Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired condition that affects 12 to 13 per 1,000,000 people.1 Patients with PNH experience nocturnal hemolysis which manifests as dark brown urine upon awakening that slowly resolves during waking hours. Nonspecific symptoms such as fatigue, weakness, and shortness of breath are common complaints among patients and are attributed to chronic hemolysis. Serious complications of PNH include arterial thromboembolisms (which may result in stroke), pulmonary hypertension, smooth muscle dystonia, and organ failure.2
The current consensus is that PNH is due to an acquired vulnerability to complement following a somatic mutation to the PIGA gene in hematopoietic stem cells (HSC). PIGA codes for a specific glycolipid structural anchor named glycosyl-phosphatidyl inositol (GPI). The defect to GPI affects anchoring of 2 complement-regulatory proteins, namely CD59/MIRL (membrane inhibitor of reactive lysis) and CD55/DAF (decay accelerating factor) which are normally found on the surface of red blood cells, white blood cells, and platelets.
CD55 regulates early complement activation and is an inhibitor of C3 and C5 convertase. Meanwhile, CD59 inhibits the formation of the membrane attack complex by blocking the incorporation of C9 onto the C5b-C8 complex.3 Serum acidification increases complement activation, which is why the intravascular hemolysis associated with PNH occurs during sleep, when carbon dioxide levels rise; although, an exact explanation for nocturnal exacerbations is still debated.3,4 This is where the disease gets its name. There are paroxysms of hemolysis during nocturnal hours resulting in dark brown urine due to the presence of hemoglobin—paroxysmal nocturnal hemoglobinuria.
This appears to be a convenient, elegant, and complete account of the pathophysiology of the disease; however, a few observations have called into question whether this is actually the whole story.
Firstly, attempts to replicate the disease in murine models have been unsuccessful. Introducing embryonic HSCs with the PIGA mutation to murine bone marrow did not result in the PNH phenotype, as the PNH clone did not seem to have any intrinsic proliferative advantage to overcome normal hematopoiesis.3
Perhaps the most perplexing of these observations is that circulating granulocytes harboring the PNH phenotype (absent GPI-APs) have been demonstrated in healthy individuals. In fact, it appears that PIGA mutations are relatively common in normal hematopoiesis. It seems the granulocytes in question are derived from colony-forming cells (CFCs), not HSCs.5 However, the presence of the mutation on CFCs is not enough to trigger clonal expansion as it appears to do in HSCs. This begs the question: What advantage does the PIGA mutation provide to HSCs?
Putting the Pieces Together
The first piece of the puzzle is to understand why HSCs that acquire a PIGA mutation proliferate and rival normal progenitors in the first place. This question is important because PNH is a clonal disorder, not a neoplasm. The acquired mutation is not to growth factors or tumor suppressor genes, but rather to anchoring proteins for complement regulators. Cells produced by the new abnormal lineage are vulnerable to complement-mediated lysis. This appears to be a disadvantage. So, why do these mutated progenitors succeed, proliferate, and rival normal HSCs?
Perhaps an even deeper understanding of the consequence of GPI loss is required. A loss of GPI does not only result in a loss of CD55 and CD59; rather, it results in a loss of all GPI-anchored proteins (GPI-AP) of which more than 25 have been discovered. We currently have an in-depth understanding of some of their roles (eg, CD55 and CD59) and a rudimentary understanding of others. Further investigation into the physiologic importance of these surface proteins might shed light on this mystery or even bring about novel therapies.
Read about the clinical presentation of PNH.
It is postulated that a loss of certain GPI-APs on platelets may explain the tendency for platelet aggregation and thrombogenesis. Additional theories include platelet stimulation, activation of the coagulation cascade, inhibition of fibrinolysis, and endothelial activation and dysfunction. The underlying cause may not be limited to complement activation, but may also be explained by free hemoglobin toxicity, nitric oxide depletion, and absence/reduced expression of GPI-APs with fibrinolytic or anticoagulant properties (yet to be identified).6
The reason for clonal expansion as a consequence of deficient GPI-APs is still a mystery. It has been hypothesized that GPI-linked immune targets exist that are necessary for elimination of mutated stem cells. Its absence in PNH may explain the immune escape of the progenitor clone due to a reduced sensitivity to immune effector mechanisms. No concrete evidence for these receptors exists to date.3
It has recently been revealed that several clones are present in patients with PNH, each with distinct somatic mutations, excluding the PIGA mutation. In other words, PNH is an oligoclonal and not monoclonal disorder. It has been suggested that these additional somatic mutations may be significant and perhaps provide some sort of advantage.7
The Theory of Dual Pathophysiology
Alternative pathophysiologic explanations have been developed in an attempt to answer why PIGA-mutated HSCs undergo clonal expansion. They have been called the “relative advantage” or “escape theory.” It has been established that PIGA mutations are relatively common during hematopoiesis and, according to this theory, this mutation indeed does not confer any intrinsic proliferative advantage to the mutants. In order for the mutated HSC to clonally expand, an extrinsic trigger is necessitated that creates a favorable environment for this expansion to occur.
At face value, this doesn’t seem to really offer any insights; however, the reasoning behind these alternative theories is drawn from some curious observations. For example, there is a close association between aplastic anemia (AA) and PNH. As a matter of fact, most patients with PNH demonstrate some degree of bone marrow failure, and it is not uncommon for patients with AA to have PIGA mutations. Turns out, up to 20% of patients with myelodysplasia have concomitant PNH.8 This is not to say that the PIGA mutation drives bone marrow failure. Rather, the theory suggests that the immune mechanisms responsible for the pathogenesis of AA (which are also poorly understood) spare PIGA-mutated HSCs.3
We still know very little about the precise underlying mechanism behind PNH. Some authors have suggested an antigen-driven immune response targeting the marrow tissue as the cause. If this is the case, it would correlate nicely with the dual theory above because if the target on HSC membranes is a GPI-linked molecule, then PNH HSCs may escape this injury while normal HSCs are destroyed. The result: dominance of PNH HSCs with little-to-no normal HSCs.3
There is one problem with this theory: PIGA is expressed ubiquitously throughout the body. It doesn’t make sense why a global immune attack would be restricted to normal HSCs. Furthermore, if the underlying phenomenon was immunological, it would be expected that immunosuppression has, at the very least, a modest therapeutic effect. In reality, immunosuppressive therapy has no effect on reestablishing normal hematopoiesis.9
Spontaneous Remission
As mysterious as the pathophysiology of PNH is, the unexplainable and sudden disappearance of the PNH clones is perhaps the most bewildering phenomenon. Different studies have reported varying incidences of spontaneous remission, ranging from 3-15%.10 Some authors have suggested that PNH clones have a finite life span, similar to normal somatic cells.11
The disappearance of the clone has intrigued scientists for decades. It has been proposed that the underlying mechanism is an obscure immune mechanism in which antibodies target unique epitopes on GPI-anchor deficient cells. Other authors suggest that treatment with biologic agents like eculizumab has curative potential in a select few individuals for unknown reasons.7
Read more about PNH prognosis
While it would be logical to expect that the extinction of the PNH clone would result in a return to normal hematopoiesis, that is not always the case. Patients must be carefully monitored following spontaneous remission because of the possibility of developing leukemia and aplastic anemia.7
Neutral Drift
Perhaps the reason for clonal expansion of PNH cells and the possibility of their disappearance are simply probabilistic and the evolutionary concept of neutral drift alone is sufficient. Neutral drift goes hand in hand with Darwinian principles of survival of the fittest. Neutral drift refers to the random changes and mutations that biological molecules undergo with time.9
One group of researchers applied Markovian stochastic dynamics alone to develop a model that successfully predicts the current incident rate and probable age of diagnosis in the global population. To put it simply, the researchers believe that cell PIGA mutations have no fitness advantage and the expansion of the clone is a mathematical game of probabilities. In fact, the authors propose that PNH is perhaps the first disease where neutral drift alone may be responsible for clonal expansion leading to a clinical problem.9
If this really is just a game of probability, I can’t help but think about the poor interns laboring over all the mysteries seemingly hidden beneath the PIGA mutation that will never be answered. Maybe we might never put this issue to bed, but isn’t that the best part of science? The pursuit of knowledge.
References
1. Jalbert JJ, Chaudhari U, Zhang H, Weyne J, Shammo JM. Epidemiology of PNH and real-world treatment patterns following an incident PNH diagnosis in the US. Blood. 2019;134(Supplement_1):3407. doi:10.1182/blood-2019-125867
2. Brodsky RA. Paroxysmal nocturnal hemoglobinuria. Blood. 2014;124(18):2804-2811. doi:10.1182/blood-2014-02-522128
3. Risitano AM, Rotoli B. Paroxysmal nocturnal hemoglobinuria: pathophysiology, natural history and treatment options in the era of biological agents. Biologics. 2008;2(2):205-222. doi:10.2147/btt.s1420
4. Farooq Q, Saleem MW, Khan ZU, Hadi N. Paroxysmal nocturnal hemoglobinuria: a diagnostic “zero-sum-game”. Cureus. 12(12):e11956. doi:10.7759/cureus.11956
5. Hu R, Mukhina GL, Piantadosi S, Barber JP, Jones RJ, Brodsky RA. PIG-A mutations in normal hematopoiesis. Blood. 2005;105(10):3848. doi:10.1182/blood-2004-04-1472
6. Lima M. Laboratory studies for paroxysmal nocturnal hemoglobinuria, with emphasis on flow cytometry. Pract Lab Med. 2020;20:e00158. doi:10.1016/j.plabm.2020.e00158
7. Korkama ES, Armstrong AE, Jarva H, Meri S. Spontaneous remission in paroxysmal nocturnal hemoglobinuria—return to health or transition into malignancy? Front Immunol. 2018;9:1749. doi:10.3389/fimmu.2018.01749
8. Dunn DE, Tanawattanacharoen P, Boccuni P, et al. Paroxysmal nocturnal hemoglobinuria cells in patients with bone marrow failure syndromes. Ann Intern Med. 1999;131(6):401-408. doi:10.7326/0003-4819-131-6-199909210-00002
9. Père NM, Lenaerts T, Pacheco JM, Dingli D. Evolutionary dynamics of paroxysmal nocturnal hemoglobinuria. PLOS Computational Biology. 2018;14(6):e1006133. doi:10.1371/journal.pcbi.1006133
10. Gurnari C, Pagliuca S, Kewan T, et al. Is nature truly healing itself? Spontaneous remissions in paroxysmal nocturnal hemoglobinuria. Blood Cancer J. 2021;11(11):1-3. doi:10.1038/s41408-021-00582-5
11. Hillmen P, Lewis SM, Bessler M, Luzzatto L, Dacie JV. Natural history of paroxysmal nocturnal hemoglobinuria. N Engl J Med. 1995;333(19):1253-1258. doi:10.1056/NEJM199511093331904
