遗传性FⅫ缺陷症分子机制及血小板相关疾病研究

发布时间：2018-05-13 14:20

本文选题：凝血因子FⅫ + 基因突变　；参考：《上海交通大学》2015年博士论文

【摘要】：本研究收集了5个遗传性凝血因子Ⅻ(coagulation factor Ⅻ,FⅫ)缺陷症家系,并对此开展了临床表现、家系调查、实验室检测和基因诊断等方面的研究;对所发现的3个导致CRM-遗传性FⅫ缺陷症的突变位点(c.776GA,c.799CG,c.1561GA)进行了突变蛋白的结构与功能研究,以探讨三个突变导致遗传性FⅫ缺陷症的分子机制。此外,本研究探讨了血小板膜蛋白GPIbβ胞内段调控GPIbα酶切的分子机制,并应用全外显子测序方法对一例难治性ITP进行了基因诊断研究。FⅫ是内源凝血系统的启动因子,遗传性FⅫ缺陷症是由编码FⅫ蛋白的基因F12突变所引起的,是一种常染色体隐性遗传性疾病,患者无临床出血表现,多在术前凝血筛查中因APTT延长而发现。本研究对发现的5个遗传性FⅫ缺陷症进行了基因诊断和分子机制研究。5个先证者FⅫ活性(FⅫ:C)和抗原(FⅫ:Ag)均明显降低,基因诊断发现了F12基因上存在3种基因突变,分别是c.776GA(p.G259E)、c.799CG(p.R267G)和c.1561GA(p.E521K),其中c.776GA(p.G259E)、c.799CG(p.R267G)为国际首次报道。针对发现的基因突变,构建FⅫ突变表达质粒,在体外探讨三个突变导致遗传性FⅫ缺陷症的分子机制。转染突变质粒细胞内外的FⅫ:Ag均明显下降,细胞上清中FⅫ:C也明显下降,提示三种突变是导致FⅫ缺陷的原因;Real time PCR结果显示三个FⅫ突变体均能正常转录;进一步蛋白降解抑制实验结果显示三个FⅫ突变体通过蛋白酶体进行降解是导致FⅫ表达量下降的根本原因;FⅫ突变体和野生型共转染HEK 293T细胞结果显示FⅫR267G和FⅫG259E具有dominant negative effect,然而,这种dominant negative effect的产生并不是由于FⅫ突变体和野生型形成杂合二聚体而引起的。GPIb-IX-V复合物是引起血小板活化第一步的受体蛋白,在生理止血中起着重要作用。GPIbα是GPIb-IX-V复合物中最重要的一个亚基,其酶切在血栓形成过程中起着负调控的作用,对于血小板保存及清除也至关重要。目前对于GPIb-IX-V复合物中GPIbα酶切调控机制仍不明确。我们前期研究发现的GPIbβ胞内近膜端可以通过与某个未知蛋白相互作用抑制GPIbα酶切。为明确这个未知蛋白,我们在大肠杆菌中表达并提纯野生型以及关键部位突变(R151E/R152E或R149E/L150E)的GPIbβ胞内段蛋白,利用pull-down结合蛋白质谱(MS)的方法,筛选出多种可能与GPIbβ胞内段相互作用的蛋白,包括膜突蛋白(moesin)、血小板反应蛋白(thrombospondin)等。免疫性血小板减少性紫癜(immune thrombocytopenia purpura,ITP)是一种获得性的自身抗体介导的破坏和损害血小板及其再生的血液系统疾病。临床以皮肤粘膜或内脏出血为主要表现。本研究中我们发现一例激素治疗无效的难治性ITP患者,为明确其致病因素,我们对患者外周血DNA/骨髓DNA进行了全外显子组测序,寻找仅存在于骨髓DNA而外周血DNA中为阴性的变异。经过生物信息学分析筛选出一组候选致病基因,进一步对其进行Sanger测序验证后,最终推断Ct BP2基因存在p.S240SF突变,从而导致巨核细胞成熟障碍可能是该患者血小板减少的致病原因。
[Abstract]:This study collected 5 hereditary coagulation factor (coagulation factor F) defect families, and carried out a study of clinical manifestations, family survey, laboratory testing and gene diagnosis, and the mutation protein (c.776GA, c.799CG, c.1561GA), which were found to lead to the CRM- hereditary F deficiency syndrome (c.776GA, c.799CG, c.1561GA), was carried out. The structural and functional studies were conducted to investigate the molecular mechanism of the genetic F deficiency caused by three mutations. In addition, the molecular mechanism of the GPIb beta cell segment of the platelet membrane protein in the regulation of GPIb alpha enzyme digestion was discussed, and an exon sequencing method was used for the diagnosis of a refractory ITP based on the study of the initiation of the endogenous coagulation system. Factor, hereditary F deficiency syndrome is caused by the mutation of the gene F12 that encodes the protein F protein. It is an autosomal recessive hereditary disease. The patient has no clinical bleeding and is found in the preoperation coagulation screening because of the extension of APTT. The genetic diagnosis and molecular mechanism of the 5 hereditary F defects found in this study are.5. The F activation (F) activity (C) and the antigen (F: Ag) were significantly reduced. The gene diagnosis found that there were 3 gene mutations in the F12 gene, c.776GA (p.G259E), c.799CG (p.R267G) and c.1561GA (p.E521K). The molecular mechanism of hereditary F defects caused by three mutations was investigated in vitro. The F of transfected plasmids, both inside and outside the cells of the mutant plasmid, decreased obviously, and the F of the cell supernatant decreased obviously, and C showed that the three mutations were the cause of the defect of F; the Real time PCR results showed that all of the three F mutants could be transcribed normally; further protein degradation and inhibition were suppressed. The results of the experiment showed that the degradation of the three F mutants through proteasome was the root cause of the decrease in the expression of F. The results of the F mutant and the wild type CO transfected HEK 293T cells showed that F R267G and F G259E had dominant negative effect. The.GPIb-IX-V complex, which is caused by the formation of heterozygous two polymer, is a receptor protein that causes the first step of platelet activation. It plays an important role in the physiological hemostasis..GPIb A is the most important subunit in the GPIb-IX-V complex. Its enzyme digestion plays a negative regulatory role in the process of thrombus formation and the preservation and removal of platelets. The regulatory mechanism of GPIb alpha enzyme in the GPIb-IX-V complex is still not clear. Our previous study found that the near membrane end of the intracellular GPIb beta can inhibit the GPIb alpha enzyme digestion by interacting with an unknown protein. To clarify this unknown protein, we express and purify wild type and key site mutations in Escherichia coli (R15). 1E/R152E or R149E/L150E) GPIb beta cytosolic protein, using pull-down binding protein mass spectrometry (MS), to screen out a variety of proteins that may interact with the intracellular segment of GPIb beta, including membrane protein (moesin), platelet reactive protein (thrombospondin), and immune thrombocytopenic purpura (immune thrombocytopenia purpura, ITP). An acquired autoantibody mediated destruction and damage to the blood system disease of platelets and their regeneration. Clinical manifestations of skin and mucous membrane or visceral hemorrhage are the main manifestations. In this study, we found a case of intractable ITP patients with ineffective hormone therapy. In order to identify the pathogeny of the patients, we were fully exoticing the DNA/ bone marrow DNA in the peripheral blood of the patients. The subgroup was sequenced to search for a negative mutation in the peripheral blood DNA only in the bone marrow DNA. A group of candidate genes were screened by bioinformatics analysis. After further Sanger sequencing, it was concluded that the Ct BP2 gene had a p.S240SF mutation, resulting in the megakaryocyte maturation disorder that may be the thrombocytopenia of the patient. Cause of disease.

【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R596

【相似文献】