1例遗传性凝血因子Ⅶ缺陷症家系基因突变分析

Gene mutation analysis of a family with hereditary coagulation factor Ⅶ deficiency

目的对1例姑表近亲结婚的遗传性凝血因子Ⅶ(FⅦ)缺陷症家系进行实验室表型诊断和基因突变检测,以期寻找致病基因并初步探讨其分子发病机制。方法检测先证者及其相关家系成员(共3代8人)的血浆凝血酶原时间(PT)、FⅦ活性(FⅦ:C)、FⅦ抗原(FⅦ:Ag)等指标。采用DNA直接测序法对先证者F7基因9个外显子及其侧翼序列、5'和3'非翻译区进行片段扩增测序,发现突变位点后用反向测序予以证实,并检测家系成员相应的突变位点区域。采用ClustalW软件分析氨基酸序列保守性,用PROVEAN、Pyolphen和Mutation Taster等在线生物信息学软件预测突变位点对蛋白质功能可能存在的潜在影响。利用Swiss-pdb Viewer软件分析FⅦ蛋白模型野生型和突变型局部空间构型及分子间作用力的变化。结果先证者PT为31.2 s,明显延长;FⅦ:C和FⅦ:Ag分别为4%和6%,较健康人对照降低;先证者母亲、父亲、大姐、二姐和哥哥PT值均稍延长,FⅦ:C和FⅦ:Ag均下降至健康人对照的一半左右。基因检测结果显示先证者F7基因第8外显子存在c.1224 T>G纯合错义突变导致p.His348Gln,其母亲、父亲、大姐、二姐和哥哥均存在c.1224 T>G杂合错义突变。His348位点在同源物种中高度保守;生物信息学软件预测显示该突变位点会影响蛋白质功能;蛋白质模型分析显示,该突变位点可导致His348和Thr359以及Gly360之间形成的氢键数目减少,可影响蛋白质结构的稳定性。结论先证者的F7基因存在c.1224 T>G纯合错义突变导致p.His348Gln,该突变位点分别来自其近亲婚配的父母,并与其FⅦ水平降低有关。

Objective To detect the phenotype and gene mutation in a family of cross-cousin marriage with hereditary coagulation factorⅦ(FⅦ)deficiency to find the pathogenic gene and explore its molecular pathogenesis.Methods Plasma prothrombin time(PT),FⅦactivity(FⅦ:C)and FⅦ antigen(FⅦ:Ag)were measured in proband and relative family members(8 persons of 3 generations).The 9 exons and their flanking sequences,as well as 5'and 3'non-translation regions of F7 gene in the proband were amplified and sequenced by DNA direct sequencing.The mutation loci were identified and confirmed by reverse sequencing.The corresponding mutation site regions of the family members were simultaneously detected.ClustalW software was used to analyze the conserved amino acid sequence,and online bioinformatics software,such as PROVEAN,Pyolphen and Mutation Taster,were used to predict the potential effect of the mutation sites on protein function.The changes of local spatial configurations and intermolecular forces of the wild-type and mutants of FⅦ protein model were analyzed by Swiss-pdb Viewer software.Results The PT of the proband was 31.2 s,significantly prolonged.FⅦ:C and FⅦ:Ag were 4% and 6%,respectively,which were significantly lower than those of the normal controls.The PT values of mother,father,eldest sister,second elder sister and elder brother of the proband were slightly prolonged.Both the values of FⅦ:C and FⅦ:Ag were reduced to about half of the normal control.The results of gene detection showed that the c.1224T>G homozygous missense in exon 8 of F7 gene in the proband resulted in p.His348Gln,and c.1224T>G heterozygous missense mutation presented in his mother,father,elder sister,second sister and elder brother.Bioinformatics analysis showed that the His348 site should be highly conservative in homologous species and predicted that its mutation site could have an effect on protein function.The protein model analysis showed the mutation site may reduce the number of hydrogen bonds formed between His348,Thr359 and Gly360,thus affecting the stability of protein structure.Conclusion The mutation of c.1224T>G homozygous missense in F7 gene of the proband leads to p.His348Gln.The mutation locus should come from his inbreeding parents and may be related to the decrease of FⅦ level.