中国输血杂志2025，Vol.38Issue(7)：873-878,895,7.DOI:10.13303/j.cjbt.issn.1004-549x.2025.07.002
人ITGB3基因c.598G/A突变体外表达分析及其与新生儿同种免疫性血小板减少症发生的关系探讨
In vitro expression analysis of the ITGB3 c.598G/A mutation and its association with FNAIT
摘要
Abstract
Objective To explore the role of the c.598G>A mutation of the ITGB3 gene in the occurrence of fetal and neonatal alloimmune thrombocytopenia(FNAIT)through its expression in vitro.Methods The platelet antibodies in the sera of the affected neonate and her mother were detected using commercial enzyme-linked immunosorbent assay(ELISA),solid-phase agglutination,flow cytometry and the gold standard monoclonal antibody-specific immobilization of platelet anti-gens(MAIPA).The common human platelet antigen(HPA)genotypes of the neonate and her parents were obtained using the HPA-SSP method.The presence of mutations was analyzed by sequencing the exons of the ITGB3 and ITGA2B genes.The target gene of ITGB3 was obtained by PCR amplification using the existing human platelet cDNA.The wild-type ITGB3 eukaryotic expression vector was constructed by TA cloning technology.The 598G>A mutant ITGB3 eukaryotic expression vector was obtained by point mutation,and the plasmid DNA was co-transfected with that of ITGA2B(αⅡb)into HEK293 cells.The transfected cells stably expressing GPⅡb/Ⅲa were screened and obtained.The expression of GPⅡb/Ⅲa in 598G>A mutant transfected cells and the presence of antibodies against this mutation in the serum of mother were detected by flow cytometry and MAIPA.Results Antibodies against HLA-class Ⅰ and GPⅡb/Ⅲa glycoproteins were detected in the serum of the neonate's mother,and subsequent HLA antibody-specific testing confirmed the presence of antibodies a-gainst HLA-B∗57∶01 and A∗02∶05.ITGB3 sequencing showed that the neonate and her father carried the c.598G>A point mutation,which results in the change of glutamate to lysine at position 200.Antibodies against GPⅡb/Ⅲa glycoproteins were not detected using constructed c.598G>A mutant transfected cells reacted with the maternal serum.Conclusion The in vitro expression and analysis of the ITGB3 c.598G>A mutation did not support a role for this mutation in the pathogenesis of FNAIT.The establishment of this method facilitates the discovery of new platelet low-frequency antigens,and provides a theoretical foundation for the detection of antibodies against platelet antigens associated with patients with adverse pregnancy and childbirth histories.关键词
ITGB3基因/血小板膜蛋白GPⅢa/FNAIT/体外表达Key words
ITGB3 gene/Glycoprotein Ⅲa/fetal and neonatal alloimmune thrombocytopenia(FNAIT)/expression system in vitro分类
医药卫生引用本文复制引用
丁浩强,叶欣,徐秀章,夏文杰,邓晶,刘静,陈扬凯,陈大伟,许耀日..人ITGB3基因c.598G/A突变体外表达分析及其与新生儿同种免疫性血小板减少症发生的关系探讨[J].中国输血杂志,2025,38(7):873-878,895,7.基金项目
广州市科技计划市校(院)企联合资助项目(2024A03J0374,2025A03J3369,2024A03J0375) （院）
广州市血小板遗传免疫研究重点实验室 （）
广州市医学重点学科(2025-2027) （2025-2027）
