巨血小板综合征糖蛋白Ⅸ基因异常的分子生物学研究

目的 探讨糖蛋白(GP)Ⅸ基因点突变G2113→A在导致巨血小板综合征(BSS)中的意义。方法 用限制性内切酶分析患和其直系亲属以及40名正常人等位基因；采用定点诱变技术构建含有GPⅨ点突变G2113→A的质粒PD-Ⅸ G2113A；用含有GPⅠ bα GP Ⅰ bβ和GPⅨ或GPⅨ突变型全长编码序列的质粒对中国仓鼠卵巢(CHO)细胞共转染；流式细胞仪检测转染后的CHO细胞表面GP的表达；免疫染色和免疫印迹分析转染后的CHO细胞胞浆中GPⅠ bα和GPⅨ的表达。结果 先证为纯合子，母亲和兄长均为杂合子；突变型CHO细胞膜上GPⅨ和GPⅠbα的表达显减少，但大量存在于细胞胞浆中。结论 本例BSS患的发病机制为GPⅨ Ala139(GCC)→Thr(ACC)突变。该突变不影响GP Ⅰb/Ⅸ在细胞内的合成与组装，但影响其在细胞膜表面的锚定与表达。

Molecular biological study of glycoprotein Ⅸ gene defect in Bernard-Soulier syndrome

OBJECTIVE: To identify a mutation G2113-->A in the glycoprotein (GP)IX gene associated with Bernard-Soulier syndrome (BSS) and to investigate BSS pathogenesis. METHODS: Allele-specific restriction enzyme was used to analyze the samples of patient, her mother, her brother and 40 healthy volunteers. Site-directed mutagenesis was performed to construct a expression vector PD-IXG2113A harboring the mutation G2113-->A. Chinese hamster ovary (CHO) cells were transiently cotransfected with plasmids harboring the entire coding region of GPIbalpha, GPIbeta and GPIX or mutant GPIX, respectively. Expression of GPIbalpha and GPIX in transfected CHO cells were analysed with flow cytometer. GPIbalpha and GPIX in the cytoplasma of transfected CHO cells were analysed by immunostaining and Western blotting. RESULTS: The patient was found to be homozygosity of the substitution, her mother and her brother be heterozygous. Expressions of GPIbalpha and GPIX in mutant CHO cells were remarkably reduced, but abundant in the cytoplasma. CONCLUSION: The mutation of Ala139(GCC)-->Thr(ACC) in the GPIX did not affect synthesis and assembly of GPIb/IX complex but influence its anchoring and expression on the cell surface, which was responsible for BSS.