title: Circulating PIG-A mutant T lymphocytes in healthy adults and patients with bone marrow failure syndromes.
abstract: OBJECTIVE
  Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal hematological disorder with acquired PIG-A gene mutations and absent surface expression of proteins utilizing glycosylphosphatidylinositol (GPI) anchors. PNH often follows aplastic anemia, suggesting PIG-A mutant cells have relative dominance over normal hematopoietic cells. Somatic PIG-A mutations could arise after aplasia, or healthy persons could have rare PIG-A mutant cells that expand under selection pressure.
  
  
  METHODS
  We developed an in vitro negative selection method to isolate GPI-deficient T lymphocytes using aerolysin, an Aeromonas toxin that binds GPI anchors and induces cell lysis. Peripheral blood mononuclear cells (PBMC) from normal adults and patients with PNH or other bone marrow failure syndromes were analyzed.
  
  
  RESULTS
  From healthy adults, 166 T lymphocyte clones with deficient GPI-linked surface protein expression (CD55, CD59) were isolated. The mean mutant frequency (M(f)) of aerolysin-resistant clones was 17.8 +/- 13.8 per 10(6) PBMC, range 5.0-59.6 per 10(6) cells. Clones had a Class A complementation defect and distinct PIG-A mutations. Patients with PNH had elevated aerolysin-resistant M(f) values averaging 19 x 10(-2), a 10,000-fold difference. Two patients with Fanconi anemia and two others with mild aplastic anemia had M(f) values less than 15 x 10(-6), but two with recovering aplastic anemia had M(f) values of 20 x 10(-4), representing an intermediate value between normal persons and PNH patients.
  
  
  CONCLUSIONS
  Identification of PIG-A mutant T lymphocytes in healthy adults suggests PNH could develop following intense negative selection of hematopoiesis, with clonal outgrowth of naturally occurring PIG-A mutant stem cells.
authors.affiliation: Division of Hematology/Oncology, Department of Pediatrics, Duke University Medical Center, Durham, NC 27710, USA. ware0005@mc.duke.edu
authors.forename: R E
authors.initials: RE
authors.lastname: Ware
authors.forename: C V
authors.initials: CV
authors.lastname: Pickens
authors.forename: C M
authors.initials: CM
authors.lastname: DeCastro
authors.forename: T A
authors.initials: TA
authors.lastname: Howard
mesh_terms: D000328:Adult; D000741:Anemia, Aplastic / Q000235:genetics*; D015703:Antigens, CD / Q000032:analysis; D001427:Bacterial Toxins / Q000494:pharmacology; D001855:Bone Marrow Diseases / Q000235:genetics / Q000276:immunology*; D002469:Cell Separation / Q000379:methods; D002999:Clone Cells; D018076:DNA, Complementary / Q000235:genetics; D005816:Genetic Complementation Test; D017261:Glycosylphosphatidylinositols / Q000172:deficiency; D006457:Hemoglobinuria, Paroxysmal / Q000235:genetics*; D006801:Humans; D008565:Membrane Proteins / Q000172:deficiency / Q000235:genetics*; D009154:Mutation; D009190:Myelodysplastic Syndromes / Q000235:genetics / Q000276:immunology*; D052899:Pore Forming Cytotoxic Proteins; D012016:Reference Values; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000276:immunology*
chemical_list: D015703:Antigens, CD; D001427:Bacterial Toxins; D018076:DNA, Complementary; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D052899:Pore Forming Cytotoxic Proteins; C080086:phosphatidylinositol glycan-class A protein; C032676:aerolysin