Three ABO 3′ splice-site variants including a novel one converge on a common splicing defect to attenuate blood group antigen expression
- 1Blood Grouping Reference Laboratory, Chengdu Blood Center , , ,
Abstract
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.
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BibTeX
@article{guan2026three,
title = {{Three ABO 3′ splice-site variants including a novel one converge on a common splicing defect to attenuate blood group antigen expression}},
author = {Hongli Guan and Yalan Yang and Jian Li and Huan Zhang and Xue Chen and Jialiang Gao},
journal = {Frontiers in immunology},
year = {2026},
month = oct,
doi = {10.3389/fimmu.2026.1959480},
url = {https://europepmc.org/article/PMC/PMC13672005},
note = {CC BY},
}
RIS
TY - JOUR
TI - Three ABO 3′ splice-site variants including a novel one converge on a common splicing defect to attenuate blood group antigen expression
AU - Hongli Guan
AU - Yalan Yang
AU - Jian Li
AU - Huan Zhang
AU - Xue Chen
AU - Jialiang Gao
PY - 2026
DA - 2026/10/02
JO - Frontiers in immunology
DO - 10.3389/fimmu.2026.1959480
UR - https://europepmc.org/article/PMC/PMC13672005
AB - 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.
SN - 1664-3224
ER -