一个复合杂合突变导致的遗传性凝血因子XII缺陷症家系分析

目的：对一个复合杂合基因突变导致的遗传性凝血因子XII（FXII）缺陷症家系进行实验室表型和基因突变分析，寻找致病基因并初步探讨其分子发病机制。方法：检测先证者及其家系成员（共3代5人）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）、凝血因子XI促凝活性（FXI:C）、XII促凝活性（FXII:C）和FXII抗原含量（FXII:Ag）等指标以明确诊断。采用PCR直接测序法分析先证者F12基因所有的外显子及其侧翼序列，对可疑的突变反向测序进行验证，并对家系成员的相应突变位点区域进行检测。采用ClustalX-2.1-win软件和4个在线生物信息工具（Mutation Taster、Poly-Phen-2、PROVEAN和SIFT）分析氨基酸突变位点的保守性和突变对蛋白质功能的可能影响；用Swiss-Pdb Viewer 4.0.1软件对突变位点进行蛋白模型相互作用分析。结果：先证者APTT为59.1 s，明显延长；FXII:C和FXII:Ag分别降低至4%和5%，其父亲、母亲、女儿的FXII:C和FXII:Ag降低至32%～37%。基因分析发现先证者F12基因第13号外显子存在复合杂合基因突变，即c.1561G＞A杂合错义突变（p.Glu502Lys）和c.1637T＞C杂合错义突变（p.Met527Thr）；其父亲为p.Met527Thr携带者，母亲和女儿为p.Glu502Lys携带者。保守性分析结果表明，Glu502和Met527在同源物种间高度保守；4个在线生物信息学软件对该两个突变的预测结果一致，均预示很可能是有害突变，可引起相关疾病。突变蛋白模型分析显示，野生型Glu502与His365之间形成1条氢键，Glu502替换为Lys502导致氢键的消失；野生型Met527与Leu524之间形成1条氢键，Met527替换为Thr527导致Thr527-Leu524之间增加2条氢键。结论：该先证者F12基因第13号外显子上存在p.Glu502Lys和p.Met527Thr复合杂合突变，推测该突变遗传自具有血缘关系且分别存在p.Glu502Lys和p.Met527Thr杂合子的父母，并与该家系FXII水平降低有关。

Genetic analysis of the pedigree with inherited coagulation factorXII deficiency caused by compound heterozygous mutations

Objective: To detect the laboratory phenotype and genotype of a hereditary factorXII (FXII) deficient pedigree caused by compound heterozygous mutations, which will help us to investigate the causative gene and 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), FXII activity (FXII:C), and FXII antigen (FXII:Ag) were assayed in proband and his family members (five persons in three generations) to confirm the diagnosis. All exons and flanking sequences of the F12 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 family members were detected. The conservation of amino acid mutation sites were analyzed using ClustalX-2.1-win software, the effect of mutations on protein functions were analyzed using four online bioinformatic tools (Mutation Taster, Poly-Phen-2, PROVEAN and SIFT); the mutation sites were analyzed by Swiss-Pdb Viewer 4.0.1 for molecular structure and protein interaction. Results: The APTT of the proband is 59.1 s, prolonged obviously, and the FXII:C decreased to 4%, the FXII:Ag decreased to 5%; the FXII:C and the FXII:Ag of his father, mother and daughter decreased to 32%-37%. Genetic analysis revealed that there were two compound heterozygous mutations in exon 13 of the F12 gene in the proband: one was the c.1561G>A point heterozygous missense mutation (p.Glu502Lys); another was the c.1637T>C point heterozygous missene mutation (p.Met527Thr). His father was carried the p.Met527Thr mutation, his mother and daughter were carried the p.Glu502Lys mutation. Conservative analysis showed that Glu502 and Met527 were highly conserved among homologous species. The four online bioinformatics sofwares predicted results for these mutations were consistent: all predicted that these mutations were likely to be deleterious mutations that could cause related diseases. Mutant protein model analysis showed that wild-type Glu502 forms a hydrogen bond with His365, the mutant Lys502 resulted in the disappearance of the hydrogen bond between Lys502-His365. Replacement of Met527 with Thr527 resulted in the addition of two hydrogen bonds between Thr527-Leu527. Conclusion: There were two compound heterozygous mutations p.Glu502Lys and p.Met527Thr in exon 13 of the F12 gene in the proband, which were speculated that the heterozygous mutations were inherited from his parents with the heterozygotes of p.Glu502Lys and p.Met527Thr respectively and were associated with the reduced blood FXII levels in the family.