一例FGG IVS7-12A>G剪接位点变异导致的遗传性无纤维蛋白原血症患者的表型及遗传学分析

A case of inherited afibrinogenemia caused by an IVS7-12A>G splice mutation of FGG gene

目的对1例遗传性无纤维蛋白原血症家系进行临床表型和基因型分析,探讨其分子发病机制。方法用全自动血液凝固分析仪检测先证者及其家系成员血浆凝血酶原时间(prothrombin time,PT)、活化部分凝血活酶时间(activated partial thromboplastin time,APTT)、凝血酶时间(thrombin time,TT)、纤维蛋白(原)降解产物[Fibrin(ogen)degradation products,FDPs]、D二聚体(D-dimer,D-D)、纤溶酶原活性(plasminogen activity,PLG:A)及硫酸鱼精蛋白对TT的纠正实验;分别用Clauss法和免疫比浊法检测纤维蛋白原(fibrinogen,Fg)活性和抗原含量;用PCR扩增纤维蛋白原FGA、FGB和FGG基因的所有外显子及其侧翼序列,扩增产物纯化后直接测序分析,寻找基因变异位点并排除基因多态性;利用Human Splicing Finder、MutationTaster、PROVEAN在线软件对变异引起的剪接位点改变和致病性进行预估和评分。结果先证者FDPs、D-D正常,PT、APTT、TT明显延长,纤维蛋白原活性及抗原含量明显降低(均<0.1 g/L);其妹与父母TT轻度延长(18.20~18.50 s),纤维蛋白原活性(1.27~1.54 g/L)及抗原含量(1.34~1.56 g/L)轻度降低;基因分析显示先证者携带FGG基因IVS7-12A>G(g.4147A>G)纯合变异,其妹及父母均携带FGG IVS7-12A>G杂合变异;Human Splicing Finder、Mutation Taster软件分析表明,该变异可产生新的剪接位点,导致第7外显子长度增加11个碱基,造成编码序列改变。PROVEAN预测为有害的变异。结论FGG IVS7-12A>G可能导致剪接位点的改变,是该先证者无纤维蛋白原血症的分子机制。

Objective To explore the genetic basis for a Chinese pedigree affected with inherited afibrinogenemia.Methods For the proband and his family members,prothrombin time(PT),activated partial thromboplastin time(APTT),thrombin time(TT),Fibrin(ogen)degradation products(FDPs),D-dimer(D-D),plasminogen activity(PLG:A)and the TT mixed experiment with protamine sulfate were determined with a STAGO-R automatic coagulation analyzer.The activity and antigen of fibrinogen(Fg)in plasma were measured with the Clauss method and immunonephelometry method,respectively.All exons and flanking regions of the fibrinogen genes(FGA,FGB and FGG)were amplified by PCR and directly sequenced.Human Splicing Finder software was used to predict and score the change of splicing site caused by the mutation.Results The proband showed normal FDPs and D-D but significantly prolonged TT,PT and APTT.The activity and antigen of fibrinogen in plasma were significantly decreased(<0.1 g/L).His young sister and parents showed slightly prolonged TT(18.20-18.50 s)and decreased fibrinogen activity(1.27-1.54 g/L)and fibrinogen antigenic content(1.34-1.56 g/L).Genetic testing revealed that the proband has carried homozygous IVS7-12A>G(g.4147A>G)mutations of the FGG gene,for which his parents and young sister were heterozygous.As predicted by Human Splicing Finder and Mutation Taster software,the variant may generate a new splicing site which can extend the sequence of exon 7 by 11 bp,with alteration of the coding sequence.PROVEAN suggested the variant to be deleterious.Conclusion The afibrinogenemia of the proband may be attributed to the FGG IVS7-12A>G variant,which was unreported previously.