Is there a little PNH in all of us?

Russell E. Ware

Comment on Hu et al, page 3848

Cells with PNH-like characteristics can be selected and grown in vitro from CD34+ progenitors isolated from healthy donors. These findings suggest that PIGA mutations develop during normal hematopoietic differentiation and are not sufficient for the development of PNH.

Almost 40 years ago, Lewis and Dacie1 observed important relationships between paroxysmal nocturnal hemoglobinuria (PNH) and aplastic anemia (AA). That same year in Blood, Dameshek2 postulated that both PNH and AA arise in the setting of marrow injury but following different responses. Since those sentinel manuscripts, the investigation of PNH has come a very long way, yet we still have many unanswered questions about how PNH arises in the setting of normal or injured bone marrow. First described (and named) for its dramatic paroxysms of dark urine, PNH is now known to be a complex acquired stem cell disorder characterized clinically by (1) episodic intravascular hemolysis due to erythrocyte sensitivity to autologous serum complement; (2) a propensity toward venous thrombosis; and (3) deficient hematopoiesis. The biochemical abnormality in PNH involves defective formation of glycosylphosphatidylinositol (GPI) anchors, with subsequent abnormal surface expression of proteins that use GPI anchors for membrane attachment. PNH cells, which typically coexist in the marrow and circulation along with their normal counterparts, therefore lack expression of a wide variety of surface proteins with functions ranging from complement regulation to receptor binding to cell-cell interactions. At the molecular level, the X-linked PIGA gene is involved in the first step in GPI anchor biosynthesis; acquired clonal PIGA mutations have been identified in all PNH patients reported to date. But are PIGA gene mutations both necessary and sufficient for the development of PNH?

GPI-negative blood cells are most easily identified in PNH patients by flow cytometry, using monoclonal antibodies that specifically bind GPI-linked surface proteins such as CD59. However, GPI-negative cells are not just confined to PNH; patients with aplastic anemia or myelodysplasia also can have small numbers (typically <1%-3%) of PNH blood cells. Are these PNH-like cells naturally occurring and just “leftover” after the marrow damage, are they actually part of the disease pathophysiology, or are they simply an epiphenomenon not causally associated with these related disorders? Interestingly, treatment with alemtuzumab (CAMPATH-1H), a monoclonal antibody therapy that lyses cells expressing the GPI-linked CD52 antigen, provides an in vivo “selection pressure” that leads to the emergence of GPI-negative cells with PIGA mutations, suggesting their prior existence in circulation.3 But do healthy persons really have GPI-negative blood cells in their marrow and circulation? If so, are they truly PNH-like cells with PIGA gene mutations?

Aerolysin is a potent bacterial toxin that binds specifically to GPI anchors and causes cell lysis; this unique feature can be exploited in vitro to select for the growth of GPI-negative cells. With this aerolysin-negative selection method, PIGA mutant T lymphocytes were previously identified in healthy adults at a frequency of approximately 20 per million cells.4 In this issue of Blood, Hu and colleagues extend these findings with aerolysin selection of CD34+ cells isolated from marrow or mobilized peripheral blood, with the subsequent growth of GPI-negative colony-forming cells (CFCs) containing specific but nonclonal PIGA gene mutations. These findings are noteworthy because PIGA mutant CFCs were identified from normal bone marrow at a frequency that suggests PIGA gene mutations are relatively common in normal hematopoiesis. Furthermore, the data provide convincing evidence that acquired PIGA mutations are not sufficient for the development of PNH.

Unfortunately, these important observations still do not elucidate how PNH develops in the setting of normal or injured marrow. Perhaps a toxin or immunologic insult preferentially kills normal marrow stem cells, allowing the growth advantage of PIGA mutant cells that develop naturally during normal hematopoietic differentiation. However, these results do extend our knowledge of normal hematopoiesis and improve our understanding of the pathogenesis of PIGA gene mutations that occasionally develop into the fascinating disorder known as PNH. ▪