一个A亚型常见型等位基因的发现和序列分析

Discovery and sequence analysis of a novel allele of A subtype

目的对2例ABO血型正反定型不符的患者血液标本进行基因检测,探讨其血清学特征和分子机制。方法对2例标本采用经典试管法进行血型血清学检测;采用PCR技术扩增ABO基因第6、7外显子,进行直接测序和克隆测序,确定其基因型。结果2例患者标本血清学结果显示:患者红细胞与抗-A,抗-A1和抗-B均不凝集,血清与A1细胞弱凝集,与B细胞呈现强凝集。直接测序和克隆测序结果显示这2例标本的基因型是ABO*O01.01和罕见型A等位基因的杂合子,罕见型A等位基因与ABO*A1.01序列相比在第7外显子第467位C>T变异,导致第156位脯氨酸(Pro)转变为亮氨酸(Leu),963位与964位之间插入了一个核苷酸C。结论结合血清学和基因测序结果,此2例标本为A亚型,引起该罕见型A等位基因的特异性突变为:c.467C>T和c.963_964insC。

Objective To identify the genotypes of two probands with ambiguous blood serological typing and explore their serological feature and molecular basis.Methods Serology assays including absorption-elution test were carried out.With genomic DNA extracted from peripheral blood samples,exons 6 and 7 of the ABO gene were subjected to cloning and haplotype sequencing analyses.Results Red blood cells from the probands showed no agglutination with monoclonal anti-A and anti-B,whilst their serum showed weak agglutination with A1 cells and moderate agglutination with B cells.Absorption-elution test failed in detecting A antigen on such cells.Direct cloning sequencing analysis indicated that they were heterozygous for an ABO*O01.01 allele and a novel A allele.The novel A allele contained c.467C>T(p.Pro156Leu)and insertion of a nucleotide C at position 963_964 in exon 7(c.963_964insC)compared with ABO*A1.01.Conclusion Through serological and sequencing analysis,the two samples were identified as A subgroup due to novel A alleles.