APOH基因多态性与中国人群动静脉血栓形成的关联研究

发布时间：2018-07-03 16:45

本文选题：β_2GPI蛋白 + 基因多态性　；参考：《华中科技大学》2015年博士论文

【摘要】：血栓栓塞性疾病是一种严重危害人类健康的常见疾病,包括静脉血栓栓塞性疾病(深静脉血栓形成、肺栓塞)和动脉血栓栓塞性疾病(心肌缺血、缺血性脑梗塞)。血栓形成及其并发症已成为世界范围内致残率和致死率最高的疾病之一。血栓栓塞性疾病是一种多因素性疾病,遗传性易栓背景及获得性易栓因素均在其发病中发挥着不可或缺的作用。尽管目前已有很多基因变异导致的凝血或抗凝蛋白功能失调被认为与血栓形成具有关联,但仍有许多血栓患者的遗传背景不能被目前已知的遗传性易栓背景所解释,故而探索新的可能与血栓形成相关的基因变异显得尤为重要。近来有研究表明,β2GPI是体内一种天然的抗凝蛋白,主要通过抑制凝血酶的凝血活性但不影响凝血酶对蛋白C的激活而发挥抗凝作用。目的：β2GPI编码基因为APOH,本研究以APOH基因作为候选基因,研究其多态性是否与为中国人群血栓栓塞性疾病的危险因素；并进一步探索APOH基因多态性导致β2GPI下降及血栓形成的具体机制。方法：选择26例狼疮抗凝物质阳性的患者,通过对其APOH基因的8个外显子及其剪接位点、3端及5端非翻译区进行PCR后直接测序的方法查找基因变异；随机挑选60例静脉血栓患者,缺血性脑梗塞患者及正常对照组DNA进行测序验证第一步所发现的基因变异,并大致评估其对疾病形成的风险；用Haploview4.0软件行基因变异的连锁不平衡分析；进一步扩大样本量行病例对照研究(1304例VTE vs1334例正常组；1444例IS vs1334例正常组)评估基因变异对血栓形成的风险比；挑选各类型血浆行Mestern blot及ELISA行β2GPI抗原水平检测及凝血功能试验(凝血酶原时间及凝血酶生成实验)；体外重组野生型及突变型APOH表达质粒行细胞实验,检测基因变异对p2GPI的表达及凝血功能的影响。结果：在26例狼疮抗凝物阳性的患者中主要发现了四个基因多态性,c.1004GC、 c.461GA.c4221C、c.-32CA,结果显示这4种APOH基因多态性完全连锁不平衡(D’=1,r2=1),选用c.461GA作为限制性内切酶酶切代表,形成三种单倍型H1(CTGG/CTGG),H2(CTGG/ACAC)及H3(ACAC/ACAC).酶切结果显示H2,H3单倍型对静脉血栓形成的优势比为1.5和7,而其对动脉血栓形成作用不大。血浆实验发现,H2,H3单倍型血浆β2GPI蛋白减少,凝血酶原时间缩短,凝血酶生成增多。细胞实验证实c.-32CA造成APOH基因的表达水平下降,导致细胞内外β2GPI蛋白均减少。含p.Trp335Ser、p.Arg154His、p.Ile141Thr的突变蛋白使血浆凝血酶原时间缩短,凝血酶生成能力增强;当血浆β2GPI蛋白浓度降低时也呈现出相同的作用。结论：APOH基因突变造成的β2GPI蛋白质和量的下降均有利于血栓形成,可将其作为血栓性疾病的候选基因,可用于由常规凝血及抗凝蛋白遗传背景不能解释的血栓患者的遗传背景分析。
[Abstract]:Thromboembolic disease is a common disease that seriously endangers human health, including venous thromboembolism (deep venous thrombosis, pulmonary embolism) and arterial thromboembolic disease (myocardial ischemia, ischemic cerebral infarction). Thrombosis and its complications have become one of the diseases with the highest morbidity and mortality in the world. Thromboembolic disease is a multifactor disease. Hereditary tethered background and acquired thrombolytic factors play an indispensable role in its pathogenesis. Although many genetic mutations have been associated with coagulation or anticoagulant protein dysfunction, it is considered to be associated with thrombus formation, but there are still many genetic backgrounds of patients with thrombus. It is not possible to be explained by the known genetic prone background of thrombus, so it is particularly important to explore new gene mutations that may be associated with thrombosis. Recently, studies have shown that beta 2GPI is a natural anticoagulant in the body, mainly by inhibiting thrombin activity but not affecting the activation of thrombin on protein C. Use.
Objective: the gene of beta 2GPI is APOH. This study uses APOH gene as a candidate gene to study whether the polymorphism is associated with the risk factors of thromboembolic disease in Chinese population, and to further explore the specific mechanism of APOH gene polymorphism leading to the decrease of beta 2GPI and thrombosis.
Methods: 26 patients with positive anticoagulant substance of lupus were selected to search for genetic variation by direct sequencing of 8 exons of APOH gene and their splice sites, 3 end and 5 end non translation regions, and 60 patients with venous thrombosis, ischemic cerebral infarction and normal control group DNA were randomly selected to verify the first step of sequencing. The genetic variation found and roughly assessed the risk of disease formation; linkage disequilibrium analysis of gene mutation with Haploview4.0 software; further enlargement of sample size in case control study (1304 cases of VTE vs1334 cases; 1444 cases of IS vs1334 normal group) to assess the risk ratio of gene mutation to thrombosis; select various types of genes Mestern blot and ELISA were used to detect the level of beta 2GPI antigen and blood coagulation test (prothrombin time and thrombin generation test); recombinant wild type and mutant APOH expression plasmid was used in vitro to detect the expression of p2GPI and the effect of coagulant function. Results: in 26 cases of lupus anticoagulant positive patients Four genetic polymorphisms, c.1004GC, c.461GA.c4221C, c.-32CA, were found. The results showed that the 4 APOH gene polymorphisms were completely unbalanced (D '=1, r2=1). C.461GA was selected as a restrictive endonuclease, and three haplotype H1 (CTGG/CTGG), H2 (CTGG/ACAC). The dominant ratio of vein thrombosis was 1.5 and 7, but it did not play a significant role in the formation of arterial thrombosis. Plasma experiments showed that H2, H3 haplotype plasma beta 2GPI protein decreased, prothrombin time shortened, and thrombin production increased.
Cell experiments confirmed that c.-32CA resulted in the decrease of the expression level of APOH gene and the decrease of beta 2GPI protein in and out of cells. The mutant protein containing p.Trp335Ser, p.Arg154His and p.Ile141Thr shortened the plasma prothrombin time and increased the ability of thrombin generation, and also showed the same effect when the concentration of plasma beta 2GPI protein decreased.
Conclusion: the decrease of protein and amount of beta 2GPI caused by APOH gene mutation is beneficial to thrombosis. It can be used as a candidate gene for thrombotic disease and can be used for genetic background analysis of patients with thrombus that can not be explained by conventional coagulation and anticoagulant genetic background.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R543

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