一例ABO血型系统B抗原减弱表达的分子机制

Molecular mechanism of an individual with weaken B phenotype in ABO blood group

目的研究1例B抗原减弱献血者的血清学特性和抗原减弱的分子机制。方法应用单克隆抗体检测献血者红细胞ABO血型抗原，标准A、B、O红细胞检测其血清中的ABO抗体，并检测其血清转移酶活性。采用聚合酶链反应技术扩增ABO基因全部外显子序列和5′端非编码区序列并进行测序分析，应用逆转录-PCR和克隆测序技术分析献血者ABO cDNA转录剪接情况，采用重亚硫酸盐转化直接测序法分析ABO基因启动子区CpG岛甲基化水平。结果献血者血清学表现为B抗原明显减弱，血清中无抗-B抗体，B转移酶活性降低。基因型为B101／O01，全部外显子序列和拼接接受位点碱基无任何突变。5′端非编码区序列多态性符合B101／O01的特征，启动子、增强子、负调控序列和微卫星重复序列未发现异常。献血者存在ABO基因全长cDNA转录本，未发现新的转录剪接体。与正常对照标本比较，在启动子区CpG岛内发现启动子附近有多个特异性半甲基化位点。结论启动子区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 elucidate the serological characteristics and molecular mechanism of a blood donor with weaken B antigen. Methods The ABO blood group antigens on red blood cells were identified by monoclonal antibodies, the ABO antibodies in serum were detected by standard A, B, O cells and the activity of the B glycosyltransferase was analyzed. The full-length sequence and 5Cuntranslated region （5C UTR） sequence of ABO gene were amplified by polymerase chain reaction （PCR） respectively and direct sequencing. The alternative splicing isoforms of ABO cDNA were obtained by reverse transcription-PCR （RT-PCR） and analyzed with cloning and sequencing techniques. The level of methylation of the CpG island in ABO gene promoter was analyzed by bisulfite sequencing method. Results The serological characteristic of the donor showed that the B antigen was decreased obviously without anti-B antibodies in serum and the B giycosyltransferase activity was decreased as well. The genotype of the donor was B101/O01 without any other mutations in the full-length coding sequences and splice receptor sites. The nucleotide characteristics of the 5′-UTR was consistent with B101/O01 and no any abnormity was identified in the promoter, enhancer and the negative 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, a number of methylated CpG sites were found near the promoter of ABO gene in this sample. Conclusion The methylation in the CpG island of ABO gene promoter region may cause weak expression of the B antigen.