中国人种深静脉血栓形成及脑梗死患者抗凝血酶剑桥Ⅱ基因突变发生频率及与静脉血栓形成相关性研究
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摘要
研究背景:血栓性疾病尤其是心、脑血管血栓性疾病,已成为我国人口病死原因的第一位,且发病率有增加趋势,严重危害人类健康。深静脉血栓形成(Deep Venous Thrombosis)是较常见的四肢血管血栓栓塞性疾病。近年来,本病的发病率呈逐年增加趋势。据报道,美国每年有25万—50万人患静脉血栓栓塞性疾病。我国静脉血栓形成发生率也呈逐年增多趋势,已位居继缺血性心脏病、脑卒中后的第三位。据VITAE研究结果报告,欧盟致命和非致命性症状性静脉血栓栓塞患者(VTE)每年已超过150万例,其中54.35万病例死亡。在欧洲,因静脉血栓栓塞死亡的患者人数超过死于乳腺癌、前列腺癌、艾滋病、交通意外人数的总合。静脉血栓栓塞常常是一种无症状性疾病,它的第一次表现可能是致命的,因此,深入研究静脉血栓栓塞的病因和发病机制,针对血栓形成的高危因素进行及时干预,对于降低深静脉血栓形成的发生率和死亡率具有重要的意义。
深静脉血栓形成(DVT)是一种单基因或多基因遗传性疾病。多种抗凝血或促凝蛋白基因突变与DVT发病相关。其中,抗凝血酶Ⅲ(AT-Ⅲ)基因突变与DVT发生率关系更为密切。新近,英国Correl等报道抗凝血酶剑桥Ⅱ(A384S)突变是DVT发病的主要遗传危险因子,同时也是在白种人中引起抗凝血酶缺陷症最主要的原因。
为了解抗凝血酶剑桥Ⅱ(A384S)突变在不同人种中的分布频率,本课题以60例正常人,50例DVT,60例脑梗死患者为研究对象,使用PCR及限制性内切酶酶切方法测定该基因的突变位点,并对AT-Ⅲ活性变化在DVT和脑梗死患者的分布状态进行了观察,为获得抗凝血酶剑桥Ⅱ突变在国人血栓性疾病中的分布频率及DVT的高危因素筛查提供依据。
第一部分血栓形成患者抗凝血酶剑桥ⅡA384S基因突变点鉴定
目的:获得中国人种深静脉血栓形成患者及脑梗死患者抗凝血酶剑桥Ⅱ(A384S)突变的百分率,并分析其与血栓形成的相关性。
方法:50例深静脉血栓患者,60例脑梗死患者及正常对照60例均常规抽取静脉血4.5ml,以3.8%枸橼酸钠1:9体积抗凝。用酚氯仿法抽提基因组DNA,并用前向引物5'-TGAGGAATTGCTGTGTCTGTG-3′和反向引物5'-AGAGGTGCAAAGAATAAGAA-3′进行PCR扩增,扩增片段含AT-剑桥ⅡA384S核苷酸位点,扩增后的PCR产物通过限制性内切酶PvuⅡ进行酶切消化,以确定是否存在AT-剑桥ⅡA384S突变。
结果:60例正常对照,50例DVT患者和60例脑梗死患者中均未发现抗凝血酶剑桥ⅡA384S基因突变。
结论:在中国人种的深静脉血栓形成及脑梗死患者,抗凝血酶剑桥ⅡA384S突变可能并非血栓形成的高危因素。
第二部分抗凝血酶活性在深静脉血栓形成患者及脑梗死患者中的变化及意义
目的:检测深静脉血栓形成患者及脑梗死患者血浆AT-Ⅲ活性水平,筛选与深静脉血栓形成相关的危险因素
方法:分离60例正常人,50例DVT患者及60例脑梗死患者外周血血浆,通过发色底物法测定血浆抗凝血酶Ⅲ的活性。
结果:与正常对照组相比,深静脉血栓形成患者血浆平均抗凝血酶Ⅲ活性呈显著性降低(P＜0.01),在DVT病人中,AT-Ⅲ活性降低例数为13例,占DVT组的26%;而脑梗死患者血浆AT-Ⅲ活性与正常对照相比,无显著性差异(P＞0.05),AT-Ⅲ活性降低的例数仅3例,占5%。
结论:血浆AT-Ⅲ活性降低是深静脉血栓形成的危险因素,而与脑动脉血栓形成无明显相关。
Background: The thrombotic disease, especially ischemic vessel disease of heart and brain, has become the leading cause of death in Chinese population. Deep venous thrombosis(DVT) is a common blood vessel thrombotic disease of limbs, with the incidence increasing, DVT impairs human's health severly. It was reported that, about 250,000 -500,000 people were affected by the venous thrombosis embolism disease every year. In our country , the morbidity of DVT ranks among the top three diseases following the ischemic heart disease and stroke . It was reported by VITAE that , over 1,500,000 patients suffered fatal or non-fatal venous thrombosis embolism (VTE) in European Union every year, and 543,500 cases of them died. In Europe, the number of patients died from DVT has surpassed sum of breast cancer, prostate cancer, AIDS and the traffic accidents. As it is frequently symptomless, venous thrombosis embolism may be lead to die at its first presentation. Therefore, further investigation for the etiology and pathogenesis of venous thromboembolism, and performing interventatin as soon as possible is very significant and useful to reduce the morbidity and mortality rate of DVT.
DVT is a kind of monogenic or polygene genetic diseases. Many mutations of anti-coagulation or procoagulant are related to DVT. Among these mutations, the anti-thrombin III (AT-III) mutation is reported to be closely association with DVT. Recently, Correl reported that the antithrombin Cambridge II (A384S) mutation is a major genetic risk factor of venous thrombosis in the United Kingdom, besides, it is the main antithrombin defect in the Caucasian.
To find out the distribution frequencies of A384S mutations in different ethnic, 60 normal controls, 50 cases of DVT, 60 cases of cerebral infarction were enrolled into the study. Ploymerase chain reacion(PCR) method combined with restriction endonuclease map from the PCR products were used to determine the mutation site, and the AT-III activity in DVT and cerebral infarction patients was detected by chromogenic methods. This setting provides a basis for screening the risk factors of DVT and obtaining the distribution frequency of thrombotic disease in Chinese population .
Part I Identification of the Mutation Site of Antithrombin Cambridge II (A384S) in patients with thrombotic disorder
Objective: To obtain the incidence of antithrombin Cambridge II (A384S)mutation and its association with thrombosis in Chinese deep venous thrombosis patients and cerebral infarction patients.
Method: Peripheral blood samples were collected for genome DNA extraction. DNA was extraced by standard procedure, and DNA samples were amplified by polymerase chain reaction(PCR), using primers5'-TGAGGAATTGCTGTGTCTGTG-3' and 5'-AGAGGTGCAAAGAATAAGAA -3'. The fragment containing antithrombin Cambridge IIA384S nucleotide sites, PCR products would be digested by therestriction enzyme PvuII, by which determine the existence ofantithrombin Cambridge II A384S mutation.
Results: Antithrombin Cambridge II A384S mutation was not foundamong 60 normal controls , 50 DVT patients and 60 cerebral infarctionpatients.
Conclusion: Antithrombin Cambridge II A384S mutation is not the highrisk factor of DVT and cerebral infarction patients in China.
Part II The changes And Significance of Antithrombinactivity in the Deep Venous Thrombosis and CerebralInfarction patients.
Objective: Detecting the AT-III activity level in deep venous thrombosispatients and cerebral infarction patients, screening deep venousthrombosis-related risk factors.
Methods: Separation of peripheral blood plasma were performed on 60normal controls, 50 cases of DVT and 60 cases of cerebral infarctionpatients. Determination of plasma antithrombin III activity were carryingout using chromogenic methods.
Results: Compared with the control group, the average of AT-III activity was significantly lower in DVT patients (P <0.01), and 13(26%) patiens showed reduced AT-III activity in DVT group. The level of AT-III activity in cerebral infarction patients and the ratio of who showed reduced AT-III activity(n=3, 5%) were not different from the controls with statistical significance.
Conclusion: Reduced AT-III activity is one of the risk factors of deep venous thrombosis, but is not related to the cerebral artery thrombosis.
深静脉血栓形成(DVT)是一种单基因或多基因遗传性疾病。多种抗凝血或促凝蛋白基因突变与DVT发病相关。其中,抗凝血酶Ⅲ(AT-Ⅲ)基因突变与DVT发生率关系更为密切。新近,英国Correl等报道抗凝血酶剑桥Ⅱ(A384S)突变是DVT发病的主要遗传危险因子,同时也是在白种人中引起抗凝血酶缺陷症最主要的原因。
为了解抗凝血酶剑桥Ⅱ(A384S)突变在不同人种中的分布频率,本课题以60例正常人,50例DVT,60例脑梗死患者为研究对象,使用PCR及限制性内切酶酶切方法测定该基因的突变位点,并对AT-Ⅲ活性变化在DVT和脑梗死患者的分布状态进行了观察,为获得抗凝血酶剑桥Ⅱ突变在国人血栓性疾病中的分布频率及DVT的高危因素筛查提供依据。
第一部分血栓形成患者抗凝血酶剑桥ⅡA384S基因突变点鉴定
目的:获得中国人种深静脉血栓形成患者及脑梗死患者抗凝血酶剑桥Ⅱ(A384S)突变的百分率,并分析其与血栓形成的相关性。
方法:50例深静脉血栓患者,60例脑梗死患者及正常对照60例均常规抽取静脉血4.5ml,以3.8%枸橼酸钠1:9体积抗凝。用酚氯仿法抽提基因组DNA,并用前向引物5'-TGAGGAATTGCTGTGTCTGTG-3′和反向引物5'-AGAGGTGCAAAGAATAAGAA-3′进行PCR扩增,扩增片段含AT-剑桥ⅡA384S核苷酸位点,扩增后的PCR产物通过限制性内切酶PvuⅡ进行酶切消化,以确定是否存在AT-剑桥ⅡA384S突变。
结果:60例正常对照,50例DVT患者和60例脑梗死患者中均未发现抗凝血酶剑桥ⅡA384S基因突变。
结论:在中国人种的深静脉血栓形成及脑梗死患者,抗凝血酶剑桥ⅡA384S突变可能并非血栓形成的高危因素。
第二部分抗凝血酶活性在深静脉血栓形成患者及脑梗死患者中的变化及意义
目的:检测深静脉血栓形成患者及脑梗死患者血浆AT-Ⅲ活性水平,筛选与深静脉血栓形成相关的危险因素
方法:分离60例正常人,50例DVT患者及60例脑梗死患者外周血血浆,通过发色底物法测定血浆抗凝血酶Ⅲ的活性。
结果:与正常对照组相比,深静脉血栓形成患者血浆平均抗凝血酶Ⅲ活性呈显著性降低(P＜0.01),在DVT病人中,AT-Ⅲ活性降低例数为13例,占DVT组的26%;而脑梗死患者血浆AT-Ⅲ活性与正常对照相比,无显著性差异(P＞0.05),AT-Ⅲ活性降低的例数仅3例,占5%。
结论:血浆AT-Ⅲ活性降低是深静脉血栓形成的危险因素,而与脑动脉血栓形成无明显相关。
Background: The thrombotic disease, especially ischemic vessel disease of heart and brain, has become the leading cause of death in Chinese population. Deep venous thrombosis(DVT) is a common blood vessel thrombotic disease of limbs, with the incidence increasing, DVT impairs human's health severly. It was reported that, about 250,000 -500,000 people were affected by the venous thrombosis embolism disease every year. In our country , the morbidity of DVT ranks among the top three diseases following the ischemic heart disease and stroke . It was reported by VITAE that , over 1,500,000 patients suffered fatal or non-fatal venous thrombosis embolism (VTE) in European Union every year, and 543,500 cases of them died. In Europe, the number of patients died from DVT has surpassed sum of breast cancer, prostate cancer, AIDS and the traffic accidents. As it is frequently symptomless, venous thrombosis embolism may be lead to die at its first presentation. Therefore, further investigation for the etiology and pathogenesis of venous thromboembolism, and performing interventatin as soon as possible is very significant and useful to reduce the morbidity and mortality rate of DVT.
DVT is a kind of monogenic or polygene genetic diseases. Many mutations of anti-coagulation or procoagulant are related to DVT. Among these mutations, the anti-thrombin III (AT-III) mutation is reported to be closely association with DVT. Recently, Correl reported that the antithrombin Cambridge II (A384S) mutation is a major genetic risk factor of venous thrombosis in the United Kingdom, besides, it is the main antithrombin defect in the Caucasian.
To find out the distribution frequencies of A384S mutations in different ethnic, 60 normal controls, 50 cases of DVT, 60 cases of cerebral infarction were enrolled into the study. Ploymerase chain reacion(PCR) method combined with restriction endonuclease map from the PCR products were used to determine the mutation site, and the AT-III activity in DVT and cerebral infarction patients was detected by chromogenic methods. This setting provides a basis for screening the risk factors of DVT and obtaining the distribution frequency of thrombotic disease in Chinese population .
Part I Identification of the Mutation Site of Antithrombin Cambridge II (A384S) in patients with thrombotic disorder
Objective: To obtain the incidence of antithrombin Cambridge II (A384S)mutation and its association with thrombosis in Chinese deep venous thrombosis patients and cerebral infarction patients.
Method: Peripheral blood samples were collected for genome DNA extraction. DNA was extraced by standard procedure, and DNA samples were amplified by polymerase chain reaction(PCR), using primers5'-TGAGGAATTGCTGTGTCTGTG-3' and 5'-AGAGGTGCAAAGAATAAGAA -3'. The fragment containing antithrombin Cambridge IIA384S nucleotide sites, PCR products would be digested by therestriction enzyme PvuII, by which determine the existence ofantithrombin Cambridge II A384S mutation.
Results: Antithrombin Cambridge II A384S mutation was not foundamong 60 normal controls , 50 DVT patients and 60 cerebral infarctionpatients.
Conclusion: Antithrombin Cambridge II A384S mutation is not the highrisk factor of DVT and cerebral infarction patients in China.
Part II The changes And Significance of Antithrombinactivity in the Deep Venous Thrombosis and CerebralInfarction patients.
Objective: Detecting the AT-III activity level in deep venous thrombosispatients and cerebral infarction patients, screening deep venousthrombosis-related risk factors.
Methods: Separation of peripheral blood plasma were performed on 60normal controls, 50 cases of DVT and 60 cases of cerebral infarctionpatients. Determination of plasma antithrombin III activity were carryingout using chromogenic methods.
Results: Compared with the control group, the average of AT-III activity was significantly lower in DVT patients (P <0.01), and 13(26%) patiens showed reduced AT-III activity in DVT group. The level of AT-III activity in cerebral infarction patients and the ratio of who showed reduced AT-III activity(n=3, 5%) were not different from the controls with statistical significance.
Conclusion: Reduced AT-III activity is one of the risk factors of deep venous thrombosis, but is not related to the cerebral artery thrombosis.
引文
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