Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal hematopoietic stem cell disorder that is caused by asomatic mutation of the PIG-A gene. The biochemical consequence of PIG-A mutations is a global loss ofglycosylphosphatidyl-inositol anchored proteins (GPI-AP). Two GPI-AP (CD55 and CD59) are importantcomplement regulatory proteins, their absence explains the complement-mediated intravascular hemolysisand thrombosis and that occur in PNH patients; however, the mechanism by which PNH cells achieve clonaldominance is not completely understood. All PNH patients have been shown to harbor PIG-A mutations;however, rare PIG-A mutations can also be found in healthy control subjects; thus, the biologic relevance ofPIG-A mutations in healthy controls remains to be determined. The overall objective of these studies is tostudy the relevance of GPI-anchor deficiency in PNH, normal, and malignant lymphohematopoietic cells.Our preliminary data demonstrates that hematopoietic cells can acquire a PNH phenotype (no cell surfaceGPI-AP) without PIG-A mutations. The mechanism of GPI-AP deficiency in these cells is throughtranscriptional silencing of genes required for GPI anchor biosynthesis. We have also established a PIGAnullCD34+ cell line with the PIG-A gene under the control of an inducible promoter. This cell line serves asa valuable model to study the mechanism of clonal dominance in PNH. Our preliminary data shows that PNHcells are relatively resistant to T cell mediatied apoptosis, and that the growth advantage of the PNH cells isamplified in the setting of an immune attack. Why PNH cells are resistant to T cell mediated killing is unclear,but our preliminary data suggest it may be related to perturbed signaling through membrane rafts andreduced ability to generate the proapoptotic second messenger, ceramide. Specifically, we will:1) Investigate the relevance of GPI-AP deficient cells in normals.Hypothesis 1.1: GPI-AP deficiency in lymphocytes from normals can result from a novel mechanism thatdoes not involve PICA mutations.Hypothesis 1.2: In healthy controls, a subset of HSPC in are GPI-APIo/neg, but do not harbor PIG-Amutations.2) Study the role of GPI-anchor deficiency in cellular resistance to apoptosis.Hypothesis 2.1: GPI-APIo/neg cells are resistant to immune attack via global resistance to apoptosis.Hypothesis 2.2:GPI-anchor expression plays a role in the response of normal cells to apoptotic stimuli.Hypothesis 2.3: Lack of GPI-AP expression disrupts lipid raft signaling.
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