三个遗传性血小板无力症家系的表型和基因型诊断分析

Molecular analysis of phenotypes and genotypes in three Chinese pedigrees with Glanzmann thrombasthenia

目的对3个遗传性血小板无力症(GT)家系进行临床表型和基因型诊断,探讨其分子发病机制。方法通过凝血指标检测、血小板(PLT)聚集试验、出血时间测定和流式细胞仪检测,明确3个遗传性GT家系的诊断,用PCR扩增先证者的αⅡb及β3基因的所有外显子及其侧翼序列,利用直接测序法进行基因序列分析。针对先证者的基因突变位点,对其家系成员进行相应外显子基因检测,同时选择100名健康人对照以排除基因多态性。结果 3个家系的先证者血小板计数及凝血四项检测均正常,而出血时间(BT)延长;血小板对多种诱聚剂反应低下,而对瑞斯托霉素反应基本正常;流式细胞术检测结果显示,家系2及家系3先证者为Ⅰ型GT,家系1先证者为Ⅱ型GT。基因检测结果显示3个家系的基因突变均位于αⅡb基因,分别是17号外显子14502C>T导致R(Arg)553X(stop)纯合无义突变;26号外显子18859C>T导致Q(Gln)860X(stop)纯合无义突变;15号外显子14103-14104insT、14105A>C、14106G>C、14107A>T导致氨基酸移码突变;其家系部分成员检测到相应位点的杂合突变。结论纯合无义突变18859C>T、纯合插入突变14103-14104insT及3个纯合性错义突变14105A>C、14106G>C、14107A>T是导致先证者2及先证者3发生Ⅰ型GT的原因,而14502C>T纯合无义突变是先证者1发生Ⅱ型GT的原因。其中,纯合性插入突变14103-14104insT及纯合性点突变14105A>C、14106G>C、14107A>T为国际首次报道的新突变。

Objective To investigate the phenotypes and genotypes in three Chinese pedigrees with Glanzmann thromboasthenia（GT）,and to identify the molecular mechanism of Glanzmann thromboasthenia（GT）.Methods The diagnosis was based on clinical features and laboratory tests including coagulation test,platelet aggregation test,bleeding time（BT）and flow cytometry.PCR amplification were used to analyze all the exons and flanking sequences of αⅡb and β3 gene of proband,PCR products analysis was by direct sequencing;The corresponding gene sites of family members was detected acocording to the gene mutation sites,gene polymorphism was excluded by direct sequencing of one hundred healthy subjects selected as controls.Results All the probands had normal platelets count and coagulation profiles,but their bleeding time prolonged and platelets response to various aggregation inducers including ADP,collagen,adrenaline and arachidonic acid impaired,and response to ristocetin was relatively normal.Flow cytometry showed that the proband 2 and proband 3 had type I GT,while proband 1 had type II GT.Gene analysis revealed all mutations were located in αⅡb gene,homozygous nonsense mutations 14502CT in exon 17,homozygous nonsense mutations18859CT in exon 26,homozygous mutation 14103-14104insT,14105AC,14106GC,14107AT in exon 15 leading amino acids frameshif .And some of the three family members were detected with the heterozygotic gene mutation.Conclusion Homozygous nonsense mutations 18859CT and homozygous insertion mutation ,three missense mutations14105AC,14106GC and14107AT are the gene defects of Type I GT.While homozygous nonsense mutations 14502CT are the gene defects of Type II GT.Homozygous mutation 14103-14104insT,14105AC,14106GC,14107AT have not been described before.