Tamiko Nakajima1, Rie Sano, Yoichiro Takahashi, Rieko Kubo, Keiko Takahashi, Yoshihiko Kominato, Junichi Tsukada, Haruo Takeshita, Toshihiro Yasuda, Makoto Uchikawa, Kazumi Isa, Kenichi Ogasawara. 1. Department of Legal Medicine, Gunma University Graduate School of Medicine, Maebashi, Japan; Cancer Chemotherapy Center and Hematology, University of Occupational and Environmental Health, Fukuoka, Japan; Department of Legal Medicine, Shimane University School of Medicine, Shimane, Japan; Division of Medical Genetics and Biochemistry, Faculty of Medicine, University of Fukui, Fukui, Japan; Japanese Red Cross Tokyo Blood Center, Tokyo, Japan; Japanese Red Cross Central Blood Institute, Tokyo, Japan.
Abstract
BACKGROUND: The ABO blood group is important in blood transfusion. Recently, an erythroid cell-specific regulatory element has been identified in the first intron of ABO using luciferase reporter assays with K562 cells. The erythroid cell-specific regulatory activity of the element was dependent upon GATA-1 binding. In addition, partial deletion of Intron 1 including the element was observed in genomic DNAs obtained from 111 Bm and ABm individuals, except for one, whereas the deletion was never found among 1005 individuals with the common phenotypes. STUDY DESIGN AND METHODS: In this study, further investigation was performed to reveal the underlying mechanism responsible for reduction of B antigen expression in the exceptional Bm individual. Peptide nucleic acid-clamping polymerase chain reaction was carried out to amplify the B-related allele, followed by sequence determination. Electrophoretic mobility assays and promoter assays were performed to examine whether a nucleotide substitution reduced the binding of a transcription factor and induced loss of function of the element. RESULTS: Sequence determination revealed one point mutation of the GATA motif in the element. The electrophoretic mobility shift assays showed that the mutation abolished the binding of GATA transcription factors, and the promoter assays demonstrated complete loss of enhancer activity of the element. CONCLUSION: These observations suggest that the mutation in the GATA motif of the erythroid-specific regulatory element may diminish the binding of GATA transcription factors and down regulate transcriptional activity of the element on the B allele, leading to reduction of B antigen expression in erythroid lineage cells of the Bm individual.
BACKGROUND: The ABO blood group is important in blood transfusion. Recently, an erythroid cell-specific regulatory element has been identified in the first intron of ABO using luciferase reporter assays with K562 cells. The erythroid cell-specific regulatory activity of the element was dependent upon GATA-1 binding. In addition, partial deletion of Intron 1 including the element was observed in genomic DNAs obtained from 111 Bm and ABm individuals, except for one, whereas the deletion was never found among 1005 individuals with the common phenotypes. STUDY DESIGN AND METHODS: In this study, further investigation was performed to reveal the underlying mechanism responsible for reduction of B antigen expression in the exceptional Bm individual. Peptide nucleic acid-clamping polymerase chain reaction was carried out to amplify the B-related allele, followed by sequence determination. Electrophoretic mobility assays and promoter assays were performed to examine whether a nucleotide substitution reduced the binding of a transcription factor and induced loss of function of the element. RESULTS: Sequence determination revealed one point mutation of the GATA motif in the element. The electrophoretic mobility shift assays showed that the mutation abolished the binding of GATA transcription factors, and the promoter assays demonstrated complete loss of enhancer activity of the element. CONCLUSION: These observations suggest that the mutation in the GATA motif of the erythroid-specific regulatory element may diminish the binding of GATA transcription factors and down regulate transcriptional activity of the element on the B allele, leading to reduction of B antigen expression in erythroid lineage cells of the Bm individual.
Authors: Aoi Wakabayashi; Jacob C Ulirsch; Leif S Ludwig; Claudia Fiorini; Makiko Yasuda; Avik Choudhuri; Patrick McDonel; Leonard I Zon; Vijay G Sankaran Journal: Proc Natl Acad Sci U S A Date: 2016-04-04 Impact factor: 11.205