五例遗传性凝血因子V缺陷症的基因分析

目的 分别对5例遗传性凝血因子V(FV)缺陷患者进行基因分析和家系调查,鉴定导致FV缺陷症的基因突变.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测序并与基因文库比对以确定基因异常,限制性内切酶酶切分析或直接测序患者直系家属及正常人相应的等位基因排除多态性影响.结果 5例患者血浆FV活性和抗原含量均明显降低,基因分析共发现6个致病突变,分别为G69969T(G2079V)、C45533T(R712Ter)、C46796T(R1133Ter)、G45366A(C656Y)、C46253T(R952C)及G16088C(D68H),后3个是新的突变位点,其中C46253T(R952C)是国际上首次发现的位于B区的错义突变.通过对直系亲属和正常人相应的等位基因进行扩增测序或酶切分析证实了3个新的突变不是基因多态性所致.结论 鉴定了5例Ⅰ型遗传性FV缺陷症的基因突变,其中包括2种无义突变和4种错义突变;错义突变导致FV蛋白稳定性降低,引起血浆中FV的含量减少.

Gene analysis of five inherited factor V deficiency cases

OBJECTIVE: To identify gene mutations involved in five cases of inherited factor V (FV) deficiency. METHODS: Activity of FV was determined by one-stage clotting assay using FV-deficiency plasma, and FV antigen by an ELISA assay. All the exons and exon-intron boundaries of FV gene were amplified by PCR and then DNA sequencing. Restriction enzyme analysis was used to analyze the probands, their family members and healthy volunteers. RESULTS: Both activity and antigen of FV in the 5 patients were extremely lower compared with that of normal mixed plasma. Six mutations were identified in these 5 patients, G69969T (G2079V), C45533T (R712Ter), C46796T (R1133Ter), G45366A (C656Y), C46253T (R952C) and G16088C (D68H), the latter three were novel mutations reported for the first time and the C46253T (R952C) was the first missense mutation reported in B domain. The result of sequencing or restriction enzyme analysis showed that the three novel missense mutations were not caused by single nucleotide polymorphisms. CONCLUSION: Gene mutations in 5 type I inherited FV deficiency of patients including 2 nonsense mutations and 4 missensense mutations identified which led to the instability of FV protein and the reducing of FV: Ag in the plasma.