Genetic and mechanistic evaluation for the weak A phenotype in A_el blood type with IVS6 + 5G>A ABO gene mutation

Background and Objectives: A_el is a rare blood type that is characterized by weak agglutination of RBCs when reacts with anti-A antibody in adsorption-elution test. Although IVS6 + 5G→A mutation is known to associate with the A_el blood type, genetic and mechanistic evaluation for the weak agglutination of A_el with IVS6 + 5G→A mutation has not yet been completely addressed. Materials and Methods: In this study, five cases of confirmed A_el individuals were analysed. The cDNAs for the A^el alleles were obtained by cloning method for sequence analyses. The erythroleukemia K562 cells were used as the cell study model and were transfected with the A^el expression construct. Flow cytometry analysis was then performed to determine the levels of surface antigen expression. Results: The results indicated that IVS6 + 5G→A attributes to all cases of A_el. RT-PCR analyses revealed the presence of at least 10 types of aberrant A^el splicing transcripts. Most of the transcripts caused early termination and produced non-functional protein during translation. Nevertheless, the transcript without exons 5-6 was predicted to generate functional A_el glycosyltransferase lacking 57 amino acids at the N-terminal segment. When the exons 5-6 deletion transcript was stably expressed in the K562 cells, weak agglutination of the cells can be induced by adding anti-A antibody followed by adsorption-elution test. Conclusion: This study demonstrates that aberrant splicing of A transcripts contributes to weak A expression and the weak agglutination of A_el-RBCs, adding to the complexity for the regulatory mechanisms of ABO gene expression.