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.