Three ABO 3′ splice-site variants including a novel one converge on a common splicing defect to attenuate blood group antigen expression

Background ABO subtypes are characterized by attenuated antigen expression, rendering them susceptible to serological misclassification and posing substantial risks to transfusion safety. Splice-site variants represent one of the mechanisms underlying ABO subtype formation; however, previous mechanistic investigations have predominantly focused on the 5′ splice-site(5′SS), leaving the functional consequences of 3′ splice-site (3′SS) variants largely unexplored. Methods Serological phenotyping of a proband with ABO grouping discrepancy was performed using tube agglutination, microcolumn gel agglutination, and absorption-elution assays. Genotyping was resolved by long-range PCR coupled with Nanopore sequencing. The splicing impact of this novel variant and two previously reported variants, c.29-3C>G and c.29-10T>A, was predicted in silico and validated by in vitro minigene assay. Computational saturation mutagenesis was further deployed to systematically delineate a preliminary framework of regulatory principles governing 3′SS variation. Results A novel 3′SS variant, c.29-2A>G, was identified in an ABO*A1.02 background in a proband with an A el phenotype. This novel variant and two previously reported variants disrupted the canonical 3’SS of intron 1, converging on exon 2 skipping as the predominant aberrant splicing event. Additionally, c.29-10T>A activated a cryptic acceptor site, generating a secondary transcript with an 8-bp intronic retention. All aberrant transcripts disrupt the open reading frame and introduce premature termination codons. Through computational saturation mutagenesis, we further established an initial position- and base-dependent risk stratification model for 3′SS variants. Conclusion This study provides the first direct mechanistic evidence linking ABO 3′SS variants to subtype pathogenesis, demonstrating that exon 2 skipping constitutes a shared molecular lesion across intron 1 3′SS variants, ultimately leading to attenuated blood group antigen expression. The preliminary computationally derived risk stratification framework established herein offers a clinically valuable reference for interpreting 3′SS variants, with implications that extend beyond blood group genetics to the broader landscape of splicing-mediated human diseases.