A novel ABO splice site variant underlying the A₃ phenotype: immunogenetic basis and functional dissection

Accurate ABO blood group typing is essential for transfusion safety. However, ABO subtypes resulting from genetic variation can complicate this process. In contrast to the extensively studied exonic variants in the ABO gene, splice-site variants are rarely reported, and the mechanisms underlying their contribution to ABO subtypes remain poorly characterized.

Four unrelated, healthy blood donors exhibiting mixed-field agglutination with anti-A reagents were subjected to Sanger sequencing. Long-range PCR coupled with nanopore sequencing was further performed to resolve the phased haplotype across the entire ABO locus. The impact of the identified variant on splicing was predicted in silico and validated by an in vitro minigene assay; its effects on protein structure were assessed using bioinformatic tools. Short tandem repeat (STR) analysis was also conducted to exclude blood group chimerism.

Four unrelated Chinese blood donors exhibiting mixed-field agglutination with anti-A reagents were identified. Sanger sequencing and nanopore sequencing revealed that all four probands harbored a novel c.239 + 6T>C variant in the ABO*A1.01 allele, with short tandem repeat (STR) analysis ruling out chimerism as the cause of the mixed-field agglutination. In silico analysis predicted activation of a cryptic splice site, leading to retention of a 55-bp intronic fragment in the mRNA transcript. Further, the minigene splicing assay validated this predicted aberrant splicing pattern and additionally revealed trace production of the normally spliced transcript. Secondary and three-dimensional structural modeling further predicted that the resulting aberrant transcript encodes a severely truncated and catalytically compromised glycosyltransferase A, providing a molecular basis for the observed A antigen expression defect.

In conclusion, a novel ABO variant (c.239 + 6T>C) responsible for the A₃ phenotype was identified, and the mechanism underlying this phenotype was elucidated. By elucidating how a splice site variant disrupts glycosyltransferase function and alters antigen expression, this study contributes to a deeper understanding of the immunogenetic basis of ABO variation, with implications for transfusion safety and personalized immunohematology.