八个遗传性血小板无力症家系基因型和表型分析

Analysis of phenotype and genotype in eight Chinese pedigrees with glanzmann thrombasthenia

目的对8个遗传性血小板无力症（GT）家系进行血小板膜糖蛋白（GPⅡb／Ⅲa）临床特性分析和基因突变检测。方法采用血小板聚集试验观察血小板对各种诱聚剂的反应，流式细胞术检测血小板表面αⅡb和β3的含量；PCR法对先证者GPⅡb／Ⅲa基因所有外显子及其侧翼序列进行扩增；PCR产物纯化后直接测序，检测其基因突变。对突变位点进行直接测序，以排除基因多态性。结果8个家系的先证者PLT计数均正常，血小板形态分散，出血时间（BT）延长，凝血象正常；对二磷酸腺苷（ADP）、凝血酶、肾上腺素、胶原、花生四烯酸等多种诱聚剂反应低下，而对瑞斯托霉素反应基本正常；流式细胞仪检测结果显示，除家系2先证者为Ⅲ型GT和家系6先证者为Ⅱ型GT外，其余家系各先证者均为I型GT。基因分析共发现12种不同类型的突变，分别为G10A、G1412T、G1199A、1525delC、G2223T、C2671T、2930delG、tVS15（-1）delG、A2334C、C1750T、69-79del和C470A。家系3先证者在GPⅡb／Ⅲa基因未检测到突变。结论GPⅡb／Ⅲa基因突变是导致血小板无力症的主要原因。G10A、69—79del、C470A、G1412T、G2223T、C2671T和1525delC是导致遗传性血小板无力症的新的基因突变位点。

Objective To study the GP Ⅱb/Ⅲ a gene mutations of eight glanzmann thrombasthenia （GT） pedigrees. Methods Responses of eight probands to different agonists were observed by platelet aggregation test and the amount of αⅡb and β3 was determined by flow cytometry. All the exons of Ⅱb and Ⅲa genes were amplified by PCR followed by sequencing for mutational screening. Further analysis of the normal population excluded the possibility of mutational sites as a polymorphism. Results Eight probands showed normal PLT counts, dispersion of the platelet particles without aggregation, prolonged bleeding time and severely reduced platelet aggregation in response to the physiological agonists ADP, epinephrine, and collagen, but relatively normal aggregation of PLT in response to ristocetin. Flow cytometry showed that all probands were Ⅰ type GT, except that proband 2 was Ⅲ type GT and proband 6 was Ⅱ type GT. The sequencing results showed that twelve different types mutations were present in eight probands, including G10A, G1412T, Gl199A, 1525delC, G2223T, C2671T, 2930delG, IVS15 （- 1 ） delG, A2334C, C1750T, 69-79del and C470A. We were not able to detected any mutations in GP Ⅱb/Ⅲa gene on proband 3. Conclusions GT is mainly caused by GP Ⅱb/Ⅲa gene mutations. G10A, 69-79del, G1412T, G2223T, C2671T and 1525delc were the novel mutations causing GT.