A novel mutation +5904 C>T of RUNX1 site in the erythroid cell-specific regulatory element decreases the ABO antigen expression in Chinese population.

BACKGROUND: An erythroid cell-specific regulatory element (+5·8-kb) in the first intron of ABO is responsible for the antigen differential expression and the regulatory activity of the element was affected by the nucleotide mutation in the +5·8-kb region. Currently, many individuals with ABO subgroups were found in the Chinese population, but there was little information about the function of +5·8-kb region in these individuals. Here, we studied the mechanism of the mutation in the +5·8-kb region responsible for reducing of antigen expression in 30 ABO subtype Chinese individuals without mutation in the coding region or splicing site.

MATERIALS AND METHODS: The nucleotide sequence of the partial intron 1 covering the +5·8-kb site was amplified and directly sequenced. The haplotype with the novel mutation was obtained by the TOPO TA cloning. Both of the ABO promoter and the +5·8 kb regulatory element were subcloned into the basic luciferase reporter plasmid using the double endonuclease digestion. The promoter activity was examined by the dual-luciferase report vector with K562 cells.

RESULTS: A novel nucleotide substitution +5904 C>T located at RUNX1-binding site in the +5·8 kb site was identified from three individuals with B subtypes. +5890 T>G were found in three Bel and one Ael phenotypes. Cotransfection and luciferase assays demonstrated that the +5904 C>T could obviously reduce activity of the +5·8 kb site.

CONCLUSION: The study suggested that the transcriptional activity of the +5·8 kb site could be downregulated by the single point mutation of RUNX1 motif, leading to reduction in A or B antigen expression.