title: Impaired hematopoiesis in paroxysmal nocturnal hemoglobinuria/aplastic anemia is not associated with a selective proliferative defect in the glycosylphosphatidylinositol-anchored protein-deficient clone.
abstract: Paroxysmal nocturnal hemoglobinuria (PNH) results from somatic mutations in the PIG-A gene, leading to poor presentation of glycosylphosphatidylinositol (GPI)-anchored surface proteins. PNH frequently occurs in association with suppressed hematopoiesis, including frank aplastic anemia (AA). The relationship between GPI-anchored protein expression and bone marrow (BM) failure is unknown. To assess the hematopoietic defect in PNH, the numbers of CD34+ cells, committed progenitors (primary colony-forming cells [CFCs]), and long-term culture-initiating cells (LTC-ICs; a stem cell surrogate) were measured in BM and peripheral blood (PB) of patients with PNH/AA syndrome or patients with predominantly hemolytic PNH. LTC-IC numbers were extrapolated from secondary CFC numbers after 5 weeks of culture, and clonogenicity of LTC-ICs was determined by limiting dilution assays. When compared with normal volunteers (n = 13), PNH patients (n = 14) showed a 4.7-fold decrease in CD34+ cells and an 8.2-fold decrease in CFCs. LTC-ICs in BM and in PB were decreased 7.3-fold and 50-fold, respectively. Purified CD34+ cells from PNH patients had markedly lower clonogenicity in both primary colony cultures and in the LTC-IC assays. As expected, GPI-anchored proteins were decreased on PB cells of PNH patients. On average, 23% of monocytes were deficient in CD14, and 47% of granulocytes and 58% of platelets lacked CD16 and CD55, respectively. In PNH BM, 27% of CD34+ cells showed abnormal GPI-anchored protein expression when assessed by CD59 expression. To directly measure the colony-forming ability of GPI-anchored protein-deficient CD34+ cells, we separated CD34+ cells from PNH patients for the GPI+ and GPI-phenotype; CD59 expression was chosen as a marker of the PNH phenotype based on high and homogeneous expression on fluorescent staining. CD34+ CD59+ and CD34+ CD59-cells from PNH/AA patients showed similarly impaired primary and secondary clonogeneic efficiency. The progeny derived from CD34+ CD59- cells were both CD59- and CD55-. A very small population of CD34+ CD59- cells was also detected in some normal volunteers; after sorting, these CD34+ CD59- cells formed normal numbers of colonies, but their progeny showed lower CD59 levels. Our results are consistent with the existence of PIG-A-deficient clones in some normal individuals. In PNH/AA, progenitor and stem cells are decreased in number and function, but the proliferation in vitro is affected similarly in GPI-protein-deficient clones and in phenotypically normal cells. As measured in the in vitro assays, expansion of PIG-A- clones appears not be caused by an intrinsic growth advantage of cells with the PNH phenotype.
authors.affiliation: Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.
authors.forename: J P
authors.initials: JP
authors.lastname: Maciejewski
authors.forename: E M
authors.initials: EM
authors.lastname: Sloand
authors.forename: T
authors.initials: T
authors.lastname: Sato
authors.forename: S
authors.initials: S
authors.lastname: Anderson
authors.forename: N S
authors.initials: NS
authors.lastname: Young
mesh_terms: D000293:Adolescent; D000328:Adult; D000741:Anemia, Aplastic / Q000473:pathology / Q000503:physiopathology*; D018952:Antigens, CD34 / Q000032:analysis; D001853:Bone Marrow / Q000473:pathology; D018957:CD59 Antigens / Q000032:analysis; D002452:Cell Count; D002455:Cell Division; D002478:Cells, Cultured; D002999:Clone Cells / Q000473:pathology*; D005260:Female; D017261:Glycosylphosphatidylinositols / Q000172:deficiency*; D006410:Hematopoiesis; D006457:Hemoglobinuria, Paroxysmal / Q000473:pathology / Q000503:physiopathology*; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000172:deficiency / Q000235:genetics; D008875:Middle Aged; D009190:Myelodysplastic Syndromes / Q000473:pathology / Q000503:physiopathology
chemical_list: D018952:Antigens, CD34; D018957:CD59 Antigens; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; C080086:phosphatidylinositol glycan-class A protein