3个遗传性抗凝血酶缺陷症家系的表型和基因型关系的研究

Study on the relationship between phenotypes and genotypes in three Chinese pedigrees with inherited antithrombin deficiency

目的探讨遗传性抗凝血酶缺陷症家系表型和和基因型的关系。方法对3个遗传性抗凝血酶缺陷症家系（家系1~3）作表型和基因型诊断：常规检测活化部分凝血活酶时间（APTT）、凝血酶原时间（PT）、纤维蛋白原（Fg）、凝血酶时间（TT）以筛查凝血功能,发色底物法检测蛋白C、蛋白S和抗凝血酶活性（PC∶A,PS∶A及AT∶A）,免疫比浊法检测抗凝血酶抗原（AT∶Ag）,Western blot检测血浆中抗凝血酶的分子量和含量;PCR扩增AT基因所有外显子及侧翼序列,DNA测序并进行基因分析。针对新基因突变,在100例正常人中检测相应突变以排除多态性,用TA克隆PCR产物测序鉴定杂合碱基缺失突变。结果 3名先证者均为反复发作的静脉血栓患者,凝血指标及PC∶A和PS∶A都正常,AT∶A分别为正常人（100%）的60%、52%和60%,AT∶Ag分别为16.9、14.1和11.4 mg/dl,Western blot显示3位先证者的血浆AT蛋白分子量正常（58 kD）而含量低于正常;基因分析发现3名先证者各携带1个杂合突变,分别为g.8263 C〉T（Leu340Phe）、g.5894-6 del TTC（Phe122del）和g.5898 T〉G（Phe123Cys）。3个家系中与先证者表型相似的成员,则携带有相同的AT基因突变;但除家系2先证者的父亲有静脉血栓外,家系1和3中含有相应AT基因突变的家庭成员均无血栓发生。结论 3名先证者遗传性抗凝血酶缺陷症分别由Leu340Phe、Phe122del和Phe123Cys突变所致,其中Leu340Phe和Phe123Cys突变为国际上首次报道。

Objective To investigate the relationship between phenotypes and genotypes in three Chinese pedigrees with inherited antithrombin deficiency.Methods The routine coagulation tests including activated partial thromboplastin time（APTT）,prothrombin time（PT）,fibrinogen（Fg） and thrombin time（TT） were performed.The activities of protein C（PC：A）,protein S（PS：A）,and antithrombin（AT：A） were tested with chromogenic substrate assay.The antigen of AT（AT：Ag） was tested with immunoturbidimetry methods.The molecular weight（MW） and amount of AT was assessed by Western blot.The sequences of all the exons and exon-intron boundaries of the AT gene were amplified by PCR and analyzed by direct sequencing.The novel mutation in AT gene was tested in 100 normal persons to exclude polymorphism.The heterozygous deletion was checked and identified by sequencing the PCR products constructed in TA cloning vector.Results Three probands had recurrent venous thrombosis.The levels of APTT,PT,Fg,TT,PC：A and PS：A were normal,AT：A in the plasma was 60%,52% and 60% of the normal levels in each of the 3 probands,with respective AT：Ag of 16.9 mg/dL,14.1 mg/dL and 11.4 mg/dL.Western blot showed that their AT MW was normal（58 kD） but the antigen levels were lower than normal.Three heterozygous mutations of Leu340Phe,Phe122del and Phe123Cys were identified in the 3 probands.In each of the 3 pedigrees,the same genotypes（heterozygous mutations） were identified in the family members with similar low level of AT as the probands.However,only the proband′s father in pedigree 2 had venous thrombosis once,there were no other family members having had thrombosis in both pedigree 1 and 3.Conclusion The three probands with inherited antithrombin deficiency were each caused by mutations of Leu340Phe,Phe122del and Phe123Cys.Both Leu340Phe and Phe123Cys were internationally first reported.