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目的:对1例Ⅰ型抗凝血酶(AT)缺陷症患者及其家系进行表型诊断、基因分析及分子发病机制研究。方法:常规筛查检测AT缺陷症患者及其家系人员的凝血功能,采用血栓弹力图评估其高凝状态,用ELISA法检测抗心磷脂抗体(ACA)、抗β2GPI抗体,稀释的蝰蛇毒法检测狼疮抗凝物质(LA),发色底物法检测抗凝血酶(AT:A)和蛋白C活性(PC:A),凝固法检测蛋白S活性(PS:A),荧光偏振免疫法测定同型半胱氨酸(Hcy),免疫比浊法定量检测抗凝血酶抗原(AT:Ag),蛋白印迹分析血浆中AT的含量及相对分子质量,PCR法扩增AT基因的全部外显子及侧翼序列,DNA直接测序法进行基因分析。针对新的非经典剪接位点突变,提取患者的RNA,通过反转录反应结合巢式PCR扩增法,检测患者外周血中的AT异位转录本。结果:先证者的凝血筛查指标均正常,血栓弹力图指标显示其存在高凝状态,ACA、抗β2GPI抗体、LA、PC:A、PS:A及Hcy检测均正常;而其AT:A和AT:Ag同等下降,分别为45%和145 mg/L。血浆蛋白印迹检测结果显示,先证者血浆AT含量较正常混合血浆减少,但相对分子质量正常;基因分析发现,在5号内含子3’端剪接位点发生了杂合突变(g.13776_13777insAGGT);巢式PCR产物直接测序检测到异位转录本,TA克隆分析发现,异常转录本与正常转录本的比例为1∶5。结论:剪接位点突变g.13776_13777insAGGT可导致AT基因异常转录,影响AT蛋白正常表达,导致该家系发生Ⅰ型遗传性AT缺陷症,此剪接位点突变为国际首次报道。
OBJECTIVE: To investigate the phenotype, gene analysis and molecular pathogenesis of a type Ⅰ antithrombin (AT) deficiency patient and their pedigree. Methods: The coagulation function of patients with AT deficiency and their pedigree was determined by routine screening. The hypercoagulability state was evaluated by thromboelastography. Anti-cardiolipin antibody (ACA) and anti-β2GPI antibody were detected by ELISA. L-ascorbic acid (LA), anti-thrombin (AT) and protein C activity (PC: A) were detected by chromogenic substrate assay. Protein S activity was measured by coagulation assay (PS: A) Hcy, antithrombin antigen (AT: Ag) were detected by immunoturbidimetric assay, AT content and relative molecular mass in plasma were analyzed by Western blotting. All exons of AT gene were amplified by PCR And flanking sequences, DNA direct sequencing for gene analysis. Aiming at the mutation of new non-classical splice site, the RNA of patients was extracted. The ectopic transcripts of AT in peripheral blood of patients were detected by reverse transcription reaction and nested PCR amplification. Results: The clotting indexes of the probands were all normal. The index of thrombus elastography showed hypercoagulable state. The ACA, anti-β2GPI antibodies, LA, PC: A, PS: A and Hcy were all normal. And AT: Ag decreased by 45% and 145 mg / L, respectively. The result of plasma western blot showed that plasma AT content in probands decreased compared with normal mixed plasma, but the relative molecular mass was normal. Gene analysis showed that there was a heterozygous mutation at the 3 ’end of intron 5 (g.13776_13777insAGGT ). Ectopic transcripts were detected by direct sequencing of nested PCR products. TA cloning analysis showed that the ratio of abnormal transcripts to normal transcripts was 1: 5. CONCLUSION: The site of splicing site mutation g.13776_13777insAGGT can lead to abnormal transcription of AT gene and affect the normal expression of AT protein, resulting in type I inherited AT deficiency in this pedigree. This mutation was the first reported in the world.