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Paroxysmal nocturnal haemoglobinuria (PNH) results from acquired mutations in the PIG-A gene of an haematopoietic stem cell, leading to defective biosynthesis of glycosylphosphatidylinositol (GPI) anchors and deficient expression of GPI-anchored proteins on the surface of the cell's progeny. Some laboratory and clinical findings have suggested genomic instability to be intrinsic in PNH; this possibility has been supported by mutation analysis of hypoxanthine-guanine phosphoribosyltransferase ( HPRT) gene abnormalities. However, the HPRT assay examines lymphocytes in peripheral blood (PB), and T cells may be related to the pathophysiology of PNH. We analysed the molecular and functional features of HPRT mutants in PB mononuclear cells from eleven PNH patients. CD8 T cells predominated in these samples; approximately half of the CD8 cells lacked GPI-anchored protein expression, while only a small proportion of CD4 cells appeared to derive from the PNH clone. The HPRT mutant frequency (Mf) in T lymphocytes from PNH patients was significantly higher than in healthy controls. The majority of the mutant T lymphocyte clones were of CD4 phenotype, and they had phenotypically normal GPI-anchored protein expression. In PNH patients, the majority of HPRT mutant clones were contained within the V β2 T cell receptor (TCR) subfamily, which was oligoclonal by complementarity-determining region three (CDR3) size analysis. Our results are more consistent with detection of uniform populations of expanded T cell clones, which presumably acquired HPRT mutations during antigen-driven cell proliferation, and not due to an increased Mf in PNH. HPRT mutant analysis does not support underlying genomic instability in PNH. [ABSTRACT FROM AUTHOR]

: ObjectiveRecently, phenotypically normal CD34 cells from the marrow of patients with paroxysmal nocturnal hemoglobinuria (PNH) were reported to show impaired growth and elevated Fas receptor expression as compared to glycophosphatidylinositol-anchored protein (GPI-AP)-deficient CD34 cells and CD34 cells from normal individuals. These results are consistent with the theory that PNH cells have an intrinsic growth advantage, but their superior expansion in vitro could also be the outcome of selective extrinsic pressure in vivo.: Material and MethodsGrowth characteristics, competitive features, and susceptibility to apoptosis of sorted normal or GPI-AP–deficient CD34+ cells derived from PNH patients were assessed in suspension and methylcellulose cultures.: ResultsWhen we directly compared the growth of patients'' CD34 cells, separated based on expression of GPI-AP CD55 and CD59, in most of the patients studied, mutant CD34 cells showed higher progeny production and outgrew phenotypically normal CD34 cells derived from PNH patients in mixing experiments. However, their proliferation rate did not exceed that of control CD34 cells. To determine whether deficient growth of phenotypically normal CD34 cells in PNH was secondary to a pre-existing in vivo insult, we determined the fraction of apoptotic cells within fresh normal and PNH CD34 cells. Normal CD34 cells from PNH patients showed a high proportion of apoptotic cells and higher Fas expression, while GPI-AP–deficient and control CD34 cells showed similar, low rates of apoptosis. After correction for pre-existing apoptosis, the proliferation potential of normal and PNH CD34 cells was similar.: ConclusionsThese results strongly suggest that clonal expansion of GPI-AP–deficient progenitor cells from PNH patients is due to their selection in the hostile marrow environment of the patient. [Copyright &y& Elsevier]

: ObjectiveCellular prion protein (PrPc) is a glycosylphosphatidylinositol (GPI)-anchored protein (GPI-AP) constitutively expressed by neurons but also in hematopoietic cells. In trasmissible spongiform encephalopathies, the protease-resistant form of prion (PrP s c) converts the host PrPc into the pathologic form. We have investigated PrPc expression in hematopoietic cells from paroxysmal nocturnal hemoglobinuria (PNH). In this disease, due to somatic mutations in PIG-A gene, biosynthesis of the (GPI)-anchor is impaired and affected cells lack membrane expression of all GPI-AP.: MethodsNormal and PNH hematopoietic progenitors and paired wild-type (WT) and PIG-A mutant cell lines were used for analysis of intracellular and surface PrPc expression using flow cytometry and Western blot.: ResultsBy flow cytometry, PrPc was constitutively present on normal CD34+ cells, including more immature CD38dim cells, as well as hematopoietic cell lines. Similar results were obtained in purified CD34+. Phospholipase C treatment confirmed that PrPc was expressed on the membrane via the GPI-anchor. In PNH patients, GPI-AP-deficient CD34+ cells lacked PrPc membrane expression. PIG-A-mutated cell lines (Jurkat, K562, CEBV, AEBV), in contrast to their normal counterparts, did not express surface PrPc. However, we detected intracellular PrPc at approximately equivalent levels in both normal and PIG-A-mutated cells using intracellular flow cytometry and Western blotting.: ConclusionCells and cell lines with PNH phenotype together with their normal counterparts may be a suitable system to explore the function of membrane PrPc in the hematopoietic system. Conversely, PrPc is a good model to elucidate the fate of GPI-AP in PIG-A-deficient cells. [Copyright &y& Elsevier]

Objective: Investigate the contribution of PIG-A mutations to clonal expansion in paroxysmal nocturnal hemoglobinuria (PNH). Materials and Methods: Primary CD34+ hematopoietic progenitors from PNH patients were assayed for annexin-V positivity by flow cytometry in a cell-mediated killing assay using autologous effectors from PNH patients or allogeneic effectors from healthy controls. To specifically assess the role of the PIG-A mutation in the development of clonal dominance and address confounders of secondary mutation and differential immune attack that can confound experiments using primary cells, we established an inducible PIG-A CD34+ myeloid cell line, TF-1. Apoptosis resistance was assessed after exposure to allogeneic effectors, NK92 cells (an interleukin-2−dependent cell line with the phenotype and function of activated natural killer [NK] cells), tumor necrosis factor (TNF)−α, and γ-irradiation. Apoptosis was measured by annexin-V staining and caspase 3/7 activity. Results: In PNH patients, CD34+ hematopoietic progenitors lacking glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-AP−) were less susceptible than GPI-AP+ CD34+ precursors to autologous (8% vs 49%; p < 0.05) and allogeneic (28% vs 58%; p < 0.05) cell-mediated killing from the same patients. In the inducible PIG-A model, GPI-AP− TF-1 cells exhibited less apoptosis than induced, GPI-AP+ TF-1 cells in response to allogeneic cell-mediated killing, NK92-mediated killing, TNF-α, and γ-irradiation. GPI-AP− TF-1 cells maintained resistance to apoptosis when effectors were raised against GPI-AP− cells, arguing against a GPI-AP being the target of immune attack in PNH. NK92-mediated killing was partially inhibited with blockade by specific antibodies to the stress-inducible GPI-AP ULBP1 and ULBP2 that activate immune effectors. Clonal competition experiments demonstrate that the mutant clone expands over time under proapoptotic conditions with TNF-α. Conclusion: PIG-A mutations contribute to clonal expansion in PNH by conferring a survival advantage to hematopoietic progenitors under proapoptotic stresses. [Copyright &y& Elsevier]