一例A3血型样本的分子机制

摘要

Objective To explore the molecular mechanism for a blood sample with mixed-field hemagglutination upon determination of ABO blood group.Methods Serological techniques were employed to identify the erythrocyte phenotype.The A and B antigens were detected by flow cytometry.The preliminary genotype of ABO gene was assayed with sequence-specific primer-polymerase chain reaction (PCR-SSP).Exons 6 and 7 of the ABO gene were amplified with PCR and analyzed by direct sequencing.Haplotypes of the ABO gene were analyzed by cloning sequencing as well.Results The serological reaction pattern has supported an O phenotype when all the tubes were centrifuged for the first time.However, a mixed-field hemagglutination of red blood cells (RBCs) with anti-A antibodies was present after the tube was centrifuged five times later.A antigens were detected on the surface of partial red blood cells of the sample by flow cytometry.PCR-SSP results have shown that the preliminary ABO genotype was A/O.Analysis of the fragments of exons 6 and 7 of the ABO gene has indicated that heterozygosis lied as follows:261G/A, 425T/T, 467C/T, 646A/T, 681A/G, 745C/T, 771C/T, 829A/G, conjecturing the genotype to be A307/O02, which was confirmed by haplotype sequence analysis.Compared with A101 allele, A307 allele has two missense mutations, 467C＞ T and 745C＞ T, which have resulted in substitutions Pro156Leu and Arg249Trp in the A glycosyltransferase polypeptide chain.Conclusion A variant allele (A307) has been identified for the first time in mainland China, which is responsible for the formation of A3 phenotype.%目的 研究1例ABO定型时出现混合外观凝集特征个体的分子遗传机制.方法 应用血清学方法和流式细胞术鉴定其ABO红细胞表型,序列特异性引物聚合酶链反应(sequence-specific primerpolymerase chain reaction,PCR-SSP)方法进行ABO基因型的初步检测,对ABO基因第6、7外显子进行聚合酶链反应和DNA序列分析,并进一步通过克隆测序法鉴定ABO基因单倍型.结果 样本在首次离心时正定型呈现O型的血清学表型,多次离心后红细胞与抗A血清出现混合凝集外观;红细胞表面ABO抗原的流式细胞术检测结果显示仅有部分红细胞表达A抗原;PCR-SSP检测结果显示ABO基因型为A/O,ABO基因第6、7外显子测序结果的多态性位点碱基分布为261G/A、425T/T、467C/T、646A/T、681A/G、745C/T、771C/T、829A/G,单倍型序列分析结果为A307/O02杂合子,A307等位基因与A101相比,在第7外显子的467C＞T、745C＞T错义突变,导致A糖基转移酶多肽链Pr0156Leu、Arg249Trp替换.结论 在中国大陆人群中首次检出一种A变异型的等位基因A307,是引发A3血清表型的重要原因.