两例遗传性凝血因子Ⅺ缺乏症患者的基因分析

The Genetic Analysis of two Patients with Congenital Coagulation Factor Ⅺ Deficiency

目的对两例遗传性凝血因子Ⅺ缺陷症的患者进行FⅪ基因突变分析,探讨其分子发病机制。方法两例患者在常规凝血检查时发现活化部分凝血活酶时间(aPTT)明显延长,进行aPTT纠正试验和凝血因子活性检测,发现均为凝血因子Ⅺ活性(FⅪ:C)降低。收集患者外周血,分别提取DNA,使用聚合酶链式反应分别扩增两例患者FⅪ的所有外显子及其侧翼序列,并将扩增产物进行测序。测序结果应用Chromas软件与FⅪ正常序列(NG_008832.1)进行比对;有突变的片段进行反向测序并比对。结果病例1:aPTT 109.6 s,FⅪ:C 0.4%;病例2:aPTT 50.1 s,FⅪ:C6.7%;两例患者aPTT纠正试验和其余凝血因子活性均正常。FⅪ基因测序结果显示:病例1在Exon2上发生18 del A纯合缺失,导致移码突变(Val-13 Trpfs X15),该突变为新发现的突变点;病例2在Exon7上发生738 G>A杂合突变、导致Trp228 stop,同时在Exon9上发生1021 G>A杂合突变、导致Glu323 Lys。结论上述突变可能分别是导致两例患者FⅪ缺乏的分子致病机制。

Objective To analyze genetic mutation and explore its molecular pathogenesis for two pa- tients with congenital coagulation factor Ⅺ deficiency. Methods Two patients were found to have prolonged activated partial thrombin time （aPTT） , then their activity of coagulant factors were tested and aPTT mixing test with incubation were performed. Low activity of the F Ⅺ （ F XI ： C ） was found in both. Two patients＇ blood was collected, and their DNA was extracted. Polymerase Chain Reaction （PCR）was used to amplify all exons and exon-intron boundaries, and then the products of PCR were sequenced directjy. The mutation was deter- mined through alignment of the normal F Ⅺ genomic DNA sequence by software chromas. The segment with mutation was sequenced backward. Results aPTT and FXI ： C was 109.6 s and 0. 4% for patient 1 and 50. 1 s and 6.7 % for patient 2, respectively. The results of aPTT mixing correction test and activities of other coagulant factors were all in normal range for both patients. The results of sequencing showed that a de novel homogenous nueleotide 18 A deletion（ 18 delA）mutation in exon2 was detected in patientl ,leading frame shift mutation（ Val-13 TrpfsX15 ） , and that combined heterogenous mutation, a nucleotide 738 G 〉 A transition in exon7 and a nucleotide 1021 G 〉 A transition in exon9 ,resulting a Trp228 stop substitution and a Glu323 Lys substitution, respectively, were detected in patient 2. Conclusion These mutations might be the molecular pathogenesis for two patients with congenital coagulation factor Ⅺ deficiency, respectively.