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目的 探讨遗传性凝血因子Ⅶ (FⅦ )缺陷症分子发病机制。方法 检测凝血指标以明确诊断 ;用DNA直接测序法对患者FⅦ基因的全部外显子和其侧翼以及 3′ ,5′非翻译区进行分析 ,寻找基因突变 ,对有突变的序列反向测序证实 ;发生在非经典的剪接位点突变用外周血单个核细胞异位转录的RT PCR方法确定其剪接方式。结果 患者在 8号外显子 10 96 1位发生T→G杂合突变 ,导致 348位His被Gln替代 ;在 1号内含子 5′端的非经典的剪接位点有Ggtgcg >Ggtgca(IVS1a+5g >a)杂合突变 ,RT PCR结果揭示剪接过程中去除了 2号外显子 ,却把 3号内含子包含进去 ,在 3号外显子起始处出现了移码突变 ,编码与原来氨基酸完全不同的 9个氨基酸后出现了终止信号 ,产生了一个只有 30个氨基酸组成的截短型蛋白。结论 患者FⅦ基因中发现了两种杂合突变 (10 96 1位T→G、IVS1a +5g >a) ,其中后一种非经典的剪接位点突变导致的异常剪接方式为国际首次报道。
Objective To investigate the molecular pathogenesis of hereditary factor Ⅶ (FⅦ) deficiency. Methods The clotting indexes were detected to confirm the diagnosis. All the exons of FⅦ gene, their flanks and the 3 ’and 5’ untranslated regions of the patients were analyzed by DNA direct sequencing to find out the gene mutations. Reverse sequencing of the mutated sequences was confirmed ; Mutations occurred at non-classical splicing sites were determined by RT PCR using heterologous transcripts of peripheral blood mononuclear cells. Results T → G heterozygous mutation occurred in exon 8 of exon 8, resulting in the substitution of His at position 348 with Gln. In the non-canonical site of splicing at exon 1, Ggtgcg> Ggtgca (IVS1a + 5g > a) heterozygous mutations, RT PCR results revealed that exon 2 was removed during splicing but inclusion of introns No. 3 revealed a frameshift mutation at the beginning of exon 3, encoding a protein that was completely homologous to the original amino acid A different signal appears after the nine amino acids, producing a truncated protein of only 30 amino acids. Conclusions Two heterozygous mutations (10 96 1 T → G, IVS 1 a + 5 g> a) were found in FⅦ gene. The first non-canonical splicing site mutation was the first reported in the world.