c.98 + 3A>G and c.155 + 1G>T splice-site variants in the ABO*B.01 allele lead to weak antigen expression in the Chinese individuals

BackgroundMany 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.MethodsABO 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.ResultsThe 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.