[Functional study of abnormal fibrinogen caused by Arg275His mutation in fibrinogen γ chain]

To investigate the function of abnormal fibrinogen in two inherited dysfibrinogenemia pedigrees.Routine coagulation tests were conducted in the probands and related family members. The antigen and activity levels of fibrinogen were detected by immunoturbidimetry assay and clauss assay, respectively. All the exons and exon-intron boundaries of the three fibrinogen genes and antithrombin gene（AT3）were analyzed by PCR amplification and direct sequencing. Routine thrombelastography (TEG) test and functional fibrinogen TEG test were both used to make a comprehensive evaluation of coagulation status and functional fibrinogen level in patients. The molecular weights of the three peptides from fibrinogen were measured by Western blot. The function of abnormal fibrinogen was assessed by fibrinogen dynamic polymerization and fibrinolysis velocity.The coagulation routine tests were normal in two probands except for prolonged thrombin time (TT) and reptilase time (RT), as well as reduced activity levels of 0.5 g/L and 0.6 g/L fibrinogen, respectively. The antigen levels of fibrinogen were 2.32 g/L and 2.66 g/L in two probands, which were in the normal reference range. The genotype analysis showed that Arg275His in fibrinogen γ chain (γ Arg275His) existed in both probands and patients in these two pedigrees. Meanwhile, proband B's grandfather and aunt also carried heterozygote g.5876TC (Ser116Pro) mutation in AT3. The results of routine TEG test demonstrated that the α values of proband B and his father were close to and lower than the lower limit of reference range, respectively, while the MA values were normal in both of them. However, functional fibrinogen TEG test revealed obviously reduced MA value. All the probands and patients demonstrated prolonged lag-off time and reduced peak value in fibrinogen dynamic polymerization tests. Meanwhile, most of fibrin formed from the patients' plasma could not be dissolved completely by plasminogen (PLG) and urokinase-typeplasminogenactivator (u-PA) at a certain time.We first reported cases of inherited dysgibrinogenemia associated with inherited AT deficiency. γArg275His mutation caused the abnormal fibrinogen in terms of fibrin mono polymerization and possibly in fibrinolysis. Combined use of routine TEG test and functional fibrinogen TEG test with comprehensive analyses of the parameters in both tests could better evaluate the level of functional fibrinogen and predict the risk of hemorrhage and thrombosis in patients with inherited dysfibrinogenemia.目的 对两个遗传性异常纤维蛋白原血症家系的突变纤维蛋白原（Fg）进行功能研究。方法 常规筛查凝血功能;Fg抗原和活性分别用免疫比浊法和Clauss法测定;抽提DNA,对Fg 3个基因（FGA、 FGB和FGG）以及抗凝血酶基因（AT3）所有外显子及侧翼序列进行PCR扩增、测序及分析;采用常规血栓弹力图（TEG）和功能性Fg TEG检查对家系B先证者及其父亲进行凝血功能的综合评价及血浆功能性Fg评估;应用Western blot 检测血浆Fg肽链分子量;采用Fg动态聚集曲线和纤维蛋白溶解曲线实验检测血浆Fg的功能。结果 2例先证者凝血酶原时间（TT）和爬虫酶凝固时间（RT）明显延长,Fg活性仅为0.5 g/L和0.6 g/L,但其抗原均正常,分别为2.32 g/L和2.66 g/L。两个家系先证者均存在γ链Arg275His杂合突变,家系B先证者的祖父和姑母同时检出AT3 g.5876TC (Ser116Pro)杂合突变。家系B先证者及其父亲TEG检测结果中α值分别接近和低于正常参考值范围下限,但最大波幅（MA值）均为正常;在功能性Fg TEG检测中,MA值明显偏低。Fg动态聚集曲线中先证者和家系患者的起跳时间明显延长、峰值明显降低。纤维蛋白溶解曲线中多数患者的纤维蛋白在特定时间内不能被纤溶酶原完全溶解。结论 首次发现遗传性异常纤维蛋白原血症合并AT缺陷的患者。γ链 Arg275His突变使Fg在纤维蛋白单体聚合以及纤维蛋白溶解方面出现异常。联合应用常规TEG和功能性TEG检测,可以更好地评估异常纤维蛋白原血症患者Fg的功能。