遗传性凝血因子Ⅺ缺陷症一例家系基因缺陷研究

Investigation on gene defects in a pedigree with inherited coagulation factor XI deficiency

目的分析1例遗传性凝血因子Ⅺ（FXI）缺陷症家系表型和分子遗传学特征，对凝血因子基因（F11）进行基因缺陷研究。方法通过检测先证者及家系成员的活化部分凝血活酶时间（APTT）、凝血酶原时间（PT）、FⅪ促凝活性（FⅪ：C）和FⅪ抗原（FⅪ：Ag）等进行临床表型诊断，应用PCR对先证者F11基因15个外显子及其侧翼序列进行扩增，对PCR产物进行直接测序。对先证者及其父母、100名健康对照者DNA相应区域进行PCR扩增，内切酶BssSI酶切，以排除基因多态性并确认突变位点。应用分析软件SignaIP对信号肽切割点及位置进行预测。结果先证者APTT为69．58，PT为12．3s，FⅪ：C为2．6％，FⅪ：Ag为2．5％，交叉反应物质（CRM）为阴性；健康对照组APTT为35s，PT为13s，FⅪ：C为100％，FⅪ：Ag为100％。测序发现，先证者F11基因外显子区共有4处与GenBank AY191837序列不同的位点，其中外显子2的G3733C杂合变异导致编码信号肽的氨基酸G-1R替换，该突变同时导入1个新的BssSI酶切位点。100名健康对照者的BssSI酶切结果排除了该变异为F11基因多态性。外显子8的C16642T杂合突变导致1个终止密码（Q263Term）产生。结论F11基因G-1R和Q263Term双重杂合突变是导致先证者遗传性凝血因子Ⅺ缺陷症的分子发病机制。

Objective To investigate the gene defects of a pedigree with inherited coagulation factor Ⅺ （FⅪ） deficiency by analyzing its phenotype and molecular genetic characteristics. Methods A pedigree with inherited FⅪ deficiency was enrolled in this study. The activated partial thromboplastin time （APTF）, prothrombin time （PT） , FⅪ activity （FⅪ： C） and FⅪ antigen （FⅪ： Ag） were determined for phenotype diagnosis. Fifteen exons and their flanks of Fll gene from the proband＇s genomic DNA were amplified by polymerase chain reaction （PCR） , and the PCR products were directly sequenced to analyze the F11 gene mutation. The PCR products amplified from genomic DNA from the proband, her parents and 100 healthy donors were digested with restriction enzyme BssSI to exclude gene polymorphism and confirm the mutation site. The cleavage site in the signal peptide was predicted by the SignalP software. Results The values of APTT, PT, FⅪ： C and FⅪ： Ag of the proband were 69.5 s, 12.3 s, 2.6% and 2.5%, respectively, indicating that this case was cross-reacting material （ CRM ） negative. The same values of healthy controls were 35 s, 13 s, 100% and 100% , respectively. As compared with Genbank AY191837 sequence, four variants in F11 exons were found. G3733C heterozygous mutation in exon 2 caused Gly to Arg substitution at-1 amino acid position in signal peptide （G-1R）. The G3733C mutation in exon 2 introduced a new BssSI enzyme digestion site. Further analysis of the 100 randomly collected DNA samples from the normal population excluded the possibility of G3733C as a polymorphism. C16642T heterozygous mutation in exon 8 introduced a premature stop codon at 263 amino acid position （Q263Term）. Conclusions G-1R mutation and Q263Term compound heterozygous mutation in Fll gene are the mechanism of F Ⅺ proband. G-1R mutation is a novel F11 gene mutation causing inherited FⅪ deficiency.