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

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 + 3A>G and c.155 + 1G>T 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.