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目的对一个遗传性凝血因子Ⅴ缺乏症家系进行凝血因子Ⅴ(factorⅤ,FⅤ)基因突变的检测,探讨先天性凝血因子Ⅴ缺乏症的分子发病机理。方法PCR结合直接测序方法对先证者的FⅤ基因全部25个外显子及其旁侧序列进行分析,鉴别其中可能存在的基因变异。应用PCR产物反向测序法或PCR限制性内切酶分析技术对突变位点进行确证。随机选择100名健康体检者作为正常对照。结果先证者FⅤ基因存在两种突变,分别是第11外显子区的A1763C错义突变和位于第16内含子3′端剪接位点的G→T突变;家系分析表明这两个突变是双杂合子型,前者遗传自父亲,后者遗传自母亲。100名健康对照者均未发现A1763C错义突变。结论第11外显子A1763C错义突变和第16内含子3′端的剪接位点突变使先证者呈现双重杂合子型,可能是先证者FⅤ先天性缺乏的原因。
Objective To detect the mutation of factor Ⅴ gene in a pedigree of hereditary factor Ⅴ deficiency and to explore the molecular pathogenesis of congenital factor Ⅶ deficiency. Methods All 25 exons and their flanking sequences of FV gene in proband were analyzed by PCR and direct sequencing to identify possible gene variants. The mutation sites were confirmed by reverse PCR or PCR restriction endonuclease analysis. 100 healthy subjects were randomly selected as normal control. Results There were two mutations in the FV gene of proband, which were A1763C missense mutation in exon 11 and G → T mutation in the 3 ’end of intron 16, respectively. The pedigree analysis showed that these two mutations Is a two-hybrid type, the former inherited from the father, the latter inherited from the mother. 100 healthy controls were found no A1763C missense mutation. Conclusions The missense mutation of exon 11 A1763C and the splice site of 3 ’end of intron 16 make the proband appear double heterozygote, which may be the cause of congenital deficiency of FV in probands.