The small population of PIG-A mutant cells in myelodysplastic syndromes do not arise from multipotent hematopoietic stem cells.
BACKGROUND
Patients with paroxysmal nocturnal hemoglobinuria harbor clonal glycosylphosphatidylinositol-anchor deficient cells arising from a multipotent hematopoietic stem cell acquiring a PIG-A mutation. Many patients with aplastic anemia and myelodysplastic syndromes also harbor small populations of glycosylphosphatidylinositol-anchor deficient cells. Patients with aplastic anemia often evolve into paroxysmal nocturnal hemoglobinuria; however, myelodysplastic syndromes seldom evolve into paroxysmal nocturnal hemoglobinuria. Here, we investigate the origin and clonality of small glycosylphosphatidylinositol-anchor deficient cell populations in aplastic anemia and myelodysplastic syndromes.


METHODS
We used peripheral blood flow cytometry to identify glycosylphosphatidylinositol-anchor deficient blood cells, a proaerolysin-resistant colony forming cell assay to select glycosylphosphatidylinositol-anchor deficient progenitor cells, a novel T-lymphocyte enrichment culture assay with proaerolysin selection to expand glycosylphosphatidylinositol-anchor deficient T lymphocytes, and PIG-A gene sequencing assays to identify and analyze PIG-A mutations in patients with aplastic anemia and myelodysplastic syndromes.


RESULTS
Twelve of 15 aplastic anemia patients were found to harbor a small population of glycosylphosphatidylinositol-anchor deficient granulocytes; 11 of them were found to harbor a small population of glycosylphosphatidylinositol-anchor deficient erythrocytes, 10 patients were detected to harbor glycosylphosphatidylinositol-anchor deficient T lymphocytes, and 3 of them were detected only after T-lymphocyte enrichment in proaerolysin selection. PIG-A mutation analyses on 3 patients showed that all of them harbored a matching PIG-A mutation between CFU-GM and enriched T lymphocytes. Two of 26 myelodysplastic syndromes were found to harbor small populations of glycosylphosphatidylinositol-anchor deficient granulocytes and erythrocytes transiently. Bone marrow derived CD34(+) cells from 4 patients grew proaerolysin-resistant colony forming cells bearing PIG-A mutations. No glycosylphosphatidylinositol-anchor deficient T lymphocytes were detected in myelodysplastic syndrome patients.


CONCLUSIONS
In contrast to aplastic anemia and paroxysmal nocturnal hemoglobinuria, where PIG-A mutations arise from multipotent hematopoietic stem cells, glycosylphosphatidylinositol-anchor deficient cells in myelodysplastic syndromes appear to arise from more committed progenitors.
Division of Hematology, Department of Medicine, School of Medicine, Johns Hopkins University, Baltimore, MD, USA.
Jeffrey J
JJ
Pu
Rong
R
Hu
Galina L
GL
Mukhina
Hetty E
HE
Carraway
Michael A
MA
McDevitt
Robert A
RA
Brodsky
D000328:Adult; D000368:Aged; D000741:Anemia, Aplastic / Q000235:genetics / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006457:Hemoglobinuria, Paroxysmal / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008875:Middle Aged; D009154:Mutation; D009190:Myelodysplastic Syndromes / Q000235:genetics* / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism; D055815:Young Adult
D008565:Membrane Proteins; C080086:phosphatidylinositol glycan-class A protein