Xia Yan, Ding Qiu-lan, Xu Guan-qun, Zhang Li-wei, Dai Jing, Lu Ye-ling, Wang Xue-feng, Xi Xiao-dong, Wang Hong-li
State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, China.
Zhonghua Xue Ye Xue Za Zhi. 2011 Dec;32(12):848-53.
To investigate the clinical phenotype, genotype and molecular mechanism of recurrent venous thrombosis in two Chinese pedigrees with type I antithrombin (AT) deficiency.
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 immunosorbent 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 supernatant and cell lysates were collected and measured for AT:Ag by ELISA and Western blot.
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 g.3291C→T(Thr98Ile), g.13863C > A(Ala404Asp) were identified in the probands, respectively. No proband had venous thrombosis associated gene polymorphisms. Expression in vitro showed that AT:Ag in culture media and lysates of Ala404Asp are 4.8% and 60.6% of that of WT, respectively.
Thr98Ile and Ala404Asp mutation of AT gene significantly correlate with recurrent venous thrombosis in the two probands, respectively. Ala404Asp has not been described before. The mutant Ala404Asp protein can not be expressed due to impaired secretion and increased intracellular degradation, resulting in type I AT deficiency.
研究两例I型抗凝血酶（AT）缺乏的中国家系中复发性静脉血栓形成的临床表型、基因型及分子机制。
进行常规凝血筛查试验，采用凝血酶生成试验评估高凝状态。分别用酶联免疫吸附测定（ELISA）和稀释蝰蛇毒时间测定（DVVT）检测抗心磷脂抗体（ACA）和狼疮抗凝物（LA）。用发色底物法或凝固法检测蛋白C、蛋白S和AT（PC:A、PS:A、AT:A）的活性。用免疫比浊法检测AT抗原（AT:Ag）。采用蛋白质印迹法分析AT:Ag的分子量（MW）和血浆水平。通过聚合酶链反应（PCR）扩增所有7个外显子及其侧翼序列。通过直接DNA测序分析AT基因的突变及血栓形成倾向相关基因多态性。基于pcDNA 3.1载体中包含的野生型（WT）AT cDNA，采用定点突变法构建Ala404Asp突变体的表达质粒，并利用人胚肾293T细胞进行WT和Ala404Asp突变体的瞬时表达。收集培养上清液和细胞裂解物，用ELISA和蛋白质印迹法检测AT:Ag。
两名先证者的常规凝血试验结果正常，凝血酶生成试验表明先证者1呈现高凝状态，其内源性凝血酶潜力（ETP）和峰值高度分别比正常高2.8倍和1.5倍。PC:A、PS:A、ACA和LA水平正常。先证者1和先证者2的AT:A分别为45%和32%，AT:Ag几乎是正常水平的一半（分别为121 mg/L和158 mg/L）。蛋白质印迹结果显示，两名先证者血浆中AT:Ag水平均低于正常混合血浆，且MW正常。在先证者中分别鉴定出g.3291C→T（Thr98Ile）、g.13863C>A（Ala404Asp）两个杂合突变。先证者均无静脉血栓形成相关基因多态性。体外表达显示，Ala404Asp培养基和裂解物中的AT:Ag分别为WT的4.8%和60.6%。
AT基因的Thr98Ile和Ala404Asp突变分别与两名先证者的复发性静脉血栓形成显著相关。Ala404Asp此前未见报道。突变体Ala404Asp蛋白因分泌受损和细胞内降解增加而无法表达，导致I型AT缺乏。
