两个遗传性异常纤维蛋白原血症家系分析

Pedigree analysis of two families with congenital dysfibrinogenemia

目的对两个因FGG基因杂合突变导致的遗传性异常纤维蛋白原血症(CD)家系进行表型和基因型分析,探讨CD的分子发病机制。方法检测两个先证者及其家系成员PT、APTT、纤维蛋白原活性(Fa)、Fib、TT、D-二聚体(D-D)、纤维蛋白(原)降解产物(FDPs)等凝血功能指标。采用PCR法扩增先证者编码Fib的FGA、FGB、FGG等3个基因,纯化产物后测序,寻找突变发生位点;采用反向测序法验证突变,同时检测其他家系成员相同基因位点;使用Polymorphism Phenotyping v2和Mutation Taster生物信息学软件预测突变位点对Fib功能的影响,利用PyMOL软件构建Fib的蛋白空间模型,预测突变对Fib结构的影响。结果两个先证者PT、APTT和TT均正常或略高于正常值,Fa明显降低(分别为0.60、0.61 g/L),Fib基本正常(分别为2.31、1.97 g/L),D-D、FDPs均在正常范围内。基因分析显示家系1先证者FGG基因存在c.952G>A杂合突变,导致292位甘氨酸突变为丝氨酸(Gly292Ser),其姐姐同样存在此基因突变;家系2先证者FGG基因存在c.902G>A杂合突变,导致275位精氨酸突变为组氨酸(Arg275His),其父亲和祖父存在相同的基因突变。Polymorphism Phenotyping v2和Mutation Taster分析提示该两种突变会引起Fib功能变化,有致病性。PyMOL突变模型提示,家系1中Fib的γ链发生Gly292Ser,突变氨基酸与周围氨基酸相互作用的氢键数量增加;家系2中Fib的γ链发生Arg275His,突变氨基酸与周围氨基酸相互作用的氢键数量减少,蛋白结构的空间稳定性均发生改变。结论家系1 FGG基因存在c.952G>A杂合突变,家系2 FGG基因存在c.902G>A杂合突变,导致翻译的Fib分子结构与功能发生致病性变化,是引起CD的主要分子机制。

Objective To analyze the phenotype and genotype of two families with congenital dysfibrinogenemia(CD) caused by heterozygous mutation of FGG gene,and to explore the molecular pathogenesis of CD.Methods The prothrombin time(PT),activated partial thromboplastin time(APTT),fibrinogen activity(Fa),fibrinogen(Fib),thrombin time(TT),D-dimer(D-D),fibrinogen degradation products(FDPs)and other coagulation function indexes of two CD probands and their family members were detected.The FGA,FGB and FGG genes encoding Fib in probands and family members were amplified by PCR,and directly sequenced to detect mutation sites.The mutations were verified by reverse sequencing.Polymorphism Phenotyping v2 and Mutation Taster bioinformatics software were used to predict the impact of mutation sites on Fib function,and PyMOL software was used to build a protein spatial model of Fib to predict the impact of mutation on Fib structure.Results PT,APTT and TT of two probands were normal or slightly higher than normal values,Fa(0.60 and 0.61 g/L,respectively)was significantly reduced,Fib(2.31 and 1.97 g/L,respectively)was basically normal,and D-D and FDPs were within normal range.Gene analysis showed that the proband in family 1 had a heterozygous mutation of c.952G>A in FGG gene,which led to the mutation of 292 glycine to serine(Gly292Ser).His sister also had this gene mutation;FGG gene of proband in family 2 existed c.902G>A heterozygous mutation resulting in the mutation of arginine at 275 to histidine(Arg275His).Her father and grandfather had the same gene mutation.Polymorphism Phenotyping v2 and Mutation Taster analysis showed that the two mutations would cause changes in Fib function and have pathogenicity.PyMOL mutation model suggested that the number of hydrogen bonds between the mutant amino acid and the surrounding amino acid increased significantly due to Gly292Ser of FGG in Family 1.The number of hydrogen bonds between the mutant amino acid and the surrounding amino acid decreased significantly caused by Arg275His of FGG in Family 2,and the spatial stability of the protein structure changed.Conclusion The c.952G>A heterozygous mutation and c.902G>A heterozygous mutation of FGG gene detected in two CD families can lead to pathogenic changes in the molecular structure and function of Fib,which may be the main molecular pathogenesis of CD.