两例先天性凝血因子V缺乏症基因突变的鉴定

Identification of Gene Mutations in Two Coagulation Factor V Deficiency Patients

目的探讨先天性凝血因子Ⅴ(FⅤ)缺乏症的分子发病机制。方法采用一期法检测血浆FⅤ活性, ELISA法检测血浆FⅤ的抗原含量,PCR法扩增FV基因的25个外显子及其侧翼序列,DNA测序并与Genebank校对确定基因异常。结果两例患者血浆FⅤ活性和抗原含量均为2％。病例1在FⅤ基因23外显子G 69969 T的纯合突变,导致G 2107 V;病例2由13外显子双杂合突变所致,其中一突变C 45533 T导致R 740 Ter,另一突变位点G 45366 A导致C 684 Y,这是国际上第一个位于13外显子的错义突变位点。分子结构分析表明684 Cys突变Tyr后,不能与603 Cys形成二硫键,影响了A2区β环状结构的形成,导致FⅤ的稳定性降低。结论FⅤ基因G 69969 T、C 45533 T及G 45366 A突变与先天性FⅤ缺乏症有关。

Objective To explore the molecular mechanisms involved in the patients with congenital F V deficiency. Methods Activity of F V was determined by a onestage clotting assay using F V--deficiency plasma, and F V antigen was measured by an ELISA assay. All the exons and exonintron boundaries of the F V genes were amplified by PCR and then DNA sequencing was performed. Results Activity and antigen of F V was about 2% in the two patients compared with normal mixed plasma. A homozygous missense mutation G 69969 T resulting in G 2107 V was revealed in the first patient, and a heterozygous mutation of C 45533 T leading to R 740 Ter and G 45366 A resulting in C 684 Y in the second patient. C ys 684 Tyr mutation was a new mutation, which disturbed the disulfide bridge between 684 Cys and 603 Cys that form the β-ring of A2 region. Conclusion G 69969 T,C 45533 T and G 45366 A mutation of FVgene is related to congenital FV deficiency.