二例Ⅰ型抗凝血酶缺陷症患者反复发生静脉血栓的分子机制研究  

Molecular mechanisms of recurrent venous thrombosis in two pedigrees with type I antithrombin deficiency

作　　者：夏燕[1] 丁秋兰[2] 许冠群[2] 张利伟[2] 戴菁[2] 陆晔玲[2] 王学锋[2] 奚晓东[1] 王鸿利[1] 

机构地区：[1]上海交通大学医学院附属瑞金医院、上海血液学研究所；医学基因组学国家重点实验室,200025 [2]上海交通大学医学院临床输血科,200025

出　　处：《中华血液学杂志》2011年第12期848-853,共6页Chinese Journal of Hematology

摘　　要：目的对2例Ⅰ型抗凝血酶（AT）缺陷症家系进行表型诊断、基因分析，并探讨其静脉血栓发生的分子机制。方法常规筛查凝血功能，凝血酶生成试验评估高凝状态。用ELISA法及稀释的蝰蛇毒法分别检测抗心磷脂抗体（ACA）及狼疮抗凝物质（LA）。用发色底物法或凝固法检测蛋白C、蛋白S和抗凝血酶活性（PC：A，PS：A及AT：A），用免疫比浊法检测抗凝血酶抗原（AT：Ag）。Western blot方法分析血浆中AT的含量及相对分子质量。用PCR方法扩增AT基因的全部外显子及侧翼序列，用DNA直接测序法进行基因分析，并对静脉血栓相关的基因多态性进行筛查。用定点突变法构建ATAla404Asp突变体表达质粒，脂质体介导瞬时转染293T细胞，检测细胞上清液和裂解液中AT：Ag。结果2例先证者的凝血筛查指标均未见异常，凝血酶生成试验显示先证者1的凝血酶生成潜力（ETP）和生成峰值分别为正常人的2．8倍和1．5倍，表明体内呈现明显高凝状态。PC：A、PS：A、ACA及LA检测均未见异常。先证者1、2的AT：A分别为45％和32％，AT：Ag分别为121mg／L和158mg／L。血浆Western blot结果显示2例先证者AT含量较正常混合血浆减少，但相对分子质量正常。基因分析发现2例先证者各携带一个杂合突变，分别为g．3291c—T（Thr9811e）和g．13863C→A（Ala404Asp），未发现静脉血栓相关的基因多态性。体外转染实验结果显示Ala404Asp突变质粒转染细胞的上清液和裂解液中的AT：Ag分别为野生型的4．8％和60．6％。结论Thi9811e和Ala404Asp两种AT突变与先证者1、2反复静脉血栓发生明显相关。其中Ala404Asp突变为国际首次报道。其分子发病机制研究显示Ala404Asp突变蛋白存在分泌障碍和细胞内降解增多，导致Ⅰ型AT缺陷症。Objective To investigate the clinical phenotype, genotype and molecular mechanism of recurrent venous thrombosis in two Chinese pedigrees with type I antithrombin （AT） deficiency. Methods The routine coagulation screening tests were detected, thrombin generation tests was performed to evaluate the hypercoagulation. Anticardiolipin antibody （ACA） and lupus anticoagulant （LA） were detected with enzyme-linked immunosorhent assay （ELISA） and diluted viper venom time assay （DVVT）, respectively. The activities of protein C, protein S and AT （PC： A, PS： A, AT： A） were tested with chromogenic substrate assay or clotting method. The antigen of AT （AT： Ag） was performed with immunoturbidimetry methods. Western blot was used to analyze the molecular weight （MW） and the plasma levels of AT： Ag. All 7 exons and the flanking sequences were amplified by PCR. The mutation of AT gene and thrombophilia associated gene polymorphisms were analyzed by direct DNA sequencing. The expression plasmid of Ala404Asp mutant was constructed with site-directed mutagenesis method based on the wild-type （WT） AT cDNA contained in pcDNA 3.1 vector, and transiently expression of AT WT and the Ala404Asp mutant was performed using HEK293T cells. Cultured supematant and cell lysates were collected and measured for AT： Ag by ELISA and Western blot. Results The results of routine coagulation tests in two probands were normal, thrombin generation tests indicated that proband 1 presented hypercoagulable state with 2.8 and 1.5 times higher of the endogenous thrombin potential （ETP） and peak height compared with that of normal, respectively. The levels of PC： A, PS： A, ACA and LA were normal. AT： A in proband 1 and proband 2 were 45% and 32% , and AT： Ag were almost half of the normal （ 121 mg/L and 158 mg/L）, respectively. The results of Western blot showed that both probands＇ plasma levels of AT： Ag were lower than the normal pooled plasma and MW was normal. Two heterozygous mutations of

关 键 词：抗凝血酶类 血栓形成倾向 DNA突变分析 静脉血栓形成 

分 类 号：R558[医药卫生—血液循环系统疾病]