INTRODUCTION — Paroxysmal nocturnal hemoglobinuria (PNH) was one of the first hematologic disorders with a clear clinical description; the defining symptom, dark urine at night, was distinctive and easily observed. Subsequent investigations have clarified much of the pathogenesis of the disease, including the genetic defect and the mechanism of complement-mediated hemolysis.
This topic discusses the pathogenesis of PNH. The clinical manifestations, diagnosis, and treatment of PNH are presented in detail separately. (See "Clinical manifestations and diagnosis of paroxysmal nocturnal hemoglobinuria" and "Treatment and prognosis of paroxysmal nocturnal hemoglobinuria".)
PNH STEM CELL — PNH originates from an acquired genetic defect (mutation) in a multipotent hematopoietic stem cell, or in a hematopoietic progenitor cell that acquires stem cell properties and is able to survive, expand, and self-renew. PNH can arise de novo or in the setting of an underlying bone marrow disorder such as aplastic anemia (AA), myelodysplastic syndrome (MDS), or primary myelofibrosis (PMF) .
PIGA gene mutation — The acquired mutation in PNH occurs in the PIGA gene (phosphatidylinositol glycan anchor biosynthesis, class A; PIG-A; MIM311770), which is responsible for the first step in the synthesis of the glycosylphosphatidylinositol (GPI) anchor that attaches a subset of proteins to the cell surface (figure 1) .
The PIGA gene is located on the X chromosome. Therefore, a single "hit" (ie, a mutation in only one allele of the gene) will generate a PNH phenotype because males have only one X chromosome and females undergo X chromosome inactivation (lyonization) in every somatic cell, including hematopoietic stem cells .
A large spectrum of acquired PIGA mutations has been described [3-13]. The majority involve a frame-shift that creates a premature stop codon, resulting in a truncated protein product that is unstable and rapidly degraded. With few exceptions, mutations are unique to a patient, suggesting the absence of a mutational hot spot within the gene.
The mechanism of PIGA mutation is unknown. Mutagenic events such as exposure to excessive radiation, administration of mutagenic chemotherapy, or underlying defects in DNA repair are seldom seen in individuals with PNH. The close association between PNH and acquired AA (an autoimmune disease) suggests that the immune attack on hematopoietic stem cells provides a conditional survival advantage to the PNH clone. (See 'Clonal selection and expansion' below.)
Interestingly, many individuals, including healthy blood donors, have rare circulating blood cells with a PIGA mutation . However, in contrast to patients with PNH and those with AA and a small PNH clone, in healthy individuals the PIGA mutations appear to arise in hematopoietic precursor cells that lack the capacity for self-renewal, rather than in hematopoietic stem cells or earlier progenitor cells [15,16].
Germline PIGA null mutations are lethal during embryogenesis [17,18]. However, inherited PIGA mutations that lead to partial PIGA expression (hypomorphs) cause a syndrome known as multiple congenital anomalies-hypotonia-seizure syndrome 2 (MCAHS2, MIM300818) [19,20]. Children with MCAHS2 present with severe intellectual disability, dysmorphic facial features, and seizures, and they have a markedly shortened lifespan. In MCAHS2, the GPI anchor protein deficiency is most conspicuous on granulocytes. Red blood cells (RBCs) from these patients have little to no GPI anchor deficiency and no hemolysis. (See 'GPI anchor' below.)
Other initiating mutations — Lack of the complement inhibitor CD59 on the RBC surface is mostly responsible for the clinical manifestations in PNH. Rare cases of inherited mutations in CD59 leading to loss of CD59 on the RBC surface have been well documented. These patients manifest with chronic intravascular hemolysis, paroxysmal flares of hemolysis, and a propensity for thrombosis. Unlike patients with PNH due to PIGA mutations, those with inherited CD59 deficiency also have relapsing immune-mediated peripheral neuropathy . In the classical form of PNH, the CD59 deficiency is only found on the blood cells; in patients with germline CD59 mutations, CD59 is deficient in all cells in the body.
Alternative genetic defects leading to GPI deficiency and PNH symptoms have also been reported. As an example, a patient with a compound heterozygous mutation involving the PIGT gene presented with classical PNH symptoms and responded to treatment with terminal complement inhibition .
Clonal selection and expansion — To cause PNH, the hematopoietic stem cell carrying a PIGA mutation must undergo clonal expansion. (See 'Progression to pancytopenia/aplastic anemia' below.)
The mechanism(s) by which the PNH clone expands is unknown. Three major hypotheses have been proposed, which are not mutually exclusive; indirect supportive data exist for all three.
●Immune escape – One hypothesis posits that PIGA mutations do not offer a selective advantage, but they do protect cells from immune mediated destruction, as occurs in AA. Thus, expansion of a PNH clone requires both PIGA mutation and growth suppression of unaffected hematopoietic cells. This is supported by finding of PNH in patients with other underlying bone marrow disorders that impair hematopoiesis (eg, MDS, AA) .
●Selective advantage for clonal expansion – Another hypothesis proposes that PNH cells acquire an additional mutation(s) or other modifications that give them a selective advantage, either due to accelerated proliferation or reduced apoptosis (programmed cell death). However, the mutation rate in PNH cells is similar to that of non-PNH cells [24-27].
●Neutral evolution – A third possibility is "neutral evolution" in which PNH clones do not have a survival advantage, but one or more clones may expand stochastically during periods of bone marrow regeneration. Neutral evolution is supported by several lines of evidence including mathematical modeling simulations of hematopoietic stem cell evolution; the finding of PNH mutations in a large number of unaffected blood donors; and the occasional finding of clonal extinction and spontaneous remission of PNH [14,17,24].
Progression to pancytopenia/aplastic anemia — Acquired AA and PNH are closely related diseases. Small clonal populations of PNH cells can be found in up to 65 percent of patients with newly diagnosed AA, and many patients with de novo PNH have some degree of underlying bone marrow failure.
The management of pancytopenia in PNH, including the role of immunosuppressive therapies and hematopoietic stem cell transplantation, is presented separately. (See "Treatment and prognosis of paroxysmal nocturnal hemoglobinuria", section on 'Hematopoietic cell transplantation'.)
Molecular structure — The PIGA protein is involved in the synthesis of the glycosylphosphatidylinositol (GPI) anchor, a glycolipid that links dozens of cell-surface proteins to the plasma membrane on hematopoietic cells, including blood group antigens, adhesion molecules, and complement regulatory proteins (figure 1) . The significance for hematopoietic cell function is known for some, but not all, of these proteins.
GPI is a large molecule synthesized in the endoplasmic reticulum; over 25 genes and at least 10 biochemical reactions are involved in its synthesis . Once formed, the intact GPI anchor is transferred to the carboxyl terminus of proteins that have a GPI-attachment signal peptide, and the GPI-linked protein is then transported to the plasma membrane, where it is retained in microdomains known as lipid rafts.
The GPI anchor consists of a molecule of phosphatidylinositol (PI), a glycan core containing a molecule of N-glucosamine and three molecules of mannose, and a molecule of ethanolamine . On the cell surface, the PI end of the anchor is inserted into the lipid bilayer of the plasma membrane and the ethanolamine end is attached by an amide bond to the carboxyl end of a protein molecule. The PIGA protein is involved in the first step in GPI biosynthesis, in which N-acetytlglucosamine is transferred to phosphatidylinositol to form GlcNac-PI.
Cellular effects — The best-described and most clinically significant consequence of reduced GPI-anchored proteins pertaining to PNH is increased complement-mediated hemolysis. (See 'Complement-mediated hemolysis' below.)
Separate populations of RBCs have been defined according to the abundance of GPI-anchored proteins on the cell surface:
●PNH type I cells – Normal levels
●PNH type II cells – Partial absence
●PNH III cells – Complete absence
Cells of partial GPI deficiency (PNH type II cells) are found in approximately half of patients with PNH examined. In some patients, they appear to represent a distinct population of cells; in others, they seem to be "transitional" between the cells bearing normal amounts of protein and those bearing none. In some cases, a missense mutation in the PIGA gene, which changes the amino acid sequence of the protein, causes limited production of the protein or a protein with diminished activity. Thus, in patients with both PNH type II and PNH type III cells, each population may be derived from a separate PNH clone. (See 'PIGA gene mutation' above.)
In almost all patients with PNH, cells completely lacking in GPI-linked proteins (PNH type III cells) may be found as a discrete population. These cells are the most sensitive to complement-mediated lysis and the most readily lysed in vivo. The proportion of these cells varies; they can be depleted by a recent hemolytic crisis or diluted by a recent blood transfusion.
ANEMIA — Anemia in PNH is often multifactorial and may result from a combination of red blood cell (RBC) destruction (hemolysis) and lack of production due to underlying bone marrow failure, iron deficiency, or other bone marrow insults. Hemolysis is the major mechanism in patients with classical PNH. (See "Clinical manifestations and diagnosis of paroxysmal nocturnal hemoglobinuria", section on 'Diagnostic criteria and classification'.)
Repeated episodes of hemolysis can also lead to iron deficiency as scavenged iron is shed in renal tubular epithelial cells.
PNH may also be diagnosed in the context of aplastic anemia (ie, PNH/aplastic anemia overlap). These patients tend to have pancytopenia, a low reticulocyte count and a hypocellular bone marrow. (See "Clinical manifestations and diagnosis of paroxysmal nocturnal hemoglobinuria", section on 'Pancytopenia'.)
Complement-mediated hemolysis — Complement-mediated intravascular hemolysis is the most prominent clinical feature in classical PNH. Evidence of intravascular hemolysis includes the presence of free hemoglobin in the serum or urine, decreased serum haptoglobin, and elevated serum lactate dehydrogenase (LDH). Elevation of the LDH level to >1.5 times the upper limit of normal can be seen with as few as 3 percent PNH RBCs . Hemolysis in PNH is not exclusively nocturnal, and may be seen continuously by sensitive methods including the serum haptoglobin and LDH.
Clinically significant anemia in untreated patients with PNH is largely due to intravascular hemolysis; however, both intravascular and extravascular hemolysis occur. These are both mediated by complement-dependent RBC lysis rather than an antibody/autoimmune mechanism. Thus, untreated patients with PNH have a Coombs-negative hemolytic anemia. (See "Diagnosis of hemolytic anemia in the adult" and "Pathogenesis of autoimmune hemolytic anemia: Warm agglutinins and drugs", section on 'Antibody detection: the direct Coombs test'.)
RBCs in individuals without PNH are protected from complement lysis by GPI-anchored proteins on their surface that block complement activation (figure 2). Intravascular hemolysis is blocked by CD59, and extravascular hemolysis by CD55. (See 'Intravascular hemolysis from reduced CD59' below and 'Extravascular hemolysis from reduced CD55' below and "Regulators and receptors of the complement system".)
Hemolysis occurs in individuals with PNH because these complement inhibitors are missing. In the absence of these inhibitors, complement proteins that bind mammalian cell membranes (self) through the alternative complement pathway can lyse self-cells as if they were bacteria (figure 3). (See "Complement pathways".)
The degree of hemolysis depends on several factors, including the number of GPI-deficient cells, the degree of GPI deficiency, and the presence of additional precipitating events. Examples of precipitating factors include the following:
●Administration of iron to an iron-deficient patient is thought to result in delivery of a large number of complement-sensitive cells to the circulation .
●Exposure to microorganisms (viruses or bacteria) is thought to increase complement activation, especially in the gastrointestinal tract. Nocturnal hemolysis has been attributed to the intestinal absorption of lipopolysaccharide, a potent complement activator .
●A surgical procedure may precipitate complement activation via inflammatory mediators.
Importantly, patients with PNH treated with eculizumab (a monoclonal antibody that blocks terminal complement) often develop a Coombs-positive hemolytic anemia that is C3-positive, IgG-negative, because eculizumab prevents the membrane attack complex from forming on the PNH red cells and prevents intravascular hemolysis (figure 2).
Intravascular hemolysis from reduced CD59 — The major mechanism of hemolysis in PNH is complement-dependent intravascular hemolysis (ie, hemolysis of RBCs in the circulation) . Intravascular hemolysis is normally blocked by CD59 (also called membrane inhibitor of reactive lysis [MIRL], protectin, homologous restriction factor, and membrane attack complex inhibitory factor [MACIF]), which prevents the final stage of complement assembly, production of the membrane attack complex [MAC] that forms a pore in the target cell (figure 3) . Intravascular hemolysis occurs in PNH because RBCs lack the GPI anchor required to attach CD59 to their surface [28,33-50].
Intravascular hemolysis leads to release of free hemoglobin into the blood. Free hemoglobin in turn can cause various toxic effects, including hypercoagulability, changes in vascular tone from reduction of circulating nitric oxide, and renal damage. (See 'Thrombosis' below and 'Smooth muscle dystonia' below and "Clinical features and diagnosis of heme pigment-induced acute kidney injury".)
Free hemoglobin is detected in laboratory testing by observation of pink/red serum or urine, and measurement of reduced haptoglobin, increased LDH, and increased reticulocyte count in iron-replete individuals. Urinalysis will show a positive dipstick for heme but no red blood cells in the sediment. (See "Clinical manifestations and diagnosis of paroxysmal nocturnal hemoglobinuria", section on 'Hemolysis'.)
Extravascular hemolysis from reduced CD55 — Extravascular hemolysis also occurs in PNH, due to complement mediated destruction of RBCs by reticuloendothelial macrophages in the liver and spleen. Extravascular hemolysis is normally blocked by CD55 (also called decay accelerating factor [DAF]), which prevents assembly of the C3 and C5 convertases upstream of MAC formation in the complement cascade. CD55 is normally attached to the RBC surface via a GPI anchor [51-55]. CD55 protects RBCs from hemolysis by inhibiting C3 convertases such as the C4b2a complex, which enzymatically cleaves and activates C3 [28,56]. Accumulation of C3 on the RBC surface in the absence of CD55 identifies (opsonizes) RBCs for reticuloendothelial destruction. (See "Regulators and receptors of the complement system".)
Extravascular hemolysis can persist in individuals treated with the monoclonal antibody eculizumab, because C3 fragments can accumulate on RBCs that are not destroyed by the MAC intravascularly, and these fragments opsonize the RBCs (figure 2) . (See "Treatment and prognosis of paroxysmal nocturnal hemoglobinuria", section on 'Eculizumab'.)
Aplastic anemia — Aplastic anemia (AA; pancytopenia due to hematopoietic stem cell deficiency/injury) may precede the diagnosis of PNH or, less commonly, may develop in the setting of preexisting PNH. In many cases, PNH becomes apparent after treatment of AA with immunosuppressive therapies. (See 'Progression to pancytopenia/aplastic anemia' above.)
The mechanism of PNH appearance after therapy for AA likely involves escape of PNH cells from the immune attack associated with acquired AA. Interestingly, PNH virtually never arises from inherited forms of AA . Thus, in children and young adults, the presence of a small PNH clone in the setting of a hypocellular bone marrow virtually excludes the diagnosis of an inherited bone marrow failure disorder. (See 'Clonal selection and expansion' above.)
THROMBOSIS — PNH is characterized by thrombosis in atypical locations. Venous thrombosis is significantly more common than arterial thrombosis, although instances of arterial thrombosis have been reported (eg, cerebral, coronary) [59-63]. Sites of venous thrombosis include intraabdominal vessels such as the hepatic vein (ie, Budd-Chiari syndrome); portal, mesenteric, and splenic veins; inferior vena cava; and cerebral vessels. (See "Clinical manifestations and diagnosis of paroxysmal nocturnal hemoglobinuria", section on 'Thrombosis'.)
The pathogenesis of thrombosis in PNH is multifactorial and incompletely understood; there are several hypothesized mechanisms [64,65]. A role for hemolysis in promoting thrombosis is suggested by the observation that complement inhibition reduces both hemolysis and thrombotic complications. In the absence of terminal complement inhibition, the risk of thrombosis correlates with the size of the PNH clone; however, it is not known whether this merely reflects the degree of hemolysis or other factors .
Specific contributing factors to the hypercoagulability in PNH may include the following:
●Free hemoglobin – Hemoglobin is released from lysed red blood cells (RBCs) during episodes of hemolysis. Free hemoglobin in the circulation scavenges nitric oxide (NO), which in turn leads to vasoconstriction. Free hemoglobin may also have direct effects on the vasculature such as endothelial cell activation, which may lead to expression of tissue factor, forming a nidus of thrombogenic factors on the vessel wall .
●Procoagulant microparticles – Procoagulant microparticles may be released from platelets undergoing complement-mediated attack. These particles are analogous to the procoagulant microparticles seen in disseminated intravascular coagulation (DIC) and antiphospholipid antibody syndrome (APS); they may contain a variety of prothrombotic factors that can activate platelets.
●Deficiency of anticoagulant and fibrinolytic factors – Several proteins involved in fibrinolysis (clot dissolution) are GPI-anchored; these appear to be deficient in PNH cells such as monocytes. Examples include tissue factor pathway inhibitor (TFPI), proteinase 3 (PR3), and urokinase plasminogen activator receptor (u-PAR) .
●Increased levels of C5a – The complement component C5a, which is increased in PNH, contributes to a hypercoagulable state by generating proinflammatory and prothrombotic cytokines such as interleukin-6, interleukin-8, and tumor necrosis factor-alpha [59,68].
The reason thrombosis occurs in atypical locations rather than typical locations such as deep veins of the leg is not well understood. Relatively low flow rates in intraabdominal vessels have been postulated as a potential mechanism.
The use of anticoagulants in the management and prevention of thrombosis in PNH is discussed in detail separately. (See "Treatment and prognosis of paroxysmal nocturnal hemoglobinuria", section on 'Thrombosis'.)
SMOOTH MUSCLE DYSTONIA — A variety of diverse symptoms in PNH are thought to be due to vasospasm or smooth muscle spasm caused by reduced levels of circulating nitric oxide (NO) . Nitric oxide is scavenged by free hemoglobin, which is released from red blood cells (RBCs) as they hemolyze. NO acts as a vasodilator in many vascular beds; thus, reduced NO levels in PNH lead to increased vascular and smooth muscle tone. The following symptoms have been suggested as a consequence of reduced NO:
This mechanism is further supported by the finding of similar symptoms in volunteers who were infused with intravenous free hemoglobin during early attempts to produce artificial blood, and in individuals who were treated with a NO synthase inhibitor .
●Paroxysmal nocturnal hemoglobinuria (PNH) originates from an acquired mutation in the PIGA gene (phosphatidylinositol glycan anchor biosynthesis, class A). To cause PNH, PIGA mutations must occur in a self-renewing hematopoietic stem cell, and that cell must undergo clonal expansion. Small clonal populations of PNH cells can be found in up to 65 percent of patients with newly diagnosed aplastic anemia (AA), and many patients with de novo PNH have some degree of underlying bone marrow failure. (See 'PNH stem cell' above.).
●The PIGA protein is involved in the first step in the synthesis of the glycosylphosphatidylinositol (GPI) anchor, a glycolipid that links dozens of cell-surface proteins to the plasma membrane on hematopoietic cells (figure 1). Complement-mediated intravascular hemolysis is largely due to lack of the GPI-linked complement inhibitor CD59 on the surface of red blood cells (RBCs) (figure 2). (See 'GPI anchor' above.)
●Anemia in PNH is largely due to intravascular hemolysis; however, other factors may contribute, including extravascular hemolysis, bone marrow failure, and iron deficiency. (See 'Anemia' above.)
●The pathogenesis of thrombosis in PNH is multifactorial and incompletely understood. Free hemoglobin released from RBCs scavenges nitric oxide (NO), which in turn leads to vasoconstriction and possibly endothelial cell activation and expression of tissue factor. Procoagulant microparticles may be released from platelets undergoing complement-mediated attack. Deficiency of proteins involved in fibrinolysis, and increases in proinflammatory/prothrombotic cytokine levels may also contribute. (See 'Thrombosis' above.)
●A variety of diverse symptoms in PNH such as abdominal pain, erectile dysfunction, fatigue, and esophageal spasm, are thought to be due to vasospasm or smooth muscle spasm caused by reduced levels of NO. (See 'Smooth muscle dystonia' above.)
ACKNOWLEDGMENT — UpToDate would like to acknowledge Wendell F Rosse, MD, who contributed to earlier versions of this topic review.