Comparison of Gene Expression in Dominant and Non-Dominant PIG-A Mutant Clones in Paroxysmal Nocturnal Hemoglobinuria (PNH).

PNH is an acquired disorder of clonal hematopoiesis in which the affected hematopoietic stem cells (HSC’s) have a mutation of the X-linked gene PIG-A. Mutant PIG-A explains the clinical manifestations of PNH that result from deficiency of glycosyl phosphatidylinositol anchored proteins but does not explain why the mutant clones are selected or expand. The peripheral blood of patients with PNH is a mosaic of normal and abnormal cells, and molecular studies have demonstrated that the abnormal cells may be derived from multiple, discrete PIG-A mutant clones. Further, the contribution of the mutant clones to hematopoiesis varies greatly, but usually one clone is dominant. Together, these observations suggest that factors independent of mutant PIG-A influence clonal expansion. To investigate the basis of clonal expansion and clonal dominance, the pattern of gene expression among T cell clones developed from an informative patient with PNH was compared. The samples analyzed represented the following 3 categories of interest: (1) PIG-A mutant, dominant; (2) PIG-A mutant, non-dominant; (3) PIG-A normal. All samples were developed at the same time. Total RNA from the clones was amplified using the RiboAmp kit (Ambion). The antisense RNA was labeled, and the transcription profile of the dominant PIG-A mutant clone was compared with that of both the non-dominant PIG-A mutant clone and the PIG-A normal clone using microarray analysis. RNA from the original samples was used to verify results by PCR. Components of the AP-1 family of transcription factors (particularly FOSB) were differentially expressed by the PIG-A mutant dominant clone compared to both the PIG-A mutant non-dominant and the PIG-A normal clones. The major downstream effect of AP-1 upregulation appears to be on prostaglandin synthesis as the dominant clone also differentially expressed COX-2. Our experiments also showed that the pool of GPI-AP deficient T cells from a second patient with PNH but not the pooled GPI-AP positive T cells from the same patient expressed COX-2. Further, COX-2 expression was not observed in cultured T cells derived from any of 3 volunteer donors. COX-2 is mainly the inducible form of cyclooxygenase, with expression regulated by inflammatory cytokines. Studies by others using knockout mice suggest that COX-2 plays a key role in accelerated hematopoiesis following marrow injury. COX-2 also appears to function as an anti-apoptosis factor in lymphoproliferative disease, and inhibition of COX-2 has been reported to activate apoptosis through both defined and novel, BCL-2 independent, mitochondrial pathways. We have discovered COX-2 overexpression in the PIG-A mutant, dominant T cells through comparative gene expression analysis. COX-2 appears to be vital to hematopoietic recovery after marrow insult, conceivably as a consequence of anti-apoptotic activity. We hypothesize that an undefined marrow insult leads to clonal selection of PIG-A mutant HSC’s because of deficiency of one or more GPI-AP. Clonal expansion and clonal dominance, however, may be dependent on the pattern of expression of genes other than PIG-A (e. g., COX-2) that enhance proliferation, survival or both. Studies designed to evaluate further the role of COX-2 in clonal expansion are in progress.