Abstract
Abstract 1987Poster Board I-1009Paroxysmal nocturnal hemoglobinuria (PNH) is caused by a somatic mutation of PIG-A gene in one or few hematopoietic stem cells and subsequent clonal expansion of mutant stem cells that leads to development of symptoms. It is known that PIG-A mutation is insufficient to account for the clonal expansion required for clinical manifestation of PNH. We are proposed a 3-step model of PNH pathogenesis. Step 1 involves the generation of a GPI-deficient hematopoietic stem cell by somatic mutation of the PIG-A gene. Step 2 involves the immunological selection of GPI-deficient hematopoietic stem cells. Based on the close association of PNH with aplastic anemia, it has been suggested that the selection pressure is immune mediated. However, in spite that over 60% of patients with aplastic anemia have subclinical population of GPI-deficient hematopoietic cells at diagnosis, only 10% develop clinical PNH, suggesting that step-1 and 2 are insufficient to cause PNH. Under immune mediated selection pressure, GPI-deficient cells not only survive, but also must proliferate much more frequently than usual to compensate for anemia. This elevated proliferation rate may increase a chance that additional genetic mutations are acquired, in turn leads to step 3. Step 3 involves a second somatic mutation that bestows on PIG-A mutant stem cell a proliferative phenotype. According to this hypothesis, we searched for the candidate gene for step 3. We reported 2 patients with PNH whose PIG-A mutant cells had an acquired rearrangement of chromosome12, making the break within the 3' untranslated region in HMGA2. This gene encodes an architectural transcription factor which is deregulated in many benign mesenchymal tumors (Blood. 2006 vol.108 no.13, p4232). Based on these, we consider HMGA2 as a candidate gene, ectopic expression of which causes proliferation of PIG-A mutant cells. We have analyzed the expression of HMGA2 by quantitative RT-PCR in peripheral blood and bone marrow from healthy volunteers and PNH patients. The samples from patients had significantly higher expression of HMGA2 than those from normal volunteers in the peripheral blood (relative mRNA expression, 4.8±2.4 vs 1.3±0.3, p<0.05) but this was not the case in the bone marrow samples. To investigate the upstream and downstream of HMGA2 upregulation, we used oligonucleotide microarrays in analysis of peripheral blood from PNH patients and normal volunteers. Bioinformatic analyses showed Wnt pathway is significantly upregulated in PNH patients. To confirm this result, we analyzed the expression of cyclin D1 and β-catenin by quantitative RT-PCR and Western blotting, respectively. Expression of cyclin D1 and β-catenin in patients' bone marrow were higher than normal volunteers'. It was reported that Wnt signaling is required for normal growth of HSCs but dysregulation of this pathway leads to various cancers such as chronic myelocytic leukemia. It is possible that HMGA2 is upregulated by enhanced expression of c-Myc, which is the main target of Wnt pathway. How Wnt pathway is dysregulated is the big question, which is common to other hematopoietic malignancies. These results are consistent with our 3-step model of PNH pathogenesis, that is, clonal expansion is caused not only by survival advantage from immunological attack but also by tumorigenic proliferation. Disclosures:Shichishima:Alexion Pharmaceuticals: Research Funding. Kinoshita:Alexion: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.
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