Written by Giacomo Deiro
Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired hemolytic anemia characterized by the increased sensitivity of red cells to complement, leading to intravascular hemolysis and hemoglobinuria. Other clinical features are cytopenias caused by bone marrow failure and an increased risk of thrombosis. If unrecognized and not treated appropriately, PNH is often associated with a substantial morbidity and mortality. PNH is caused by the expansion of a hematopoietic progenitor cell that caries a somatic mutation in the X-linked phosphatidylinositol glycan complementation group A (PIGA) gene. In pluripotent hematopoietic stem cells, this leads to a deficiency of glycosylphosphatidylinositol (GPI)-anchors and GPI-anchored proteins, including the complement regulators CD55 and CD59, on the surface of affected blood cells. PNH red blood cells are highly vulnerable to activation of complement and the formation of the membrane attack complex (MAC). The resulting chronic intravascular hemolysis is the underlying cause of PNH morbidities and mortality. Until recently, the treatment of PNH has been largely empirical and symptomatic with blood transfusions, anticoagulation, and supplementation with folic acid or iron. The only potentially curative treatment is allogeneic stem cell transplantation, but this has severe complications and high mortality and morbidity rates. A new targeted and disease-modifying treatment strategy is the inhibition of the terminal complement cascade with the humanized monoclonal anti-C5 antibody, eculizumab. This effectively inhibits MAC formation and intravascular hemolysis. Eculizumab has shown significant efficacy in controlled studies, with a marked decrease in anemia, fatigue, transfusion requirements, renal impairment, pulmonary hypertension, and risk of severe thromboembolic events, ultimately resulting in improving quality of life and survival.
Deficiency of the GPI anchor caused by a somatic mutation of the PIG-A gene in paroxysmal nocturnal hemoglobinuria. Cell. 1993
Treatment of paroxysmal nocturnal hemoglobinuria in the era of eculizumab. Eur J Haematol. 2011
The pathophysiology of paroxysmal nocturnal hemoglobinuria and treatment with eculizumab
*The genetic defect
*The complement cascade and hemolysis in PNH
*A standardized approach to PNH
*Clinical symptoms and the role of nitric oxide
*Conclusion and summary
Paroxysmal nocturnal hemoglobinuria is a rare hematological disorder where hemopoietic stem cells (HSCs) acquire an abnormality that is then passed on to their progeny. The red blood cells derived from these abnormal HSC
s are extremely sensitive to complement mediated lysis . Most of the symptoms experienced in this disease occur due to the absence of complement regulatory proteins on the surface of the red blood cells. Complement activation is thus not checked and causes destruction of these cells. Affected individuals have a triad of clinical associations – intravascular hemolysis, an increased risk of thromboembolism, and bone marrow failure. Prior to eculizumab, treatment was mainly supportive in nature with a median survival of 10 to 15 years for patients treated between 1940 and 1970. More recently, data from France reported a median survival of 22 years. This increase in survival may reflect improved supportive care as well as better treatment of more specific complications, such as thromboses. Eculizumab is a humanized monoclonal antibody which binds to the complement protein 5 (C5), thereby inhibiting the formation of the terminal components of the complement cascade. It was licensed by the Food and Drug Administration in March 2007 and by the European Medicines Agency in June 2007 for the treatment of paroxysmal nocturnal hemoglobinuria (PNH). It has been shown to be a well tolerated and highly effective treatment for patients with PNH.
Eculizumab prevents intravascular hemolysis, stabilizes hemoglobin levels, reduces or stops the need for blood transfusions, and improves fatigue and patient quality of life as well as reducing pulmonary hypertension, decreasing the risk of thrombosis and protecting against worsening renal function. It is not a curative therapy but has a great benefit on those with this rare debilitating condition.
The pathophysiology of paroxysmal nocturnal hemoglobinuria and treatment with eculizumab. Ther Clin Risk Manag. 2009
Eculizumab (INN and USAN,trade name Soliris) is a monoclonal antibody directed against the complement protein C5. Eculizumab has been shown to be effective in treating paroxysmal nocturnal hemoglobinuria (PNH) and is approved for this indication.
Eculizumab is a recombinant humanized monoclonal IgG2/4 antibody that specifically binds to the complement protein C5, inhibiting its cleavage by the C5 convertase which prevents the generation of the terminal complement complex C5b-9. It is this terminal complement complex that causes intravascular hemolysis in people with (PNH).
Eculizumab is a product of Alexion Pharmaceuticals and was approved by the United States Food and Drug Administration (FDA) on March 16, 2007 and the European Medicines Agency on June 20, 2007.
According to Forbes magazine, Soliris, at $409,500 a year, is the world's single most expensive drug.
The genetic defect
The disease is characterized by hemopoietic clones which harbor somatic mutations of the phosphatidylinositol glycan synthetic pathway due to inactivation of the complementation class A gene (PIG-A). The PIG
-A gene is one of a number of genes needed for the synthesis of the glycophosphatidylinositol (GPI) anchor within the endoplasmic reticulum (ER). GPI
biosynthesis occurs via a stepwise addition of sugar nucleotides and phospholipids within the ER before the completed protein is transferred to the cell surface (Figure 1). The GPI
moiety serves as a membrane anchor for a variety of cell surface proteins. Mutations of the PIG
-A gene disrupt the first step of GPI
biosynthesis leading to an absence of the GPI
anchor and, in turn, a marked deficiency of all GPI
Glycophosphatidylinositol biosynthesis: an illustration showing the stepwise addition of sugar residues and the sites at which PIG-A and PIG-M are required. Abbreviations: M, mannose; NA, N-acetylglucosamine; PI, phosphatidylinositol.
PIG-A is located on the X chromosome and is mono-allelically expressed. All the other genes involved in GPI biosynthesis are autosomal. A single mutation in the PIG-A gene is therefore sufficient to disrupt GPI assembly leading to complete loss of function. For the remainder of genes in this pathway, both alleles would need to be mutated in the same cell to affect GPI production. This explains why all cases of acquired PNH which have been examined, harbor PIG-A mutations.
The gene involved is
PHOSPHATIDYLINOSITOL GLYCAN, CLASS A; PIGA
Approved Gene Symbol: PIGA
Cytogenetic location: Xp22.2 Genomic coordinates (GRCh37): X:15,337,572 - 15,353,675 (from NCBI
Gene Phenotype Relationships
Phenotype: Paroxysmal nocturnal hemoglobinuria, somatic
Glycosylphosphatidylinositol (GPI) is a glycolipid that attaches dozens of different proteins to the cell surface. GPI
is synthesized in the endoplasmic reticulum (ER) and transferred to the C termini of proteins with GPI
attachment signal peptides. The common core structure of GPI
consists of a molecule of phosphatidylinositol (PI) and a glycan core that contains glucosamine, 3 mannoses, and an ethanolamine phosphate. Biosynthesis of GPI
anchors involves at least 10 reactions and more than 20 different genes. PIGA
is 1 of 7 proteins required for the first step of GPI
anchor biosynthesis, the transfer of N-acetylglucosamine (GlcNAc) from uridine 5-prime-diphospho-N-acetylglucosamide (UDP-GlcNAc) to PI to yield GlcNAc-PI.
Some of the genes involved in GPI
biosynthesis are represented by different complementation classes of GPI
anchor-deficient mutant cells derived from human and rodent cell lines. By expression cloning using a GPI
anchor-deficient human B-lymphoblastoid cell line belonging to complementation class A, cloned PIGA.
The predicted 484-amino acid PIGA
protein has a single transmembrane domain. Has been reported that the deduced amino acid sequence of the mouse Piga protein is 88% identical to that of the human protein. Database analysis demonstrated that a yeast gene, Spt14, is homologous and that these genes are members of a glycosyltransferase gene family.
Has been reported that the PIGA
gene is at least 17 kb long and has 6 exons. They sequenced the exon-intron and described the characteristics of the 5-prime promoter region.
has been mapped the PIGA
gene to chromosome Xp22.1.
Using human and mouse GPI
anchor-deficient cell lines, has been showed that PIGA
takes part in the synthesis of GlcNAc-PI, the first intermediate in the biosynthetic pathway of GPI
In 1994 has been found that transfection of the mouse Piga cDNA complemented the defects of both a Piga-deficient murine cell line and a PIGA
-deficient human cell line, demonstrating that functions of the mouse and human proteins are conserved.Watanabe, a researcher, in 1996 found that the PIGA
proteins form a protein complex and are subunits of the GPI
GlcNAc transferase of the ER.
They showed that PIGA
is an ER transmembrane protein with a small luminal domain and a large cytoplasmic domain. The luminal domain contains information which targets the protein to the rough ER,
while the cytoplasmic domain has homology to the bacterial GlcNAc transferase RfaK. Watanabe concluded that the first step of GPI
anchor synthesis occurs on the cytoplasmic side of the ER membrane.Using immunoprecipitation experiments, Watanabe demonstrated that PIGQ
associates specifically with PIGA, PIGC,
and that all 4 proteins form a complex that has GPI
-GlcNAc transferase (GPI-GnT) activity in vitro.
The complement cascade and hemolysis in PNH
The complement cascade is an integral part of the innate immune system. It involves sequential reactions that ultimately cause cell lysis either by opsonization and subsequent cell phagocytosis, or by the formation of a phospholipase, called the membrane attack complex (MAC) that punches holes in the cell membrane (Figure 2). CD55
(decay accelerating factor, DAF
) and CD59
(membrane inhibitor of reactive lysis, MIRL
) are widely expressed on all hemopoietic cells and are both involved in the regulation of complement activation. CD55
increases the removal of C3 convertase, thereby reducing the amount of C3 that is cleaved. CD59
inhibits C9 binding to C5b, C6,
C7 and C8 which together make up the MAC.
is then inserted into the cell membrane causing cell lysis.
The complement cascade showing the proximal and the terminal complement components which lead to the formation of the membrane attack complex which in turn causes hemolysis of erythrocytes in paroxysmal nocturnal hemoglobinuria.
The absence or reduced expression of CD55 and CD59 on PNH red blood cells leads to their increased sensitivity to complement mediated attack. This in turn causes the majority of symptoms of the disease. Based on their sensitivity to complement attack, erythrocytes in PNH have been classified into 3 groups. Type I cells are normal red blood cells, type III cells have a complete deficiency of GPI anchored proteins and type II cells have a partial deficiency (Figure 3). The degree of hemolysis suffered by individuals is relative to the proportions of the type II and III cells present. In general, the larger the proportion of type III cells, the more severe the hemolysis suffered by the affected individual.
PNH red blood cell analysis showing type III paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes which express no CD59 (shown on the x axis), type II PNH erythrocytes with reduced levels of CD59 and type I erythrocytes with normal levels of CD59.
is a rare disease, its precise incidence and prevalence has not been well documented. It is not unusual for patients to be misdiagnosed or remain undiagnosed for long periods. The most reliable data on the incidence and prevalence of the disease is from work undertaken in Yorkshire, England. In this study, the prevalence of patients with PNH
clones of any size is 15.9 per million and the incidence is 1.3 per million of the total population. Eighty-two percent of these patients had a granulocyte clone size greater than 1%, with 43% of these greater than 10% and a quarter greater than 50%. Initially, PNH
diagnosis relied on a biochemical assay, the Ham test, in which red blood cells are exposed to acidified serum. Under these conditions, complement is activated via the alternative pathway and causes lysis of PNH
cells as they are sensitive to complement attack. This test was time consuming, non-specific, insensitive and lacked standardization. It was succeeded by flow cytometry
in the 1990s. Flow cytometry is routinely performed to evaluate the size of erythrocyte and granulocyte clones and is considered the “gold standard” for diagnosing PNH.
Peripheral blood granulocyte clone size is believed to be the best marker for evaluating the extent of affected HSC
s in the bone marrow as the erythrocyte clone size can vary depending both on the degree of intravascular hemolysis present and whether an individual has had a recent red cell transfusion of normal erythrocytes. The granulocyte clone size also correlates well with the platelet clone size. Initially, flow cytometry was used to look for the absence of specific GPI
-linked proteins such as CD55
and determination of the presence of these antigens is still routinely used in evaluating the proportion of PNH
red blood cells present. The use of both of these targets in conjunction excludes rare single antigen deficiencies and allows a red cell clone as small as 0.01% to be detected. Evaluation of CD59
expression usually provides the clearest separation of type I, II and III
red blood cells (Figure 3). A wide variety of different GPI
-linked proteins have been used to assess the PNH
granulocyte clone size. The most recent development in this diagnostic field is the development of the FLAER
is a fluorochrome-labeled inactive variant of the protein aerolysin which selectively binds directly to the GPI
anchor. It cannot be used to ascertain the extent of the PNH
red cell clone size as the presence of glycophorin, a non-GPI-linked protein on erythrocytes, binds to the FLAER
can be used alone or in combination with other monoclonal antibodies to GPI
-linked antigens to evaluate the granulocyte clone size. At the moment there is no standardization for the diagnosis of PNH
with a marked diversity seen in the antibodies used between different centers. FLAER
is becoming increasingly used for granulocyte clone evaluation and provides a more accurate assessment, especially of small PNH
clones. In one of the 2 National Centers for treating PNH
patients in England, laboratorians routinely use 6 color flow cytometry with a combination of FLAER, CD16, CD24, CD33, CD15
for granulocyte analysis and CD59, CD55
a for evaluating erythrocyte clone sizes.
A standardized approach to PNH
A proposed classification to allow a standard approach to PNH
patients for clinicians is to divide them into three distinct groupings. Classical PNH
– these patients have the characteristic symptoms of PNH
with intravascular hemolysis and a cellular bone marrow. They have no evidence of any other bone marrow pathology. PNH
in the setting of another specified bone marrow disorder – these patients have symptoms of intravascular hemolysis but also have, or have previously had, an underlying bone marrow abnormality such as aplastic anemia (AA) or myelodysplasia (MDS). Subclinical PNH
– these patients have no evidence of ongoing hemolysis. They have small PNH
clones present and are often seen in patients with bone marrow failure especially in AA and MDS.
These patients have been identified due to the development of improved diagnostic flow cytometry as prior to this these small clones would have remained undetected. The significance and relevance of subclinical PNH
is unclear and will be evaluated in ongoing studies on otherwise healthy individuals. The concern with this approach relates to the overlap between those with classical PNH
that may have a small degree of aplasia and those with PNH
in the setting of another specified bone marrow disorder that may have significant intravascular hemolysis.
Clinical symptoms and the role of nitric oxide
In general, the size of the PNH
clone correlates with the degree of symptoms observed. Therefore, patients who have symptomatic PNH
tend to have larger clones of PNH
cells present. Affected individuals have chronic low grade hemolysis with episodes, or “paroxysms”, of severe intravascular hemolysis. During periods of intravascular hemolysis, free hemoglobin is released into the circulation. Haptoglobin, a protein produced mainly by hepatocytes in the liver, rapidly binds free hemoglobin and this haptoglobin–hemoglobin complex is then degraded in the liver. This process is overwhelmed in PNH
and leads to excess free hemoglobin avidly and irreversibly binding to nitric oxide (NO). NO plays an important role in the maintenance of vascular tone by relaxation of smooth muscle which consequently causes blood vessel vasodilation. Depletion of NO in individuals with PNH
leads to smooth muscle dystonia and this may be responsible for many of the symptoms of the disease. These include esophageal spasm and dysphagia, abdominal pain, severe lethargy and erectile dysfunction in men. NO depletion is associated with the development of a number of cardiovascular morbidities including pulmonary hypertension. Recently it has been shown that PNH
patients have a high prevalence of pulmonary hypertension which is likely secondary to NO depletion. The clinical outcome of patients with PNH
is highly variable from one individual to the next. Thrombosis remains the commonest cause of death in the disease, occurring in 40% of patients, with a third of these being fatal. These thromboses predominantly occur in the venous system with an increase in thromboses in typical sites such as deep vein thromboses and pulmonary emboli as well as at unusual sites, such as the hepatic, mesenteric and cerebral veins. The arterial thrombosis risk is also elevated with increased occurrences of myocardial infarctions and strokes. Hall et al reported the risk of thrombosis in patients with a 50% or greater PNH
clone to be 44%, and those with a less than 50% PNH
clone to be 5.8%. Although the risk is far greater in those with larger PNH
clones, even those with small clones (as low as 10%) have a much higher thrombotic risk when compared with the general population. The underlying mechanisms causing thromboembolism in individuals with PNH
has not been clearly defined and may be multifactorial in nature. NO depletion due to binding of NO to free hemoglobin causes both increased platelet aggregation and adhesion. Additionally, platelets lacking GPI
-linked proteins are susceptible to complement mediated attack which leads to platelet activation and the formation and exocytosis of prothrombotic microvesicles containing the MAC.
These microvesicles have been shown to be present at high levels in the blood of patients with PNH.
Another potential cause for the increased thrombotic risk is due to GPI
deficient neutrophils lacking urokinase type plasminogen activator thereby reducing plasminogen activation and causing a reduction in fibrinolysis. It is likely that the increased thrombotic risk in PNH
is due to a combination of these factors rather than a single underlying mechanism.
Allogeneic bone marrow transplantation is the only curative therapy for PNH.
However it carries a high rate of mortality and morbidity due to infection, graft versus host disease and graft failure. It is only an option for a minority of patients either because they are not suitable candidates for the procedure, a donor is unavailable or treatment with eculizumab may be deemed more appropriate. The introduction of eculizumab treatment plus the fact that around 15% of patients with PNH
undergo a spontaneous remission of the disease, result in transplantation only being undertaken in specific circumstances. Transplantation should be considered when there is an associated severe bone marrow failure, life-threatening hemolysis with no access to eculizumab, recurrent thromboses despite eculizumab treatment and in cases of syngeneic twins. Prior to eculizumab, the mainstay of treatment for patients with PNH
has been supportive in nature. Folic acid is routinely taken, as in other hemolytic anemias, in view of the increased red cell production. Many patients have become transfusion dependent in an effort to alleviate symptoms related to their anemia. Some of these patients have developed iron overload due to the number of transfusions and require treatment with iron chelation. The majority, however, remain in an iron deficient state due to their persistent hemoglobinuria and therefore need to take oral iron supplements. In those patients with a large PNH
clone, warfarin therapy has been employed, which can reduce the risk of developing thrombosis.
Eculizumab is a humanized monoclonal antibody that binds to the complement protein C5 and prevents its cleavage into C5a and C5b. It is comprised of murine complementarity-determining regions within a human antibody framework that includes IgG2 and IgG4 regions. Treatment with eculizumab therefore prevents C5b formation which is necessary to form MAC
through binding to the complement proteins C6, C7,
C8 and C9.
As the clinical features in PNH
are caused by the MAC
attack on erythrocytes, preventing its formation was likely to protect PNH
red blood cells in the circulation (Figure 4). Information on the possible effects eculizumab might have in treating PNH
was first seen in a case reported by Yonemura et al of a patient with co-existing PNH
and a deficiency of C9.
This patient was well with only mild hemolysis and only became unwell after receiving a whole blood transfusion after an operation. Transfusion of whole blood includes exogenous C9 allowing the symptoms of PNH
Eculizumab binding to C5 inhibiting formation of the membrane attack complex and release of C5a.
The importance of both the proximal and the terminal complement proteins can be shown by examining people with rare inherited complement protein deficiencies. Congenital deficiencies of proximal complement proteins result in recurrent severe infections and death early in life. Deficiencies of the terminal complement proteins, C5, C6, C7, C8 or C9 increase the susceptibility to infection with the bacteria Neisseria meningitidis. In these individuals however, the proximal complement cascade remains intact allowing the formation of C3b and its subsequent opsonization and clearance of most other bacteria.C5 is a good therapeutic target as all the proximal complement pathways converge at C5 (Figure 2). Complement blockade at C5 will therefore halt the complement cascade preventing activation of the terminal complement components no matter which initial pathway has been activated. Eculizumab was initially evaluated in the treatment of patients with rheumatoid arthritis and systemic lupus erythematosis. These early clinical studies provided information on the frequency of dosing as well as the doses required to provide complement blockade.
Conclusion and summary
Eculizumab has dramatically changed the way clinicians approach treatment for patients with PNH.
It took just 5 years from the pilot study in 2002 to the drug gaining its Food and Drugs Administration license and its European Medicines Agency license in March and June 2007 respectively. The eculizumab clinical trials have shown that it is safe and well tolerated and provides huge benefits for PNH
patients who previously received supportive therapies. It has been shown to stop intravascular hemolysis and the subsequent symptoms patients develop, reduce or abolish the need for transfusions, stabilize hemoglobin levels, improve patient quality of life, reduce fatigue, reduce the risk of developing thromboses, protect against worsening renal function and decrease pulmonary hypertension. It is likely that in the future eculizumab will improve patient mortality. Although it has many positive points, there are some negatives to eculizumab treatment. It has to be given as an intravenous infusion every 2 weeks, there is a small but definite increase in susceptibility to N. meningitidis, it costs ~US$400,000 per year and it does not cure the disease. Eculizumab is of use in treating classical PNH,
ie, where hemolysis is the predominant disease component. It does not have a role in the treatment of patients with subclinical PNH
with no evidence of hemolysis. Further research into PNH
is needed to ascertain why and how it occurs and may allow future therapies to be developed to cure the disease. For the time being, eculizumab provides patients who have previously suffered with a chronic illness the ability to lead normal family and working lives within their communities.
The pathophysiology of paroxysmal nocturnal hemoglobinuria and treatment with eculizumab. Ther Clin Risk Manag. 2009