2种F10基因新突变导致凝血因子Ⅹ缺陷症的分子发病机制研究

Molecular mechanism of coagulation factor Ⅹ deficiency caused by two novel F10 gene mutation

目的:探讨1个凝血因子Ⅹ(FⅩ)缺陷症家系的分子发病机制。方法:对先证者及家系成员凝血、抗凝及纤溶功能筛查以及凝血因子活性及抗原含量检测进行表型诊断;以Western Blotting检测血浆中FⅩ抗原含量和分子量大小;以中和试验检测FⅩ的抑制物。以PCR方法对F10基因所有外显子及侧翼序列和5’端非翻译区进行扩增,产物纯化后直接测序进行基因诊断;构建F10基因突变表达质粒,瞬时转染HEK293T细胞,测定表达产物的FⅩ促凝活性(FⅩ:C)和FⅩ抗原含量(FⅩ:Ag)。结果:先证者FⅩ:C和FⅩ:Ag分别为<1%和53.36%,中和试验结果阴性,诊断为交叉反应物质阳性(CRM+)的FⅩ缺陷症。F10基因分析发现2个杂合突变:IVS5+1G>A和Asp368del。Asp368del体外表达显示FⅩ:C和FⅩ:Ag分别为(0.52±0.04)%和(85.9±5.0)%,为CRM+突变。结论:F10基因双重杂合突变IVS5+1G>A和Asp368del导致该家系遗传性FⅩ缺陷症。剪接位点突变IVS5+1G>A导致内含子无法正常剪接,影响FⅩ正常表达。Asp368del突变蛋白能够正常表达,但功能降低。

Objective To investigate the molecular pathogenesis of a pedigree with inherited coagulation factorⅩ（FⅩ） deficiency.Methods Screen assays of coagulation,anticoagulant and fibrinolytic functions and FⅩactivity（FⅩ：C） and FⅩantigen（FⅩ：Ag） were performed in proband and pedigree members for phenotype diagnosis.The antigen level and molecule weight of the FⅩin plasma were determined by Western blotting.Neutralization test was used for detecting FⅩ inhibitors.All exons and flanking sequences,and 5＇ untranslated region of F10 gene were amplified by PCR,and the products were purified and sequenced directly to detect the mutations for gene diagnosis.Expression plasmids containing mutant F10 cDNA were constructed and transfected into HEK293T cells transiently.F Ⅹ：C and FⅩ：Ag of expressed product were assayed.Results FⅩ：C and FⅩ：Ag in proband were 1% and 53.36%,respectively.Neutralization test was negative.Cross reacting material positive（CRM＋） FⅩ deficiency was diagnosed.Two F10 gene mutations（IVS5＋1G A and Asp368del） were identified in the proband.Asp368del in vitro expression showed that FⅩ：C and FⅩ：Ag were（0.52±0.04）% and（85.9±5.0）%,respectively,and CRM＋ mutation was diagnosed.Conclusions The hereditary FⅩ deficiency of this pedigree was caused by double heterozygous mutation,IVS5＋1GA and Asp368del.Splice site mutation IVS5＋1GA causes the fail of normal splicing of introns and affects the normal expression of F Ⅹ.Asp368del mutated protein could normally express FⅩ,but with reduced function.