Transcriptional Profile of Carbohydrate Blood Group Genes in Erythroid Versus Neutrophil Differentiation of Human CD34+ Cells In Vitro.

Abstract

Carbohydrate blood groups and their corresponding antibodies are clinically important, known to be involved in conditions such as hemolytic transfusion reactions, hemolytic disease of the newborn and spontaneous abortion. However, little is understood about the developmental changes in expression of carbohydrate blood groups during hematopoiesis, with preceding studies mainly focusing on protein-based blood group molecules. We have previously identified the carbohydrate blood group A antigen as the earliest, specific marker for definitive erythroid commitment, in preference to other suggested candidates such as Kell or Glycophorin C [Br J Haematol 2004; 127:451–63]. With regard to this lineage-restriction point we mapped the gene expression of some clinically important carbohydrate blood group systems during erythroid versus neutrophil differentiation in vitro. Human bone marrow CD34+ cells from healthy donors, carrying the blood group A1 allele and functional secretor (FUT2) and Lewis (FUT3) genes, were cultured in vitro towards erythroid or neutrophil development and sorted on a flow cytometer into subpopulations according to their surface expression of blood group A antigen/CD117 and CD15/CD33. Sorted cells were cultured in clonogenic assays in methylcellulose or analyzed by TaqMan real-time reverse transcriptase PCR for gene expression of a number of carbohydrate blood group glycosyltransferases. Surface expression of the blood group A antigen coincided with commitment to erythroid differentiation and the expression of CD15 with neutrophil/monocytic differentiation. In gene expression studies the ABO, H (FUT1), I (IGnT) and Pk (A4GALT) genes were all expressed in freshly isolated and sorted CD34+ cells. The ABO and the H genes were up-regulated in erythroid differentiation and silenced in neutrophil differentiation. The ABO gene expression was markedly decreased in late stages of erythroid maturation. The I gene was expressed both during erythroid and neutrophil development with an increased expression in late erythroid differentiation. The Pk gene retained a low expression throughout neutrophil differentiation and was up-regulated several-fold during erythroid differentiation. There was no detectable expression of FUT3 and the gene suggested being responsible for biosynthesis of the Sda antigen, GALGT2, in either erythroid or neutrophil differentiation.Our data support the identification of the blood group A antigen as an early and specific marker for definitive erythroid differentiation. In mature cells of the myeloid lineage, the results of the gene expression studies are compatible with previous findings of gene and/or surface expression of the I and Pk blood groups but not of ABO and H. The marked increase in expression of the Pk gene during erythroid differentiation may well agree with the fact that the Pk antigen is the precursor structure of globoside, the most abundant neutral glycolipid in the erythrocyte membrane. The absence of hematopoietic FUT3 expression in Lewis gene positive individuals was expected whilst the relevance of undetectable GALGT2 expression in hematopoietic differentiation is uncertain. The role of the GALGT2 gene in surface expression of Sda has not been definitively proven and the molecular basis of different Sda phenotype variants is unknown.In conclusion, our data extend previous findings of carbohydrate blood group distribution, primarily obtained from mature blood cells and leukemic cell lines, to normal human hematopoiesis.