Introduction
In transfusion medicine and genetic research, accurately analyzing rare blood group subtypes and their molecular mechanisms is crucial. Variants in the ABO blood group system, especially those located in intronic regions, often have unpredictable functional consequences. A recent study using PacBio third-generation sequencing technology discovered a novel ABO gene intronic variant c.28+5G>A in a blood donor with atypical agglutination patterns (Ael phenotype). The RDDC online RNA Splicer tool played a key role in this study, with its accurate prediction of the variant's splicing effects highly consistent with subsequent minigene experimental results, successfully revealing the molecular mechanism underlying the Ael phenotype.
Research Challenge: A "Variant of Uncertain Significance" in an Intron
The study began with a healthy male blood donor exhibiting the Ael phenotype. Serological testing showed extremely weak A antigen expression on his red blood cells. To investigate the genetic basis, the research team employed PacBio long-read sequencing technology and discovered that the donor carried a novel heterozygous variant c.28+5G>A, located in intron 1 of the ABO gene, on an A2.01 allele background.
This is a typical non-canonical splice site variant (+5 position), with unknown specific effects on mRNA splicing, classified as a "variant of uncertain significance" (VUS). To understand the cause of the Ael phenotype, the function of this intronic variant must be elucidated.
RDDC's Accurate Prediction: Providing Verifiable Splicing Patterns
To obtain a clear molecular mechanism hypothesis before investing experimental resources, the research team used the RDDC online RNA Splicer tool to predict the potential splicing consequences of the c.28+5G>A variant. RDDC's analysis results were highly specific, proposing three possible splicing patterns:
- 167 bp intronic sequence insertion: Predicted that the variant would activate a cryptic splice donor site within the intron, causing a 167 bp intronic sequence to be erroneously included in the mature mRNA.
- Normal wild-type splicing: Predicted that some mRNA might still be produced through normal splicing pathways.
- Exon skipping leading to 40 bp deletion.
These predictions, particularly the possibility of coexisting 167 bp insertion and normal splicing, provided highly valuable theoretical foundation for explaining the Ael phenotype (extremely weak A antigen expression).
Perfect Confirmation by Minigene Experiments
The research team promptly validated RDDC's predictions through in vitro minigene experiments. The experimental results powerfully confirmed the core content of RDDC's predictions:
- The
c.28+5G>Avariant indeed abolished the canonical 5' splice donor site and activated a downstream cryptic site, leading to insertion of 167 bp intronic sequence in the mRNA. This aberrant mRNA would translate to produce a truncated, non-functional glycosyltransferase. - Simultaneously, the experiment also detected small amounts of wild-type transcripts produced through normal splicing.
The high consistency between in silico prediction and in vitro experimental results (both 167 bp insertion and normal splicing patterns were confirmed) clarified the molecular mechanism by which the c.28+5G>A variant causes the Ael phenotype through interference with mRNA splicing.
Case Implications
This case once again demonstrates that RDDC RNA Splicer is a powerful tool for analyzing the pathogenic mechanisms of intronic VUS (particularly variants affecting splicing). It can provide accurate, specific splicing pattern predictions, effectively guide subsequent functional experimental design, significantly improve research efficiency and diagnostic accuracy, and provide important support for understanding the genetic basis of rare blood group subtypes.
Content Source and Disclaimer
This article is a compilation and interpretation of the following scientific research, aimed at demonstrating the application of RDDC bioinformatics tools. All research data and conclusions belong to the original authors and publications.
Original Literature:
Ye L, Lin W, Xu X, et al. Characterization of a novel AEL allele harboring a c.28 + 5G>A mutation on the ABO*A2.01 background: a study utilizing PacBio third-generation sequencing and functional assays. Annals of Translational Medicine. 2024 Feb 29;12(4):114.
Literature Link: https://pubmed.ncbi.nlm.nih.gov/39763657/
