均活性及抗原的平均水平分别为对照的78.4%(P0.0001)及88.9%(P=0.0004)。构建野生型及突变型萤光素酶表达载体,分别转染HEK-293T和COS-7细胞,突变型表达载体在HEK-293T和COS-7细胞中表达的双荧光素酶活性分别为野生型的62.9%(P=0.006)和53.1%(P=0.004)。提取外周血细胞及上述两种培养细胞的总RNA做实时定量PCR显示,突变型mRNA表达量分别为野生型的35.8%(P=0.026)、57.1%(P=0.010)及52.8%(P=0.004)。凝胶迁移实验(EMSA)显示突变型探针与DNA结合蛋白的结合明显强于野生型探针。结论：SERPINC1基因5'非翻译区-11位的G碱基突变为A碱基后,突变后的DNA与转录抑制因子的结合显著增加,从而抑制了抗凝血酶基因的转录,使突变携带者体内抗凝血酶的量显著降低,导致携带者患VTE的风险显著增加。第二部分TFPI及ADTRP基因多态性与静脉血栓的关联研究目的：研究组织因子途径抑制物(TFPI)及雄激素依赖的TFPI调节蛋白(ADTRP)基因可能存在的变异与静脉血栓的关联。方法与结果：随机挑选176例年龄不超过65岁的无抗凝血酶、蛋白C及蛋白S缺乏的VTE患者,用酶联免疫吸附试验(ELISA)检测血浆中TFPI抗原浓度,以来自100例健康体检者的混合血浆作为参照,挑选出TFPI抗原浓度低于混合血浆TFPI抗原浓度50%的VTE患者48例。对这部分患者的TFPI, ADTRP基因重测序,分别发现了10种TFPI基因突变及11种ADTRP基因突变,其中位于ADTRP基因上c.154-18delT突变为首次报道。对TFPI c.662AG (rs7586970)及ADTRP c.154-18delT突变进行大样本(901例病例vs 799例对照)基因分型分析结果显示,c.662AG及c.154-18delT均不是VTE的危险因素,其OR值分别为0.85 (95% CI; 0.71-1.03; P= 0.106)及1.07(95% CI；0.998-1.14：P=0.058)。结论：在中国汉族人群中,TFPI基因上目前尚未发现增加携带者患VTE的风险的变异,而ADTRP上c.154-18delT多态性位点可能是VTE的危险因素,该结论有待进一步研究验证。第三部分血栓调节蛋白基因变异在血栓性疾病中的作用的分子遗传学研究目的：研究血栓调节蛋白(thrombomodulin, TM)基因(THBD)罕见突变在血栓性疾病中作用的可能的分子遗传学机制。方法与结果：通过基因测序,在七位血栓性疾病患者的THBD基因中发现了7种新发突变：c.376GT (p.Asp126Tyr), c.569CG (p.Ser 190Trp), c.659TG (p.Leu220X), c.1208GA (p.Arg403Lys), c.1288GA (p.Gly430Ser), c.*6_*23del和c.*23_*40del.其中c.376GT和c.*6_*23del同时出现在一发病年龄为10岁的反复发作的深静脉血栓(VTE)患者,该患者在随访一年后死亡；无义突变c.659TG在一55岁的女性心肌梗死(MI)患者及46岁的男性VTE患者中均被发现,其中该男性VTE患者在随访两年后死亡。酶联免疫吸附试验显示这些突变携带者血浆游离TM抗原水平均比对照组低。通过体外细胞实验,我们发现与野生型相比,有两个点突变显著降低了TM抗原的表达：Ser190Trp(71.2%±12.8%,P0.05),p.Leu220X(20.1%±10.4%,P0.01)；通过激活的PC水平判断,与野生型比,四个点突变(c.569CG.c.659TG.c.1208GA和c.1288GA)均使TM的辅因子活性显著降低。对于c.*6_*23de1和c.*23_*40del两个18碱基缺失突变,双荧光素酶报告基因表达水平均较野生型减低。结论：THBD基因上这7种变异都是有害突变,增加了携带者患血栓性疾病的风险。
[Abstract]:Part 1 Association of antithrombin gene mutation with venous thrombosis: To study the association of antithrombin gene polymorphism (SERPINC1) with venous thrombosis and the underlying molecular genetic mechanism. Methods and results: a random selection of 60 non acute VTE patients with age 65 years old and no protein S and protein S deficiency were selected. The SERPINC1 gene promoter region, the coding region and the flanking sequence were re sequenced, and the same heterozygous mutation c.-11GA (rs483352843) was found in the 5'non translation region of the two VTE patients. A large sample of the mutation (1760 cases VS 2200 cases) showed that the c.-11GA mutation increased significantly. The risk of venous thrombosis (0R=6.91; 95% CI:1.53-31.22; P=0.0035). The average activity of antithrombin and the average level of antigen were 78.4% (P0.0001) and 88.9% (P=0.0004) in the heterozygous mutant carriers, respectively. The wild type and mutant fluoroenzyme expression vector was constructed, and HEK-293T and COS-7 cells were transfected respectively, and the mutant expression vectors were used. The activity of double luciferase expressed in HEK-293T and COS-7 cells were 62.9% (P=0.006) and 53.1% (P=0.004) in the wild type respectively. The real-time quantitative PCR of the extraction of peripheral blood cells and the total RNA of the above two cultured cells showed that the expression of mutant mRNA was 35.8% (P= 0.026), 57.1% (P=0.010) and 52.8% (P=0.004), respectively. EMSA) showed that the combination of the mutant probe and the DNA binding protein was stronger than the wild type probe. Conclusion: after the mutation of the G base of the -11 site of the SERPINC1 gene 5'to the A base, the binding of the mutant DNA to the transcriptional inhibitor increased significantly, thus inhibiting the transcription of the antithrombin gene and the amount of the antithrombin in the mutant carrier. A significant decrease, leading to a significant increase in the risk of carriers suffering from VTE. Second the association of TFPI and ADTRP gene polymorphisms with venous thrombosis: the study of the association of the possible mutations of the tissue factor pathway inhibitor (TFPI) and the androgen dependent TFPI regulatory protein (ADTRP) gene with the venous thrombosis. Methods and results: random selection 176 patients aged 65 years of age without antithrombin, protein C and protein S deficient VTE were detected by enzyme linked immunosorbent assay (ELISA) to detect the concentration of TFPI antigen in plasma, and 48 cases of VTE patients with TFPI antigen concentration lower than the concentration of TFPI antigen of mixed plasma were selected and 48 cases of VTE patients were selected from the mixed plasma TFPI antigen concentration 50%. TFPI, ADTRP gene resequencing, 10 TFPI gene mutations and 11 ADTRP mutations were found, of which the c.154-18delT mutation located on the ADTRP gene was first reported. A large sample of TFPI c.662AG (rs7586970) and ADTRP c.154-18delT mutations (901 case vs 799 cases) 154-18delT was not a risk factor for VTE, and its OR values were 0.85 (95% CI; 0.71-1.03; P= 0.106) and 1.07 (95% CI; 0.998-1.14:P=0.058). Conclusion: in Chinese Han population, the risk of increasing VTE is not found in the TFPI gene, and ADTRP c.154-18delT polymorphic loci may be a risk factor. This conclusion needs further research and verification. Third the molecular genetic study of the role of thrombomodulin gene mutation in thrombotic diseases: Objective: To study the possible molecular genetic mechanism of the rare mutation of thrombomodulin (TM) gene (THBD) in thrombotic diseases. Methods and results: through the basis By sequencing, 7 new mutations were found in the THBD gene of seven patients with thrombotic disease: c.376GT (p.Asp126Tyr), c.569CG (p.Ser 190Trp), c.659TG (p.Leu220X), c.1208GA (p.Arg403Lys), c.1288GA (p.Gly430Ser), and simultaneously appeared at the age of 10. In patients with recurrent deep venous thrombosis (VTE), the patient died after a year of follow-up; nonsense mutation c.659TG was found in a 55 year old female myocardial infarction (MI) patient and a 46 year old male VTE. The male VTE patient died after two years of follow-up. The enzyme linked immunosorbent assay showed the free TM antigen of these mutant carriers. Compared with the control group, we found that two point mutations decreased the expression of TM antigen significantly compared with the wild type: Ser190Trp (71.2% + 12.8%, P0.05), p.Leu220X (20.1% + 10.4%, P0.01), and four point mutations (c.569CG.c.659TG.c.1208GA and c.1288GA), compared with the wild type, by the activation of PC level. The activity of the cofactor of TM was significantly reduced. For two 18 base deletion mutations in c.*6_*23de1 and c.*23_*40del, the expression level of the double luciferase reporter gene was lower than that of the wild type. Conclusion: the 7 mutations in the THBD gene are all harmful mutations, increasing the risk of thrombotic disease in the carrier.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R543

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