B弱亚型表达调控的分子机制研究

Molecular Mechanism of Expression Regulatory in B Weak Subgroup

目的研究B抗原减弱血液标本的血清学特性和抗原表达减弱的分子机制。方法选择2007年1月至2010年12月于本中心行常规ABO血型检测样本中，B抗原表达减弱的血液样本10份作为研究对象，纳入研究组；计算机随机选择5份正常B表现型的血液样本作为对照组。采用经典血清学技术检测红细胞AB0血型，聚合酶链反应（PCR）技术扩增AB0基因全部外显子序列和5′端调控（CBF／NF-Y增强子）序列，并进行测序分析，应用逆转录-PCR（RT-PCR）技术分析AB0cDNA转录剪接情况，采用重亚硫酸盐转化和克隆测序法分析AB0基因启动子区CpG岛甲基化水平。结果研究组标本的血清学表现为B抗原明显减弱，全部外显子编码序列和拼接位点碱基无任何突变。ABO基因增强子未发现异常，存在着ABO基因全长cDNA转录本，未发现新的转录剪接体。与对照组比较，研究组10份标本在启动子区CpG岛均呈现不同程度的甲基化，其中6个标本在nt-26序列位点表现半甲基化状态。结论ABO基因启动子区CpG岛中的甲基化位点可能是引起B抗原弱表达的原因。

Objective To study the serological characteristics and molecular mechanism of blood samples with weaken B antigen. Methods From January 2007 to December 2010, a total of 10 blood samples with weak B antigen which were chosen from conventional ABO blood samples tested blood samples, were included in this study as study group. And a total of 5 blood samples with normal B antigen expression were selected as control group by computer randomly. All the samples were detected ABO blood group by standard serum technique. The whole code sequences and 5＇ regulatory region containing the CBF/NF-Y enhancer of ABO gene were amplified by polymerase chain reaction （PCR） respectively and direct sequencing. The alternative splicing isoforms of ABO cDNA were analyzed by reverse transcription-PCR （RT-PCR）. The level of methylation of the CpG island in ABO gene promoter was further analyzed by bisulfite and cloning sequencing method. Results The serological characteristic of these samples showed that B antigen was obviously decreased. There was no mutation in the full-length coding sequences and splice receptor sites. The nucleotide characteristics of the 5′-UTR was consistent with common allele type and none abnormity was identified in the promoter, enhancer and regulatory sequence regions. The integrative cDNA transcript of ABO gene was obtained and no new splicing isoform was found. Compared with the normal B phenotype samples, 10 samples of study group all showed different methylation levels on the promoter CpG sites of ABO gene and nt-26 residues of 6 samples were partially methylated. Conclusions The methylation in the CpG island of ABO gene promoter region may cause weak expression of the B antigen.