五个遗传性血小板无力症家系的表型和基因型诊断

Phenotype and genotype diagnosis in five Chinese pedigrees with Glanzmann thrombasthenia

目的:对5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。方法:通过止血和凝血指标检测、血小板(PLT)聚集试验和流式细胞术明确5个GT家系的诊断,用PCR扩增结合直接测序法分析先证者的αⅡb基因和β3基因的所有外显子及其侧翼序列,突变位点经直接测序证实排除基因多态性。同时选择100名健康人作为对照进行分析。结果:5个家系的先证者PLT计数均正常,凝血象正常,而出血时间(BT)延长;PLT对多种诱聚剂反应低下,而对瑞斯托霉素反应基本正常;流式细胞术结果显示,除家系3先证者为Ⅲ型GT外,其余4个家系的先证者均为Ⅰ型GT。基因分析共发现8种突变,均发生于αⅡb基因,分别为1750C>T(Arg553Stop)、2671C>T(Gln860Stop)、235G>T(Glu48Stop)、1084T>C(Phe331Leu)、1772A>C(Asp560Ala)、69-79del(移码突变)、2333A>C(Gln747Pro)和3060G>C(Lys989Asn)。结论:αⅡb1750C>T和αⅡb2671C>T复合杂合突变是导致家系1先证者发生GT的原因;αⅡb2671C>T和αⅡb235G>T复合杂合突变是导致家系2先证者发生GT的原因;αⅡb1084T>C和αⅡb1772A>C复合杂和突变是导致家系3先证者发生GT的原因;αⅡb69-79del纯合突变是家系4先证者发生GT的原因;αⅡb2333A>C和αⅡb3060G>C是家系5先证者发生GT的原因。235G>T、1084T>C、1772A>C和3060G>C突变为国际首次报道的发生于αⅡb基因的突变。

Objective To study the phenotype and genotype diagnosis of 5 Chinese pedigrees with Glanzmann thromboasthenia （GT）. Methods The diagnosis was based on clinical features and laboratory tests including coagulation test, platelet aggregation test and flow cytometry. PCR amplification and direct sequencing were used to analyze all the exons and flanking sequences of αⅡb and 133 gene of proband; gene polymorphism was excluded by direct sequencing of mutation locus. One hundred healthy subjects were selected as controls. Results All the probands had normal platelet count and coagulation profiles, but their bleeding time prolonged and platelet response to various aggregation inducers including ADP, collagen, adrenaline and arachidonic acid impaired, and response to ristocetin was relatively normal. Flow cytometry showed that all the probands bad type Ⅰ GT, except proband 3 which had type Ⅲ GT. Gene analysis revealed eight mutations, all located in αⅡb gene, including 1750C 〉T （Arg553Stop）, 2671C〉T （Gln860Stop）, 235G〉T（Glu48Stop）, 1084T〉C（Phe331Leu）, 1772A〉C （Asp560Ala）, 69-79del （frame-shift mutation）, 2333A〉C （Gln747Pro）and 3060G〉C （Lys989Asn）. Conclusions Compound heterozygous mutations 1750C〉T and 2671C〉T of anb gene lead to type Ⅰ GT for proband 1. Compound heterozygous mutations 2671C〉T and 235G〉T of anb gene lead to type Ⅰ GT for proband 2. Compound heterozygous mutations 1084T〉C and 1772A〉C of αⅡb gene lead to type Ⅲ GT for proband 3. Homozygous mutation 69-79 del of αⅡb gene leads to type Ⅰ GT for proband 4. Compound heterozygous mutations 2333A〉C and 3060G〉C of αⅡb gene lead to type Ⅰ GT for proband 5. Among them, 235G〉T, 1084T〉C, 1772A〉C and 3060G〉C are the mutations in αⅡb gene identified for the first time internationally.