The Investigation of HMGA2 Dysregulation and Promoter Mutations in PIG-M in the Molecular Pathogenesis of Paroxysmal Nocturnal Haemoglobinuria (PNH).

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disorder of hemopoietic stem cells (HSC) that is characterized by intravascular hemolysis and venous thrombosis. The PNH HSC’s (and their progeny) have a somatic mutation of the X-linked phosphatidylinositol glycan class A gene (PIG-A) which causes a complete or partial deficiency of glycophosphatidylinositol (GPI) anchored proteins, which in turn produces the symptoms of the disease. PIG-A mutations account for all of classical acquired PNH cases thus far reported as a single acquired PIG-A mutation on the only active X-chromosome results in GPI-deficiency. A novel mutation in the promoter of the phosphatidylinositol glycan class M gene (PIG-M) has recently been reported in two unrelated families exhibiting autosomal recessive inheritance of congenital GPI-deficiency. This point mutation reduces PIG-M transcription and causes partial deficiency of GPI-anchored proteins. It is possible that this point mutation in PIG-M occurs at low frequency in the population but predisposes to mutation of the other PIG-M allele and could result in acquired PNH. To determine the prevalence of the PIG-M in classical acquired PNH the PIG-M promoter region spanning the proposed mutation was sequenced to look for heterozygotes with the C → G substitution at position −270. This mutation was not identified in 36 patients with PNH. There is convincing evidence that GPI-deficient HSC’s have no intrinsic proliferative advantage over normal HSC’s suggesting that other factors underlie the clonal expansion of PNH cells. The most likely explanation appears to be due to factors which are extrinsic to the PNH clone such as immune attack on normal HSC. Recently secondary molecular events within the PNH clone have been suggested to provide the growth advantage allowing expansion of the PNH clone. One such proposed event is a translocation affecting the HMGA2 gene, which has been described in 2 cases of PNH. HMGA2 is a member of the high motility group of proteins and acts as an architectural transcription factor. HMGA2 proteins are associated with gene activation and are mainly expressed during embryonic development. Rearrangements of HMGA2 commonly occur in benign mesenchymal tumours, but have also been identified as infrequent events in myeloid malignancies. To gain insight into the potential role of HMGA2 deregulation in the pathogenesis of PNH we evaluated the expression of HMGA2 mRNA by means of quantitative RT-PCR in peripheral blood samples of 42 PNH patients (median age: 42, range: 18–81) and ten normal controls. All PNH samples had large granulocyte clones (median size 97.81%) but showed no increase in HMGA2 mRNA. In nine of the 42 PNH patients blood samples underwent additional enrichment for CD15 positive cells to maximize the proportion of PNH granulocytes present. This again showed no aberrant expression of HMGA2 mRNA. This is the largest reported group with PNH evaluated for HMGA2 expression. Despite two case reports suggesting that the deregulation of HMGA2 may be a pathophysiological factor in occasional cases of PNH our data indicates that this mechanism accounts for the growth advantage of PNH cells in, at most, only a small minority of patients. Similarly, the PIG-M promoter mutation observed in congenital GPI-deficiency was not found in any of our patients with classical acquired PNH indicating that it has no or little role in the pathophysiology of PNH.