Minor GPI(-) granulocyte populations in aplastic anemia and healthy individuals derived from a few PIGA -mutated hematopoietic stem progenitor cells

[Background] Minor populations (0.003%-1.0%) of glycosylphosphatidylinositol-anchored protein-deficient granulocytes (GPI[-] Gs) are often detected in the peripheral blood (PB) of patients with acquired aplastic anemia (AA) and low-risk myelodysplastic syndromes and are thought to represent immune pathophysiology of bone marrow failure. We previously reported that minor GPI(-) G populations were detected in some healthy individuals (HIs) and persisted over several years at similar percentages (Katagiri T, et al. Stem Cells 2013). Several lines of evidence have suggested that small numbers of GPI(-) Gs detected in HIs are polyclonal populations mostly derived from short-lived PIGA-mutated committed progenitor cells. Minor GPI(-) G populations in AA patients may also be derived from multiple committed progenitor cells rather than from a few hematopoietic stem progenitor cells (HSPCs) with PIGA mutations. However, minor GPI(-) G populations usually persist for a long period of time at similar frequencies in AA patients, suggesting that they may instead be derived from a few HSPCs that have undergone PIGA mutations. This issue remains debated due to the inability to sequence the PIGA gene in the very few GPI(-) granulocytes available. We recently developed a sensitive method capable of detecting PIGA mutations in minor GPI(-) Gs using amplicon sequencing of GPI(-) Gs that were enriched with magnetic microbeads followed by FACS sorting. Using this method, we addressed whether minor GPI(-) G populations in AA patients and HIs are oligoclonal or polyclonal as well as which cell population they are derived from HSPCs or committed progenitor cells.