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目的:对1例有血小板膜糖蛋白(GP)Ⅰb/Ⅸ缺失的巨大血小板综合征(BSS)患者的GPⅠb/Ⅸ的基因进行系统的研究。方法:用PCR扩增GPⅠbα.GPⅠbβ与GPⅨ基因全长编码序列并做DNA序列分析。用限制性内切酶HPYCH4Ⅲ对GP Ⅸ的基因片断做酶切位点分析。用定点诱变技术制备突变质粒并通过脂质体转染技术转染至中国仓鼠卵巢(CHO)细胞。用流式细胞术比较野生型与突变CHO细胞表面的GPⅠb/Ⅸ表达。结果:在GP Ⅸ cDNA的688 bp位置发现G→A突变,导致CPⅨ跨膜区139 Ala(GCC)被Thr(ACC)取代。该突变为一种国际上尚未报道的BSS新的基因突变。限制性酶切位点分析显示患者为纯合子,而家庭的其它成员为杂合子。野生型细胞表面有大量的GPⅠb/Ⅸ,但突变型细胞几乎无GPⅠb/Ⅸ表达。结论:研究表明本例BSS患者的发病机理为GP Ⅸ Ala139(GCC)→Thr(ACC)突变,并提出GP Ⅸ跨膜区结构对维持GPⅠb/Ⅸ在血小板正常表达与可能中的重要意义。
OBJECTIVE: To study the GPⅠb / Ⅸ gene in a patient with giant platelet syndrome (BSS) with deletion of platelet glycoprotein (GP) Ⅰb / Ⅸ. Methods: The full-length coding sequence of GPⅠbα.GPⅠbβ and GPⅨ was amplified by PCR and analyzed by DNA sequence. Restriction endonuclease HPYCH4 Ⅲ GP Ⅸ gene fragments do restriction enzyme analysis. Mutagenized plasmids were prepared by site-directed mutagenesis and transfected into Chinese hamster ovary (CHO) cells by lipofection techniques. GPIb / IX expression on wild-type and mutant CHO cells was compared by flow cytometry. Results: The G → A mutation was found at the position of 688 bp of GP Ⅸ cDNA, resulting in the replacement of 139 Ala (GCC) in CPⅨ by Thr (ACC). This mutation is a new gene mutation in BSS that has not been reported in the world. Restriction enzyme analysis showed that the patient was homozygous, while the rest of the family was heterozygous. The wild-type cells have a large number of GPⅠb / Ⅸ on the surface, but the mutant cells have almost no GPⅠb / Ⅸ expression. CONCLUSION: The study shows that the pathogenesis of BSS in this case is GP Ⅸ Ala139 (GCC) → Thr (ACC) mutation. It is also suggested that the structure of GP Ⅸ transmembrane region plays an important role in maintaining the normal expression of GP Ⅰ b / Ⅸ in platelets.