title: PIG-A mutations in normal hematopoiesis.
abstract: Paroxysmal nocturnal hemoglobinuria (PNH) is caused by phosphatidylinositol glycan-class A (PIG-A) mutations in hematopoietic stem cells (HSCs). PIG-A mutations have been found in granulocytes from most healthy individuals, suggesting that these spontaneous PIG-A mutations are important in the pathogenesis of PNH. It remains unclear if these PIG-A mutations have relevance to those found in PNH. We isolated CD34+ progenitors from 4 patients with PNH and 27 controls. The frequency of PIG-A mutant progenitors was determined by assaying for colony-forming cells (CFCs) in methylcellulose containing toxic doses of aerolysin (1 x 10(-9) M). Glycosylphosphatidylinositol (GPI)-anchored proteins serve as receptors for aerolysin; thus, PNH cells are resistant to aerolysin. The frequency of aerolysin resistant CFC was 14.7 +/- 4.0 x 10(-6) in the bone marrow of healthy donors and was 57.0 +/- 6.7 x 10(-6) from mobilized peripheral blood. DNA was extracted from individual day-14 aerolysin-resistant CFCs and the PIG-A gene was sequenced to determine clonality. Aerolysin-resistant CFCs from patients with PNH exhibited clonal PIG-A mutations. In contrast, PIG-A mutations in the CFCs from controls were polyclonal, and did not involve T cells. Our data confirm the finding that PIG-A mutations are relatively common in normal hematopoiesis; however, the finding suggests that these mutations occur in differentiated progenitors rather than HSCs.
authors.affiliation: Johns Hopkins University, School of Medicine, Division of Hematology, Baltimore, MD 21205, USA.
authors.forename: Rong
authors.initials: R
authors.lastname: Hu
authors.forename: Galina L
authors.initials: GL
authors.lastname: Mukhina
authors.forename: Steven
authors.initials: S
authors.lastname: Piantadosi
authors.forename: Jamie P
authors.initials: JP
authors.lastname: Barber
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Jones
authors.forename: Robert A
authors.initials: RA
authors.lastname: Brodsky
mesh_terms: D001427:Bacterial Toxins / Q000494:pharmacology; D001854:Bone Marrow Cells / Q000187:drug effects; D002478:Cells, Cultured; D004351:Drug Resistance; D017261:Glycosylphosphatidylinositols / Q000172:deficiency; D006410:Hematopoiesis / Q000235:genetics*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics*; D009154:Mutation / Q000235:genetics*; D052899:Pore Forming Cytotoxic Proteins; D017422:Sequence Analysis, DNA
chemical_list: D001427:Bacterial Toxins; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D052899:Pore Forming Cytotoxic Proteins; C080086:phosphatidylinositol glycan-class A protein; C032676:aerolysin