抗凝血酶基因G13328A杂合突变导致血栓形成

目的对1个遗传性抗凝血酶（AT）缺陷症家系进行AT抗原（AT：Ag）、活性（AT：A）和基因突变检测并对该突变导致的AT结构和功能的变化进行研究。方法采用免疫比浊法和发色底物法分别检测AT：Ag和AT：A，用PCR法对先证者AT基因的7个外显子及其侧翼内含子序列进行扩增，PCR产物纯化后直接测序，检测其基因突变。根据基因检测结果，对家系成员相应外显子进行PCR扩增，扩增产物直接测序用大引物法构建突变的AT表达质粒并瞬时转染COS-7细胞，用ELISA法检测细胞培养上清液和细胞内提取物巾AT：Ag。结果先证者的AT：Ag和AT：A分别为179mg／L和42．3％，为Ⅰ型AT缺陷；AT基因测序显示在AT外显子6区存在G13328A杂合突变，导致Ala（GCC）404→Thr（ACC）对家系成员进行的基因测序显示有3人（Ⅱ2、Ⅱ3、Ⅲ2）存在该突变。突变质粒在细胞培养上清液和细胞内的AT：Ag分别为正常人的40％和68％。结论该家系为Ⅰ型遗传性AT缺陷症；G13328A杂合突变是导致先证者血栓形成的原因．

A heterozygous point mutation G13328A in antithrombin gene causes thrombosis

OBJECTIVE: To identify the phenotype and the gene mutation in a kindred with antithrombin (AT) deficiency. METHODS: Immuno-nephelometry and chromogenic assay were used to detect the plasma level of AT antigen (AT: Ag) and activity (AT: A), respectively. All the seven exons and intron-exon boundaries of AT gene from the propositus were amplified by PCR and direct sequencing of the PCR pro-ducts was performed. Corresponding PCR fragments from the kindred were also sequenced directly. Megaprimer method was used to construct the mutant AT cDNA expressing vector from normal plasmid pCRII AT cDNA. The normal and mutant AT plasmid were transiently transfected into Cos-7 cells and AT: Ag was detected in supernatant and lysate of transfected cell with ELISA. RESULTS: The plasma level of AT: Ag and AT: A for the propositus were 179 mg/L and 42.3%, respectively. A heterozygous G13328A missense mutation in exon 6 was identified, which led to the substitution of Thr (ACC) 404 for Ala (GCC). The sequencing results from the pedigree suggested that three other members also had the mutation. The level of AT:Ag in supernatant and lysate from cells transfected with mutant AT cDNA was 40% and 68% of that of normal AT cDNA transfected cells. CONCLUSION: This is an unreported AT gene mutation in China, which causes type I hereditary antithrombin deficiency and thrombosis in the proposita.