一个遗传性凝血因子ⅩⅢ缺陷症家系新的基因异常

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目的对1例遗传性凝血因子ⅩⅢ(ⅩⅢ)缺陷症家系进行基因分析,探讨其发病的分子机制。方法用尿素溶解法和 Berichrom kit 检测患者及其家系成员血浆 FⅩⅢ的活性,用火箭电泳和酶联免疫吸附试验测定 FⅩⅢ抗原含量。PCR 法扩增 FⅩⅢA 基因的所有外显子和侧翼序列,DNA 测序分析基因异常,用直接测序法和限制性内切酶(SfaN Ⅰ、Nsp Ⅰ)分析80名正常人相应序列的 PCR 产物以排除基因多态性。应用生物信息学软件对所鉴定的突变进行分子模建,探讨其致病的分子机制。结果患者纤维蛋白凝块住5mol/L 尿素中30min 内完全溶解,加入正常人血浆后则24 h不溶解,患者血浆FⅩⅢ活性为0,FⅩⅢA 抗原含量<2%,FⅩⅢB 抗原含量在正常范围。家系成员中其父母和外祖母血浆 FⅩⅢ活性和 FⅩⅢA 抗原约为正常人一半。在患者 FⅩⅢA 因因外显子15中发脱两处杂合异常,分别位于177 246位碱基(C→T,导致 Arg703→Trp)和177 286位碱基(A→G,导致 His716→Arg),直接测序和酶切分析两种方法均排除了基因多态性。患者的母亲与外祖母为 Arg703→Trp 杂合子,患者的父亲为His716→Aug 杂合子。分子模建分析表明,Arg03和 His716两个位点突变后均可导致 barrel 2与 core结构域之间距离和结合能力的改变,使蛋白质发生错误折叠,稳定性降低。His716→Arg 还可能影响酶的催化活性基因的空间结构形成。结论 FⅩⅢA 基因外显子15上 Arg703→Trp、His716→Arg 复合杂合突变影响了蛋白质的正确折叠和稳定性,造成患者 FⅩⅢ抗原和活性的缺失。此复合杂合错义突变是一种未报道过的新的突变类型。 Objective To analyze the genetics of a familial familial hereditary factor ⅩⅢ (ⅩⅢ) deficiency and to explore its molecular mechanism. Methods The plasma F Ⅹ Ⅲ activity in plasma of patients and their pedigrees was detected by urea dissolution and Berichrom kit. The content of F Ⅹ Ⅲ antigen was determined by rocket electrophoresis and enzyme-linked immunosorbent assay. All the exons and flanking sequences of F Ⅹ Ⅲ A gene were amplified by PCR. DNA sequencing was used to analyze the gene abnormalities. The PCR products of 80 normal individuals were analyzed by direct sequencing and restriction endonucleases (SfaN Ⅰ, Nsp Ⅰ) Gene polymorphism. Bioinformatics software was used to molecularly identify the identified mutations and to explore the molecular mechanisms of their pathogenicity. Results Fibrin clot lived in 5mol / L urea completely dissolved within 30min. After adding normal human plasma, it did not dissolve after 24 h. The plasma FXIII activity was 0, the content of FⅧⅢA antigen was less than 2%, and the content of F Ⅹ Ⅲ B antigen was within the normal range. Family members of their parents and grandmothers plasma F Ⅹ Ⅲ activity and F Ⅹ Ⅲ A antigen about half of normal people. In patients with F XIIIA exon 15 exfoliated at two heterozygotes at 177 246 bases (C → T, resulting in Arg703 → Trp) and 177 286 bases (A → G, resulting in His716 → Arg ), Direct sequencing and digestion analysis of two methods are excluded from the gene polymorphism. Patient’s mother and grandmother Arg703 → Trp heterozygous, the patient’s father His716 → Aug heterozygotes. Molecular modeling showed that the mutation of both Arg03 and His716 could result in the change of the distance and the binding ability between barrel 2 and core, resulting in the misfolding of protein and the decrease of stability. His716 → Arg may also affect the spatial structure of the catalytically active gene of the enzyme. Conclusion The mutation of Arg703 → Trp and His716 → Arg in exon 15 of F Ⅹ Ⅲ A gene affected the correct folding and stability of protein, resulting in the deletion of FⅧ Ⅲ antigen and activity in patients. This composite heterozygous missense mutation is a new type of mutation that has not been reported.
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