ITGA2B基因复合杂合突变所致遗传性血小板无力症家系分析及致病机制研究  

Pedigree Analysis and Molecular Mechanism Study of Hereditary Glanzmann Thrombasthenia Caused by Compound Heterozygous Mutation of the ITGA2B Gene

作　　者：卢晓梅 付栋彦 张耀方 赵丽东 王蕾 杨嘉 刘杰 郑嘉伟 杨林花 王刚 Lu Xiaomei;Fu Dongyan;Zhang Yaofang;Zhao Lidong;Wang Lei;Yang Jia;Liu Jie;Zheng Jiawei;Yang Linhua;Wang Gang(Institute of Hematology,The Second Hospital of Shanxi Medical University,Taiyuan 030001,China)

机构地区：[1]山西医科大学第二临床医学院(第二医院)血液科、山西医科大学血液病学研究所,太原030001

出　　处：《中华血液学杂志》2024年第4期370-377,共8页Chinese Journal of Hematology

基　　金：国家自然科学基金(81700182);山西省自然科学基金(20210302123295);山西省科技创新团队(201605D131044-05)。

摘　　要：目的对一个ITGA2B基因复合杂合突变导致的遗传性血小板无力症家系进行表型及基因型研究,并探索其分子致病机制。方法使用二磷酸腺苷、胶原、肾上腺素、花生四烯酸及瑞斯托霉素等诱聚剂进行血小板聚集试验,检测先证者及家系成员的血小板聚集率。通过流式细胞术检测血小板表面CD41(αⅡb)、CD61(β3)、CD42b(GPⅠb)的表达。采用基因测序技术进行基因鉴定。利用RT-PCR检测ITGA2B基因mRNA剪接情况,qRT-PCR检测ITGA2B基因mRNA相对水平。生物信息学分析评估突变位点的致病性及对蛋白结构和功能的影响。通过Western blot检测分析血小板总αⅡb、β3的表达。结果除瑞斯托霉素外其他4种诱聚剂均无法使先证者血小板聚集。流式细胞术检测先证者血小板表面αⅡb的表达仅为0.25%,β3弱表达为9.76%,而GPⅠb表达相对正常,其余家系成员膜糖蛋白表达基本正常。基因测序结果显示先证者存在ITGA2B基因c.480C>G与c.2929C>T复合杂合突变,其中c.480C>G突变遗传自其母亲,c.2929C>T遗传自其父亲。RT-PCR及测序结果表明c.480C>G突变导致先证者及其母亲发生c.476G-574A(p.S160-S192)共99个碱基缺失的mRNA剪接。qRT-PCR检测发现c.2929C>T突变导致先证者及其父亲ITGA2B基因mRNA水平减低。生物信息学分析提示c.480C>G突变形成了与hnRNP A1蛋白结合序列,产生了5'SS剪接位点。αⅡb亚基的蛋白三维结构模型显示,p.S160-S192缺失的β-propeller结构域第2 blade缺失两条β链和一个α螺旋;c.2929C>T无义突变使得翻译提前终止产生p.R977-E1039缺失的截短型蛋白,包括胞质域(CD)、跨膜域(TM)以及胞外Calf-2结构域一条β链的缺失。Western blot检测先证者血小板总αⅡb表达缺失、β3的相对表达量为正常人的11.36%。结论ITGA2B基因第4外显子c.480C>G与第28外显子c.2929C>T的复合杂合突变是本家系遗传性血小板无力症的致病原因。Objective The phenotype and genotype of a pedigree with Glanzmann thrombasthenia caused by compound heterozygous mutation in the ITGA2B gene and its molecular pathogenesis were explored.Methods The platelet aggregation rate of the proband and his family was detected by using a platelet aggregation test with adenosine diphosphate,collagen,epinephrine,arachidonic acid,and ristocetin.The expression levels of CD41(αⅡb),CD61(β3),and CD42b(GPⅠb)on the platelet surface was detected by flow cytometry.Gene sequencing technology was used for the genetic identification of the family.RT-PCR was used in the detection of mRNA splicing,and qRT-PCR was used in detecting the relative mRNA level of the ITGA2B gene.Bioinformatics analysis was used to evaluate the pathogenicity of mutation sites and their effects on protein structure and function.The expressions of totalαⅡb andβ3 in platelets were analyzed by Western blot.Results Except ristocetin,the other four inducers could not induce platelet aggregation in the proband.Flow cytometry showed that the expression levels ofαⅡb andβ3 were only 0.25%and 9.76%,respectively,on the platelet surface of the proband,whereas GPⅠb expression was relatively normal.The expression levels of glycoproteins in the other family members were almost normal.c.480C>G and c.2929C>T mutations were detected in the proband through gene sequencing.The c.480C>G mutation was inherited from his mother,and the c.2929C>T mutation was inherited from his father.The RT-PCR and sequencing results showed that the c.480C>G mutation caused mRNA splicing in the proband and his mother,resulting in the deletion of 99 bases in c.476G-574A(p.S160-S192).qRT-PCR showed that the c.2929C>T variant reduced the mRNA level of the ITGA2B gene in the proband and his father.Bioinformatics analysis suggested that the c.480C>G mutation might form a binding sequence with hnRNP A1 protein and generate the 5′SS splice site.The three-dimensional structural model of theαⅡb subunit showed that theβ-propeller domain of th

关 键 词：遗传性血小板无力症 ITGA2B基因 整合素αⅡbβ3 mRNA剪接 无义突变 

分 类 号：R558[医药卫生—血液循环系统疾病]