Zhou Rong-fu, Shi Guo-chao, Fu Qi-hua, Wang Wen-bin, Xie Shuang, Dai Jing, Ding Qiu-lan, Hu Yi-qun, Wang Xue-feng, Deng Wei-wu, Wang Hong-li
Shanghai Institute of Hematology, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025, China.
Zhonghua Xue Ye Xue Za Zhi. 2005 Nov;26(11):661-4.
To identify the phenotype and the gene mutation in a kindred with antithrombin (AT) deficiency.
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.
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.
This is an unreported AT gene mutation in China, which causes type I hereditary antithrombin deficiency and thrombosis in the proposita.
鉴定一个抗凝血酶（AT）缺乏家系的表型及基因突变。
分别采用免疫比浊法和发色底物法检测血浆中AT抗原（AT:Ag）水平及活性（AT:A）。对先证者的AT基因全部7个外显子及其内含子-外显子边界进行PCR扩增，并对PCR产物进行直接测序。对家系中相应的PCR片段也进行直接测序。采用大引物法从正常质粒pCRII AT cDNA构建突变型AT cDNA表达载体。将正常和突变型AT质粒瞬时转染至Cos-7细胞，用ELISA法检测转染细胞上清液和裂解液中的AT:Ag。
先证者血浆中AT:Ag和AT:A水平分别为179 mg/L和42.3%。在第6外显子中鉴定出一个杂合性G13328A错义突变，导致第404位苏氨酸（ACC）被丙氨酸（GCC）替代。家系测序结果表明另外3名成员也有该突变。转染突变型AT cDNA的细胞上清液和裂解液中AT:Ag水平分别为转染正常AT cDNA细胞的40%和68%。
这是国内未报道的AT基因突变，导致先证者I型遗传性抗凝血酶缺乏及血栓形成。
