Ying Yanling, Hong Xiaozhen, Zhang Jingjing, Ma Kairong, Liu Ying, Xu Xianguo, He Ji, Zhu Faming
Blood Center of Zhejiang Province, Key Laboratory of Blood Safety Research of Zhejiang Province, Hangzhou, Zhejiang 310052, China.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2023 Jan 10;40(1):110-113. doi: 10.3760/cma.j.cn511374-20211217-01003.
To explore the molecular mechanism for an individual with Bweak subtype.
Serological methods were used to identify the proband's phenotype. In vitro enzyme activity test was used to determine the activity of B-glycosyltransferase (GTB) in her serum. The genotype was determined by PCR amplification and direct sequencing of exons 5 to 7 and flanking sequences of the ABO gene. T-A cloning technology was used to isolate the haploids. The primary physical and chemical properties and secondary structure of the protein were analyzed with the ProtParam and PSIPRED software. Three software, including PolyPhen-2, SIFT, and PROVEAN, was used to analyze the effect of missense variant on the protein.
Serological results showed that the proband's phenotype was Bweak subtype with anti-B antibodies presented in her serum. In vitro enzyme activity assay showed that the GTB activity of the subject was significantly reduced. Analysis of the haploid sequence revealed a c.398T>C missense variant on the B allele, which resulted in a novel B allele. The 398T>C variant has caused a p.Phe133S substitution at position 133 of the GTB protein. Based on bioinformatic analysis, the amino acid substitution had no obvious effect on the primary and secondary structure of the protein, but the thermodynamic energy of the variant protein has increased to 6.07 kcal/mol, which can severely reduce the protein stability. Meanwhile, bioinformatic analysis also predicted that the missense variant was harmful to the protein function.
The weak expression of the Bweak subtype may be attributed to the novel allele of ABO*B.01-398C. Bioinformatic analysis is helpful for predicting the changes in protein structure and function.
探讨B弱亚型个体的分子机制。
采用血清学方法鉴定先证者的表型。通过体外酶活性试验测定其血清中B-糖基转移酶（GTB）的活性。采用PCR扩增及对ABO基因第5至7外显子及其侧翼序列进行直接测序来确定基因型。利用T-A克隆技术分离单倍体。使用ProtParam和PSIPRED软件分析蛋白质的一级理化性质和二级结构。使用包括PolyPhen-2、SIFT和PROVEAN在内的三种软件分析错义变异对蛋白质的影响。
血清学结果显示先证者的表型为B弱亚型，其血清中存在抗B抗体。体外酶活性测定表明该受试者的GTB活性显著降低。单倍体序列分析显示B等位基因上存在一个c.398T>C错义变异，导致一个新的B等位基因。398T>C变异导致GTB蛋白第133位发生p.Phe133S替换。基于生物信息学分析，该氨基酸替换对蛋白质的一级和二级结构没有明显影响，但变异蛋白的热力学能量增加到6.07千卡/摩尔，这会严重降低蛋白质稳定性。同时，生物信息学分析还预测该错义变异对蛋白质功能有害。
B弱亚型的弱表达可能归因于ABO*B.01-398C新等位基因。生物信息学分析有助于预测蛋白质结构和功能的变化。
