Investigation of the mechanism for clonal expansion of GPI negative cells in paroxysmal nocturnal hemoglobinuria

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1. Investigation of the mechanism for clonal expansion of GPI negative cells (Taroh Kinoshita)We reported 2 patients with PNH whose PIGA mutant cells had a concurrent, acquired rearrangement of chromosome 12. In both cases, der(12) had a break within the 3 untranslated region of HMGA2,the architectural transcription factor gene dereguated in many benign mesenchymal tumors, that caused ectopic expression of HMGA2 in the bone marrow. These observations suggest that aberrant HMGA2 expression,in concert with mutant PIGA, accounts for clonal hematopoiesis.To investigate whether ectopic expression of HMGA2 is also the case with other PNH patients without chromosomal abnormalities, we have established the method for stabilization and purification of mRNA from peripheral blood cells or bone marrow cells of patients and normal volunteers. We could detect mRNA in blood both from normal volunteers and the PNH patient who has ectopic expression of HMGA2 because of chromosomal abnormalities by Q-PCR, and its expression in the patient is significantly higher than in normal volunteers. We also analyzed the genomic sequences of 3'UTR of HMGA2 of granulocytes from four PNH patients who have no chromosomal abnormalities, and found no abnormalities in the genomic sequences.2. Regulation of expression of GPI anchored proteins by the stimulations (Yoshiko Murakami)We have identified a novel disease characterized by venous thrombosis and seizures in which deficiency of GPI is inherited in an autosomal recessive manner. In patients, a point mutation (c-g) at position -270 from the start codon of PIGM, a mannosyltransferase-encoding gene, disrupts binding of the transcription factor Sp1 to its cognate promoter motif and reduces transcription of PIGM, causing partial GPI deficiency. We have made the mouse model which has the same mutation as patients to investigate how the expression of Pigm is regulated during embryogenesis and how this down regulation causes the symptoms.