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

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.

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.

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.

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.