Abstract
目的 探讨遗传性凝血因子Ⅹ(FⅩ)缺陷症的分子发病机制.方法对在一个遗传性凝血因子Ⅹ(FⅩ)缺陷症家系中发现的两种FⅩ基因新突变剪接位点突变IVS1+1G>A和错义突变Arg347His,分别进行异位转录和体外表达的研究,通过逆转录反应结合巢式PCR扩增的方法,检测患者外周血中的FⅩ异位转录产物.针对Arg347His突变,将野生型FⅩ cDNA克隆至真核细胞表达载体,定点突变构建Arg347His突变质粒.然后将野生型质粒和突变体质粒分别转染HEK293细胞,采用一期法检测细胞培养液中的FⅩ活性;采用ELISA检测分别细胞裂解液和细胞培养液中的FⅩ抗原含量.结果对于IVS1+1G>A突变,逆转录结合巢式PCR的产物经克隆后测序只检测到了正常的转录本,而没有发现异常转录本,原因可能是由于IVS1+1G>A导致了终止密码的提前出现,从而致使异常转录的mRNA在体内很快被降解.对于Arg347His错义突变,真核细胞表达结果发现,Arg347His突变并没有影响FⅩ蛋白的合成分泌,但突变质粒转染的细胞所分泌的FⅩ蛋白促凝活性明显下降.结论异位转录和体外表达的实验进一步证实了IVS1+1G>A和Arg347His是导致本家系先证者表现为FⅩ缺陷症的原因.IVS1+1G>A突变导致了异常转录的mRNA在体内被很快降解;Arg347His突变显著降低了FⅩ的促凝活性。
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