Circulating PIG-A mutant T lymphocytes in healthy adults and patients with bone marrow failure syndromes

Objective Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal hematological disorder with acquired PIG-A gene mutations and absent surface expression of proteins utilizing glycosylphosphatidylinositol (GPI) anchors. PNH often follows aplastic anemia, suggesting PIG-A mutant cells have relative dominance over normal hematopoietic cells. Somatic PIG-A mutations could arise after aplasia, or healthy persons could have rare PIG-A mutant cells that expand under selection pressure. Methods We developed an in vitro negative selection method to isolate GPI-deficient T lymphocytes using aerolysin, an Aeromonas toxin that binds GPI anchors and induces cell lysis. Peripheral blood mononuclear cells (PBMC) from normal adults and patients with PNH or other bone marrow failure syndromes were analyzed. Results From healthy adults, 166 T lymphocyte clones with deficient GPI-linked surface protein expression (CD55, CD59) were isolated. The mean mutant frequency (Mf) of aerolysin-resistant clones was 17.8 ± 13.8 per 106 PBMC, range 5.0–59.6 per 106 cells. Clones had a Class A complementation defect and distinct PIG-A mutations. Patients with PNH had elevated aerolysin-resistant Mf values averaging 19 × 10−2, a 10,000-fold difference. Two patients with Fanconi anemia and two others with mild aplastic anemia had Mf values less than 15 × 10−6, but two with recovering aplastic anemia had Mf values of 20 × 10−4, representing an intermediate value between normal persons and PNH patients. Conclusion 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.