一个FGG杂合缺失突变导致异常纤维蛋白原血症家系分析  

Analysis of inherited dysfibrinogenemia pedigree associated with a heterozygous and deletion mutantion in the FGG gene

作　　者：谢耀盛[1] 季伟丹 李阳阳 金艳慧[1] 杨丽红[1] 程晓丽[1] 王明山[1] Xie Yaosheng;Ji Weidan;Li Yangyang;Jin Yanhui;Yang Lihong;Cheng Xiaoli;Wang Mingshan(Department of Clinical Laboratory, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325015, China)

机构地区：[1]温州医科大学附属第一医院医学检验中心,325015

出　　处：《中华检验医学杂志》2018年第4期305-311,共7页Chinese Journal of Laboratory Medicine

基　　金：浙江省自然科学基金资助项目（LY16H080005）

摘　　要：目的对一个新的FGG基因杂合缺失突变导致异常纤维蛋白原血症家系进行表型和基因突变分析，探讨其分子发病机制。方法收集2016年4月来温州医科大学附属第一医院就诊的先证者临床资料及其家系成员（共3代5人），采用凝固法检测血浆纤维蛋白原活性（Fg：C）、活化部分凝血活酶时间（APTT）、凝血酶原时间（PT）、凝血酶时间（TT）；免疫比浊法检测纤维蛋白原抗原（Fg：Ag）、D-二聚体（D-D）和纤维蛋白（原）降解产物（FDPs）含量；抽提先证者及其家系成员外周血的基因组DNA，采用PCR扩增先证者纤维蛋白原的FGA、FGB及FGG基因所有外显子和侧翼序列，以及家系成员相应的突变位点区域，产物纯化后直接测序，同时采用克隆测序及非变性聚丙烯酰胺凝胶电泳-银染色进一步验证；用ClustalX-2.1-win软件分析突变位点基因的保守性，并采用Pymol软件分析突变前后蛋白质空间结构及分子间作用力的改变。结果先证者Fg：C明显下降，而Fg：Ag含量正常，分别为0.30 g/L和2.00 g/L（参考范围均为2.00～4.00 g/L）；母亲及外婆Fg：C和Fg：Ag分别为0.42 g/L、2.09 g/L及0.47 g/L和2.42 g/L。基因分析发现先证者FGG基因8号外显子存在c.944_c.952 delCCTTTGATG杂合缺失突变，导致氨基酸289_291delAla，Phe，Asp，其母亲和外婆同样携带此突变，父亲和外公为正常野生型。同源性分析表明Ala289，Phe290，Asp291残基在同源物种间高度保守；蛋白模型分析发现在野生型FGG蛋白质中，Ala289分别与Gly287、Gly292及Thr371形成3个氢键，Phe290分别与Tyr262、Tyr278、His307及Asn308形成4个氢键，Asp291分别与Asp288及Lys302形成2个氢键，当Ala289、Phe290和Asp291发生缺失突变后，原有的氢键连接全部消失。结论本研究发现了纤维蛋白原γ链289_291delAla，Phe，Asp杂合缺失突变会引起Fg分子空间结构重排，降低了结构稳定性，其Objective To analyze the phenotype and genotype of inherited dysfibrinogenemia pedigree associated with a novel heterozygous and deletion mutation in the FGG gene, and to investigate its molecular mechanism. Methods The clinical data were collected from the proband found at our hospital and her family members in April 2016. The activity plasma fibrinogen （Fg： C ）, activated partial thromboplastin time （APTT）, prothrombin time （PT）, thrombin time （TT） were detected by coagulation method and the antigen plasma fibrinogen （ Fg ： Ag ） , D-Dimer （ D-D ） , fibrinogen degradation products （FDPs） were analyzed by immunoturbidimetry method. All of the exons and exon-intron boundaries of the genes of FGA, FGB and FGG with the fibrinogen （Fg） were amplified by PCR and followed by direct sequencing. And further verification were performed by cloning sequence and non-denatured polyacrylamide gel electrophoresis and silver staining. The conservatism of mutated gene locus were analyzed by ClustalX-2. 1-win. The change of the protein spatial structure and the intermolecular forces with mutation were analyzed by Pymol. Results The Fg：C of the proband was significantly reduced（0. 30 g/L） and the Fg：Ag of the proband was normal （2. 00 g/L）. Their Fg：C were both significantly reduced and the Fg：Ag were both normal （0. 42 g/L,2. 09 g/L ＆ 0.47 g/L,2. 42 g/L, respectively） , these were found in her mother and grandma. Genetic analysis revealed a novel heterozygous and deletion mutation with c. 944_ c. 952 delCCTFTGATG in exon 8 of FGG gene in the proband, predicting a heterozygous 289_291delAla, Phe, Asp mutation. The same mutations were carried by her mother and grandma, but her father and grandpa were normal. Homology analysis indicated that the Ala289, Phe290 and Asp291 were maintained highly conservative in homogenous species. Protein model analysis found that the original hydrogen bonds were disappeared when the deletion mutation happened with the Ala289, Phe290 and Asp291.

关 键 词：纤维蛋白原缺乏血症 系谱 杂合子丢失 纤维蛋白原 突变 误义 

分 类 号：R446.6[医药卫生—诊断学] R596.1[医药卫生—临床医学]