Circulating pig-a mutant t lymphocytes in healthy adults and patients with bone marrow failure syndromes including pnh

Paroxysmal nocturnal hemoglobinuria (PNH) is a stem cell disorder characterized by one or more hematopoietic clones with an acquired PIG-A gene mutation, which leads to reduced or absent surface expression of proteins that utilize glycosylphosphatidylinositol (GPI) anchors. The clinical observation of PNH frequently developing after aplastic anemia suggests that rare naturally occurring PIG-A mutant hematopoietic stem cells could expand under selection pressure. To date, however, these primitive PIG-A mutant cells have not been identified in normal persons. We have developed an in vitro negative selection method to isolate and grow GPI-deficient and PIG-A mutant cells using aerolysin, a toxin that binds GPI anchors and causes cell lysis. From peripheral blood mononuclear cells (PBMC), the mutant frequency (Mf) of aerolysin-resistant T lymphocytes in 16 healthy adults was 18 ±4 per 10' PBMC. In contrast, the aerolysin-resistant Mf for 11 patients with PNH was 19 ±12 per 102 PBMC, a 10,000-fold difference. Two pédiatrie patients with Fanconi Anemia and 2 with mild untreated aplastic anemia had low Mf values below 10 per 106 PBMC, but 2 others with recovering aplastic anemia had elevated Mf values of 20 per 104 PBMC, representing an intermediate value 100 times greater than normal adults but 100 less than patients with PNH. Neither of these latter 2 patients currently has clinical or laboratory evidence of PNH, but we speculate that their elevated aerolysin-resistant Mf may portend the development of clinically apparent PNH. Aerolysin negative selection allows the analysis of PIG-A mutant cells and may provide an efficient strategy to identify rare PIG-A mutant hematopoietic stem cells in normal persons. Identification of PIG-A mutant T lymphocytes in healthy adults suggests PNH could develop following intense negative selection of hematopoiesis, with clonal outgrowth of naturally occurring PIG-A mutant stem cells.

Duke Scholars

Author Carlos Manuel de Castro III Medicine, Hematologic Malignancies and Cellular Therapy