一个纯合子Tyr503Cys突变导致的遗传性凝血因子Ⅺ缺陷症家系分析

Genetic analysis of the pedigree with inherited coagulation factor XI deficiency caused by a homozygous Tyr503Cys mutation

目的:对一个纯合子Tyr503Cys错义突变导致的遗传性凝血因子XI(FXI)缺陷症家系进行表型和基因检测,寻找致病基因并初步探讨其分子致病机制。方法:检测先证者及其家系成员(共3代5人)的血浆凝血酶原时间(PT)、活化部分凝血活酶时间(APTT)、凝血因子Ⅷ促凝活性(FVIII:C)、凝血因子IX促凝活性(FIX:C)、FXI促凝活性(FXI:C)、凝血因子XII促凝活性(FXII:C)和FXI抗原(FXI:Ag)含量等指标以明确诊断。采用PCR直接测序法分析先证者F11基因所有外显子和侧翼序列,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。使用ClustalX-2.1-win软件分析氨基酸突变位点的保守性;用PolyPhen-2、PROVEAN和MutationTaster软件分析突变对蛋白质功能的影响;用Swiss-PdbViewer软件对突变位点进行蛋白模型和氨基酸相互作用分析。结果:先证者APTT为59.3s,明显延长,FXI:C降低至13%;其母亲、女儿和儿子的APTT均有不同程度延长,FXI:C降至37%~42%,该家系5人FXI:Ag均在参考值范围。基因分析发现先证者F11基因第13号外显子存在c.1562A>G纯合错义突变,导致Tyr503Cys突变;其母亲、女儿和儿子存在Tyr503Cys突变杂合子。保守性分析结果表明,Tyr503在同源物种间高度保守。3种生物信息学软件对该突变的预测结果一致,均预示此突变很可能是有害突变,可引起相关疾病。突变蛋白模型分析显示,野生型Tyr503与Ile370、Lys554形成2个氢键;突变型Cys503与Lys554之间新增了一个氢键。结论:该先证者F11基因第13号外显子存在c.1562A>G纯合错义突变,导致Tyr503Cys突变;推测该纯合突变遗传自具有血缘关系且均存在Tyr503Cys杂合子的父母,并与该家系FXI水平减低有关。

Objective: To detect the phenotype and gene of a hereditary factor XI (FXI)-deficient pedigree caused by a homozygous Tyr503Cys missense mutation in search for the causative gene and exploration of its molecular pathogenesis.Methods: Prothrombin time (PT),activated partial thromboplastin time (APTT),coagulation factor VIII activity (FVIII:C),coagulation factor IX activity (FIX:C),coagulation factor XI activity (FXI:C),coagulation factor XII activity (FXII:C),and FXI antigen (FXI:Ag) were assayed in probands and their family members (five persons in three generations) to confirm the diagnosis.All exons and flanking sequences of the F11 gene of the proband were analyzed by direct PCR sequencing.The mutation sites were confirmed by reverse sequencing and the corresponding mutation site regions of the familial members were detected.The conservation of amino acid mutation sites was analyzed using ClustalX-2.1-win software; the effect of mutations on protein function was analyzed using PolyPhen-2,PROVEAN,and Mutation Taster software; the mutation site was analyzed by Swiss-PdbViewer software for protein model and amino acid interactions.Results: The APTT of proband was 59.3 s,prolonged obviously,and the FIX:C decreased to 13%; the APTT of mother,daughter and son were all prolonged to varying degrees,the FIX:C fell to 37%-42%,and the family of five people’s FXI:Ag were in the reference range.Genetic analysis revealed that there was a c.1562A>G homozygous missense mutation in exon 13 of the F11 gene in the proband,leading to Tyr503Cys; heterozygotes of the Tyr503Cys mutation existed in the mother,daughter,and son.Conservative analysis showed that Tyr503 is highly conserved among homologous species.The three bioinformatics software’s predictive results for this mutation were consistent: both predicted that this mutation was likely to be a deleterious mutation that could cause related diseases.Mutant protein model analysis showed that wild-type Tyr503 forms two hydrogen bonds with Ile370 and Lys554; a hydrogen bond was added between mutant Cys503 and Lys554.Conclusion: There was a c.1562A>G homozygous missense mutation in exon 13 of the F11 gene in the proband,laeding to Tyr503Cys.It was speculated that the homozygous mutation was inherited from a parent with a heterozygous Tyr503Cys homozygote and that it was associated with reduced FXI levels in the family.