Paroxysmal nocturnal hemoglobinuria (PNH) is a disease as simple as it is complex,” Colden and colleagues wrote in Frontiers in Immunology. 

Simple in the sense that it is primarily characterized by hemolysis, a vulnerability for the blood to clot, and failure of the bone marrow. It was this constellation of symptoms that first led to its discovery in 1882 by Dr. Paul Strübing. 

Complex in the sense that there are multiple layers to its pathophysiology, as researchers have since found out. A century after it was first discovered, clinicians now understand PNH to be driven by the absence of a category of membrane proteins attached by glycosylphosphatidylinositol (GPI) anchors to cell surfaces. In most patients, this deficiency is determined by somatic mutations in phosphatidylinositol glycan anchor biosynthesis class A (PIGA), an X-linked gene. The result is an abundance of PIGA-mutant cells that lack GPI-anchored proteins. 

In PNH, these GPI-negative cells undergo large clonal expansions, driving hemolysis. Scientists have discovered that patients with concurrent aplastic anemia are more likely to have vastly expanded PNH clones, thereby cementing the relationship between these 2 disorders. However, the exact correlation between aplastic anemia and PNH continues to drive academic debate; the lack of PNH hematopoietic stem and progenitor cells during immune-mediated aplastic anemia have led some researchers to conclude that PNH hematopoietic stem and progenitor cells confer a survival advantage during these attacks. 

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The Significance of PNH Clones 

The increase in PNH cells in patients with aplastic anemia stands in contrast to the number in healthy individuals, in whom PNH cell populations are found to be exceedingly small (0.001%-0.005%). Aside from patients with aplastic anemia, those with immune-mediated bone marrow failure are also known to have a large population of PNH clones. More rarely, this same pathology can be detected in some patients with myelodysplastic syndrome. 

The difficulty with PNH clones is that they are known to persist over a long period of time; around one-tenth will expand in size, while one-quarter will shrink. The clinical implication is that physicians should initiate serial evaluation of PNH clonal expansion, ideally annually, with concurrent blood tests to inspect hemolytic parameters. Because of the unique relationship between aplastic anemia and PNH, it is highly advisable for patients with aplastic anemia to be monitored for PNH clonal expansion as well. 

PNH testing can also help physicians to more quickly arrive at a correct diagnosis of aplastic anemia. When a PNH clone is discovered, the differential diagnosis of aplastic anemia is elevated; in contrast, a diagnosis of aplastic anemia is unlikely in a patient with a known syndrome disorder with multiple congenital anomalies and worsening cytopenia. 

“In a hypothetical case with a low pretest probability of 2%, a positive PNH clone would raise the posttest probability of aplastic anemia into a moderate range of 65%,” Babushok of the Hospital of the University of Pennsylvania wrote in Hematology. 

Interestingly, researchers have discovered that PNH clones have a unique distribution when assessed using PNH flow cytometry: one-third of patients with PNH clones will have “classical PNH”—having a mean PNH clone size of more than 70%, overt hemolysis, an increased risk of thrombosis, and multiple PNH symptoms. Two-thirds of individuals with PNH clones have cytopenia and bone marrow failure, but no hemolysis. 

It should also be noted that PNH clones can emerge late into the disease progression, hence the importance of close monitoring of PNH clonal expansion. When PNH clones are discovered later, it once again helps physicians in identifying patients with refractory immune-mediated aplastic anemia if the diagnosis is in doubt. 

Progress in Mice Studies 

To deepen our understanding of how PNH clones drive pathology, scientists have developed PIGA-mutant mice, which was no small feat, given that it triggered embryonic lethality.

Scientists have now been able to develop mouse models with hematopoietic cell-specific PIGA deficiency. Two resulting studies have been particularly illuminating. The first involved reconstituting mouse models using donor GPI-positive and GPI-negative cells and tracking how GPI-negative cells affect various peripheral blood cell lineages; the second involved incomplete and gradual PIGA excision using a Cre/LoxP system under the control of the c-fes promoter region. 

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“Both models demonstrated stable proportions of GPI-negative blood cells over the animals’ lifetime without clonal expansion in up to 42 weeks of follow-up,” Colden and colleagues wrote. “GPI-negative cells reconstituted near-normal hematopoiesis, with no or only minimal differences in blood counts between mutants and wild-type mice.” 

In other words, a pathway to stabilize PNH clonal expansion appears to exist, offering hope for patients who have large and growing PNH clonal populations that drive hemolysis and thrombosis. Scientists are now turning their attention toward identifying external factors that might contribute to GPI-negative cell clonal expansion. Hypotheses include PNH cell resistance to immune-mediated attacks and PNH cell sensitivity to apoptosis and inflammatory cytokines. 

The recent focus on the significance of PNH clonal expansion and the therapeutic effects of stabilizing it have shown a way forward, laying wide the path of a possible approach to research that might yet yield lasting results for patients with PNH. For vulnerable patients at any stage of their disease, this day cannot come soon enough. 


Colden MA, Kumar S, Munkhbileg B, Babushok DV. Insights into the emergence of paroxysmal nocturnal hemoglobinuriaFront Immunol. 2022;12:830172. doi:10.3389/fimmu.2021.830172

Babushok DV. When does a PNH clone have clinical significance?Hematology Am Soc Hematol Educ Program. 2021;2021(1):143-152. doi:10.1182/hematology.2021000245