p.Arg352Gln突变对Bw07转移酶影响的研究

Studies on the effect of the p.Arg352Gln mutation on Bw07 transferase

目的探讨1例ABw07亚型先证者所携带的Bw07等位基因及其转移酶改变的分子生物学机制。方法选取1例2岁男性患儿为研究对象。先证者及其父母的外周血采用试管法鉴定ABO血型,并对三者进行ABO亚型PCR-SSP检测、ABO基因测序以确定其血型基因型,最后通过模拟突变、DUET结构预测、分子动力学分析和体外细胞实验验证p.Arg352Gln突变对Bw07转移酶的影响。结果先证者及其母亲血清学表型分别为ABw和Bw,父亲为正常的A型,ABO亚型PCR-SSP检测确定三者基因型分别为Bw07/A、Bw07/O、A/A,Sanger测序进一步验证了先证者及其母亲携带有Bw07基因,模拟突变显示R352Q突变后分子间作用力减弱,DUET预测该p.Arg352Gln突变可以影响Bw07转移酶的热力学稳定性,分子动力学分析证实了热力学稳定性的改变主要是与125-133、193-198、336-354区域氨基酸骨架原子出现较大的波动性有关,体外细胞实验进一步验证了Bw07转移酶合成的抗原减弱。结论ABw07亚型的形成与Bw07转移酶上高度保守的Arg352突变成Gln有关。

Objective To investigate the molecular biological mechanism of Bw07 allele and its transferase alteration carried by a proband of ABw07 subtype.Methods A 2-year-old male child was selected as the research object.The peripheral blood of the proband and his parents was identified for ABO blood type by the test tube method,and the ABO subgroup PCR-SSP detection and ABO gene sequencing were performed on the three individuals to determine their blood type genotypes.Finally,the effect of the p.Arg352Gln mutation on Bw07 transferase was verified by virtual mutation,DUET structure prediction,molecular dynamics analysis,and in vitro cellular experiments.Results The serological phenotypes of the proband and his mother were ABw and Bw,respectively,while his father was normal A.The ABO subgroup PCR-SSP assay identified the three genotypes as Bw07/A,Bw07/O,and A/A,respectively.Sanger sequencing further verified that the proband and his mother carried the Bw07 gene,and virtual mutation showed that the intermolecular forces were weakened by the R352Q mutation.DUET predicted that this p.Arg352Gln mutation could affect the thermodynamic stability of Bw07 transferase.Molecular dynamics analysis confirmed that the alteration of thermodynamic stability was mainly related to the appearance of large fluctuations in the amino acid backbone atoms in the 125-133,193-198 and 336-354 regions,and in vitro cellular experiments further verified the weakened antigen synthesis of Bw07 transferase.Conclusion The formation of the ABw07 phenotype is associated with the mutation of the highly conserved Arg352 to Gln in Bw07 transferase.