两个遗传性纤维蛋白原缺陷症家系表型与基因突变分析

Phenotype and genotype analyses of two pedigrees with inherited fibrinogen deficiency

目的对两个杂合突变导致遗传性纤维蛋白原缺陷症家系进行表型和基因突变分析,并初步探讨其分子致病机制。方法采用凝固法检测两个先证者及其各自家系成员(3代9人和2代3人)凝血酶原时间(PT)、活化部分凝血活酶时间(APTT)、凝血酶时间(TT)和纤维蛋白原活性(Fg∶C),免疫比浊法检测纤维蛋白原抗原(Fg∶Ag)。DNA直接测序法分析先证者FGA、FGB和FGG基因所有外显子和侧翼序列及家系成员相应的突变位点区域。通过凝血酶诱导进行纤维蛋白原聚集试验;用ClustalX-2、1-win软件分析突变位点的保守性;用Mutation Taster、PolyPhen-2、PROVEAN、SIFT和LRT在线生物信息学软件预测突变位点的致病性;用Swiss-pdb Viewer4.0.1分析突变前后蛋白质空间结构及分子作用力的变化。结果家系1先证者和家系2先证者Fg∶C明显下降(分别为1.28 g/L、0.98 g/L);家系1先证者Fg∶Ag正常(2.20 g/L),家系2先证者Fg∶Ag降低(1.01 g/L)。基因分析发现,家系1先证者的FGB基因第2号外显子存在c.293C>A(p.BβAla98Asp)杂合错义突变;家系2先证者的FGB基因第8号外显子存在c.1418C>G(p.BβSer473*)杂合无义突变。同源性分析表明Ala98和Ser473残基在同源物种间呈不同保守状态;在线生物信息学软件预测显示p.BβAla98Asp和p.BβSer473*突变为致病突变;蛋白模型分析显示,p.BβAla98Asp突变使氨基酸之间的氢键发生改变,p.BβSer473*突变产生了截短蛋白。结论家系1先证者的异常纤维蛋白原血症和家系2先证者的低纤维蛋白原血症可能分别与p.BβAla98Asp杂合错义突变及p.BβSer473*杂合无义突变有关。

Objective To analyze the phenotype and genotype of two pedigrees with inherited fibrinogen(Fg)deficiency caused by two heterozygous mutations.We also preliminarily probed the molecular pathogenesis.Methods The prothrombin time(PT),activated partial thromboplastin time(APTT),thrombin time(TT)and plasma fibrinogen activity(Fg∶C)of all family members(nine people across three generations and three people across two generations)were measured by the clotting method.Fibrinogen antigen(Fg:Ag)was measured by immunoturbidimetry.Direct DNA sequencing was performed to analyze all exons,flanking sequences,and mutated sites of FGA,FGB,and FGG for all members.Thrombin-catalyzed fibrinogen polymerization was performed.ClustalX 2.1 software was used to analyze the conservatism of the mutated sites.MutationTaster,PolyPhen-2,PROVEAN,SIFT,and LRT online bioinformatics software were applied to predict pathogenicity.Swiss PDB Viewer 4.0.1 was used to analyze the changes in protein spatial structure and molecular forces before and after mutation.Results The Fg∶C of two probands decreased(1.28 g/L and 0.98 g/L,respectively).The Fg∶Ag of proband 1 was in the normal range of 2.20 g/L,while it was decreased to 1.01 g/L in proband 2.Through genetic analysis,we identified a heterozygous missense mutation(c.293C>A;p.BβAla98Asp)in exon 2 of proband 1 and a heterozygous nonsense mutation(c.1418C>G;p.BβSer473*)in exon 8 of proband 2.The conservatism analysis revealed that Ala98 and Ser473 presented different conservative states among homologous species.Online bioinformatics software predicted that p.BβAla98Asp and p.BβSer473*were pathogenic.Protein models demonstrated that the p.BβAla98Asp mutation influenced hydrogen bonds between amino acids,and the p.BβSer473*mutation resulted in protein truncation.Conclusion The dysfibrinogenemia of proband 1 and the hypofibrinogenemia of proband 2 appeared to be related to the p.BβAla98Asp heterozygous missense mutation and the p.BβSer473*heterozygous nonsense mutation,respectively.This is the first ever report of these mutations.