摘要
目的:对一例遗传性凝血因子Ⅶ(FⅦ)缺陷症的患者及其家系进行基因分析,探讨其分子发病机制。方法:使用Sysmex-CS5100全自动血凝分析仪检测先证者及其家系成员(共3代8人)的凝血酶原时间(PT)、活化部分凝血活酶时间、凝血酶时间、D-二聚体、纤维蛋白降解产物以及血浆FⅦ的活性(FⅦ:C)水平;PCR法扩增先证者凝血因子Ⅶ基因(F7)所有外显子和侧翼序列,PCR产物纯化后测序,发现突变位点则反向测序给予证实;使用Clustal W软件对突变位点进行保守性分析;应用Mutation Taster和Poly Phen-2在线生物学软件评估突变氨基酸对FⅦ蛋白结构与功能的危害性;运用Swiss软件对突变建模分析。结果:凝血常规检查结果显示,先证者PT单独性延长至42.5 s;FⅦ:C明显降低,仅为2%;同样先证者外婆、母亲和妹妹的FⅦ:C都有轻度降低,分别为49%、51%和42%;父亲各指标均在正常参考范围。基因分析结果显示,先证者F7基因第6号外显子c DNA的646位发生G>A杂合突变,导致FⅦ催化区的156位甘氨酸被替换为丝氨酸(p.Gly156Ser)。F7其他外显子和侧翼序列的测序结果均正常。其外婆、母亲和妹妹均携带c.646G>A杂合突变,父亲为正常野生型。模型构建显示p.Gly156Ser突变使该位点氨基酸极性发生改变并出现侧链,从而使蛋白的不稳定性增加,可能影响所在结构域的催化活性。同时,Mutation Taster和Poly Phen-2两个在线生物信息学软件也高分预测该突变具有致病性。结论:该遗传性凝血因子Ⅶ缺陷症患者FⅦ蛋白p.Gly156Ser错义突变与血浆FⅦ:C水平降低有关。
Objective:To identify the genetic mutation of coagulation factorⅦ(F7)gene in a pedigree with coagulation factorⅦ(FⅦ)deficiency and explore the molecular pathogenesis.Methods:The prothrombin time(PT),activated partial thromboplastin time(APTT),thrombin time(TT),D-dimer(DD),fibrin degradation products(FDP)and coagulation factorⅦactivity(FⅦ:C)of the proband and her family members were detected by Sysmex-CS5100 analyzer.All exons and exon-intron boundaries of the F7 gene were amplified by PCR followed by direct sequencing.The detected mutation was confirmed by reverse sequencing.The ClustalW software was used to analyze the conservatism of the mutant site.Pathogenicity of the mutation was assessed with Mutation Taster and PolyPhen-2 online bioinformatics software.Structure of the mutant protein was analyzed using Swiss-PdbViewer software.Results:The results of routine coagulation tests showed that PT of the proband was markedly extended to 42.5 s,and her FⅦ:C significantly reduced to 2%.The FⅦ:C of her grandmother,mother and sister had slightly reduced to 49%,51%,and 42%,respectively.These coagulant parameters of her father were within the normal range.Genetic analysis reveled a heterozygous G>A change at cDNA 646 in exon 6 of F7 gene in the proband,resulting in a replacement of glycine at 156 of FⅦcatalytic region with serine(p.Gly156Ser).The sequencing results of other exons and exon-intron boundaries of her F7 gene were normal.The proband's grandmother,mother and sister were all the carriers of this missense mutation except her father.Bioinformatics analysis showed that the p.Gly156Ser mutation caused polarity change of the amino acid at this site and formation of side chains,leading to increase of protein instability,which may affect catalytic activity of structural domain.Meanwhile,both Mutation Taster and PolyPhen-2 online bioinformatics software also predicted the pathogenicity of this missense mutation with high scores.Conclusion:The heterozygous p.Gly156Ser mutation is the direct cause of the reduced FⅦin this proband.
作者
王莹宇
张永根
陈文柏
WANG Ying-Yu;ZHANG Yong-Gen;CHEN Wen-Bai(Department of Laboratory Medicine,The Affiliated Hospital of Xuzhou Medical University,Xuzhou 221300,Jiangsu Province,China;Department of Nuclear Medicine,The Affiliated Hospital of Xuzhou Medical University,Xuzhou 221300,Jiangsu Province,China)
出处
《中国实验血液学杂志》
CAS
CSCD
北大核心
2024年第3期857-861,共5页
Journal of Experimental Hematology
