1种罕见SERPINC1基因复合杂合突变及其机制研究  

A study on rare compound heterozygous mutations in SERPINC1 gene and their mechanisms

作　　者：张柯 林双女 谢海啸[1] 叶龙颖 秦朗译 潘景业[1] 杨丽红[1] 王明山[1] Zhang Ke;Lin Shuangnü;Xie Haixiao;Ye Longying;Qin Langyi;Pan Jingye;Yang Lihong;Wang Mingshan(Department of Clinical Laboratory,Key Laboratory of Clinical Laboratory Diagnosis and Translational Research of Zhejiang Province,the First Affiliated Hospital of Wenzhou Medical University,Wenzhou 325015,China)

机构地区：[1]温州医科大学附属第一医院医学检验中心,浙江省检验诊断及转化研究重点实验室,温州325015

出　　处：《中华检验医学杂志》2024年第3期301-307,共7页Chinese Journal of Laboratory Medicine

基　　金：浙江省检验诊断及转化研究重点实验室(2022E10022);温州市科技计划基金(Y20220126)。

摘　　要：目的对1个遗传性抗凝血酶(AT)缺陷症患者所携带的复合杂合突变进行分子机制研究。方法先证者因"突发晕厥并四肢抽搐一天"于2018年11月就诊于温州医科大学附属第一医院。采集先证者及其家系成员(共3代9人)外周静脉血并进行家系调查。采用发色底物法检测AT活性(AT:A),免疫比浊法检测AT抗原(AT:Ag)。对SERPINC1基因进行直接测序以确定突变位点。利用多种计算机工具预测突变的保守性和疏水性变化。构建重组质粒表达载体并瞬时转染HEK293T细胞以进行体外过表达研究。采用Western Blotting、ELISA和细胞免疫荧光实验对重组AT蛋白进行体外表征。结果先证者为一例21岁男性。AT:A为33%,AT:Ag同步下降,属于Ⅰ型AT缺陷症。基因分析显示先证者在第2和第5外显子分别携带c.318319insT(p.Asn107*)杂合插入突变和c.922G>T(p.Gly308Cys)杂合错义突变。该两个突变位点在同源物种中均完全保守;疏水性分析表明,p.Gly308Cys突变可能降低氨基酸残基307-313的亲水性。体外表达显示,重组蛋白AT-G308C在转染细胞裂解液和培养上清中的含量分别降低至46.98%±2.94%和41.35%±1.48%;经蛋白酶体抑制剂(MG132)处理后,Western Blotting分析显示在细胞质中的AT-G308C蛋白量恢复到与野生型相似的水平;在细胞裂解液和培养上清中均未检测到重组蛋白AT-N107*。结论p.Asn107*杂合插入突变和p.Gly308Cys杂合错义突变与该家系先证者AT水平降低有关。p.Asn107*杂合插入突变可能触发无义突变介导的mRNA降解,从而消除异常转录本;p.Gly308Cys杂合错义突变可能通过改变局部残基的疏水性导致AT蛋白在细胞质中发生蛋白酶体依赖性降解。Objective Molecular mechanisms underlying compound heterozygous mutations in a patient with inherited antithrombin(AT)deficiency.Methods The proband was admitted to the First Affiliated Hospital of Wenzhou Medical University in November 2018 with a one-day history of sudden syncope and limb twitching.Peripheral venous blood was collected from the proband and members of his lineages,totaling nine persons across three generations,and a family lineage survey was conducted.AT activity(AT:A)was measured using a chromogenic substrate assay,while AT antigen(AT:Ag)was detected through an immunoturbidimetric assay.Mutation sites were identified by means of Sanger sequencing of the SERPINC1 gene,and silico tools were applied to predict the mutational conservation and hydrophobicity changes.Recombinant plasmid expression vectors were constructed and transfected into HEK293T cells for in vitro overexpression studies.The recombinant AT protein was characterized using Western Blotting,ELISA,and cellular immunofluorescence assays.Results The proband was a 21-year-old man with typeⅠAT deficiency.His AT:A was 33%,along with a corresponding reduction in AT:Ag.The genetic analysis revealed there was a heterozygous insertion mutation at c.318_319insT(p.Asn107*)and a heterozygous missense mutation at c.922G>T(p.Gly308Cys)in exons 2 and 5,respectively.These mutation sites were entirely conserved among the homologous species.Additionally,hydrophobicity studies showed that the p.Gly308Cys mutation will decrease the hydrophilicity of amino acid residues 307-313.The in vitro expression studies indicated a reduction of approximately 46.98%±2.94%and 41.35%±1.48%in the amount of recombinant protein AT-G308C in transfected cell lysates and culture supernatants,respectively.Treatment with the proteasome inhibitor(MG132)restored the cytoplasmic levels of AT-G308C protein to a level similar to that of wild-type protein.However,neither cell lysate nor culture supernatant demonstrated the presence of the recombinant protein AT-N107*.Conclusion

关 键 词：抗凝血酶类 抗凝血酶缺陷症 血栓形成 复合杂合突变 体外表达 

分 类 号：R596[医药卫生—内科学]