ABO血型新等位基因O61的分子生物学研究

本研究探讨ABO新等位基因O61的分子机制。利用单克隆抗体检测标本红细胞ABO血型抗原,标准A、B、O红细胞检测标本血清中的ABO抗体。采用聚合酶链反应(polymerase chain reaction,PCR)技术扩增ABO基因的第5至7外显子序列,PCR产物经双酶切后直接测序分析6和7外显子。1例B表型标本采用基因组单链抽提技术分离杂合基因型标本的2条单体型,对分离的单体型扩增后进行ABO基因测序分析。结果表明,在417例标本中发现3个标本743位碱基发生突变,其中2例表型为O型,1例为B型。2例O型直接测序分析显示,6和7外显子有261G缺失及743G/C杂合。1例B型直接测序结果为261G/缺失及297A/G、526C/G、743G/C、657C/T、703G/A、796C/A、803G/C、930G/A杂合。采用基因组单链抽提技术将B型标本的两条单体型进行分离,对分离的单体型扩增后进行ABO基因测序分析,结果显示,2条链分别为B101和突变的O型。将突变的新O型基因提交血型抗原基因突变数据库,被正式命名为O61。结论:ABO基因7号外显子743GC为1个新的突变点,发现1个ABO新等位基因O61。

Molecular Basis of A New O61 Allele in ABO Blood Group

Objective of this study was to explore the molecular basis of a new O61 allele in ABO blood group.The ABO group antigens on red cells of the blood samples were identified by monoclonal antibodies and the ABO antibody in serum was detected by the standard A,B,O red cells.The coding region sequences of exon 5 to exon 7 in ABO gene were amplified by polymerase chain reaction（PCR） and the amplification products were purified with double enzyme digestion and directly sequenced for exon 6 and 7.The diploid of the individual with B phenotype was separated into its haploid components with a haplotype specific extraction method.The exons 6 to 7 of the two single ABO haplotypes were then amplified and sequenced separately.The results indicated that 3 samples had mutation at 743 site in total 417 individuals,in which 2 individuals were with O phenotype and 1 individual was with B phenotype.The DNA sequencing of exon 6 and 7 in 2 samples with O phenotye showed 261G delation and 743G/C heterozygotes.The DNA sequencing of exon 6 and 7 in the sample with B phenotype showed 261G/delation and 297A/G,526C/G,743G/C,657C/T,703G/A,796C/A,803G/C,930G/A heterozygotes.After separating of the 2 single strands in the B sample with haplotype specific extraction,an O and B101 allele were identified after sequencing.The novel allele was submitted to the Blood Group Antigen Gene Mutation database and is named as O61.It is concluded that 743GC is a novel mutation in exon 7 of ABO and a novel O61 allele with 743GC has been identified.