Background Many variants within the ABO gene across multiple subtypes have been identified. These variants are located in the coding region, the erythroid-specific regulatory element region of intron 1, splice sites, and other non-coding regions. However, data regarding the functional analysis of these variants remain limited. In this study, we analyzed the function of two splice-site variants in ABO subtypes. Methods ABO phenotyping was performed using conventional serology methods. The entire coding sequence of the ABO gene was characterized by polymerase chain reaction sequence-based typing, while haplotypes were determined using one-step long-range PCR combined with single-molecule real-time sequencing. The functional impact of splice-site variants was predicted using in silico tools and subsequently verified in vitro using a minigene splicing assay. Furthermore, stable cell lines expressing ABO cDNA with exon 2 or 3 deletions were established to evaluate their effects on antigen expression utilizing serology, flow cytometry, and glycosyltransferase activity assays. Results The c.98 + 3 heterozygous variants were identified in Bw and ABw individuals, while the c.155 + 1 heterozygous variant was identified in an ABw individual. In all three individuals, these splice-site variants could be assigned to the ABO*B.01 allele by haplotype analysis. These variants affected splicing as predicted by in silico analysis, resulting in exons 2 or 3 skipping, as demonstrated by minigene assays. B antigen expression was significantly weakened in cells transfected in vitro with ABO*B.01 cDNA containing deletions in exons 2 or 3, as analyzed by cytometry and serological methods. In addition, no enzymatic activity was detected in the supernatant of these transfected cells. Conclusions The c.98 + 3AG and c.155 + 1GT variants within the ABO gene may induce aberrant mRNA splicing, leading to skipping of exons 2 or 3, and resulting in the formation of a nonfunctional glycosyltransferase.
Zhang et al. (2026) studied this question.