Paroxysmal nocturnal hemoglobinuria

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Classification according to ICD-10
D59.5 Paroxysmal nocturnal hemoglobinuria [Marchiafava-Micheli], excl. Hemoglobinuria NOS
ICD-10 online (WHO version 2019)

The paroxysmal nocturnal hemoglobinuria (PNH) (synonym: Marchiafava-Micheli syndrome ) is a rare, potentially life-threatening disease of the blood , which is due to an acquired genetic defect in the destruction, especially of red blood cells (erythrocytes) by a part of the immune system, the complement system , comes.

PNH can be characterized by anemia with shortness of breath and rapid heartbeat, a tendency to thrombosis , severe exhaustion , stomach and back pain, and a darkening of the urine from hemoglobin ( hemoglobinuria ). The symptoms may worsen in a paroxysmal manner. The eponymous nocturnal hemoglobinuria occurs in only a quarter of patients. The course can vary greatly, but if left untreated, the disease is often fatal. The most common cause of death is thrombosis, often in atypical locations such as the liver veins or on the brain ( sinus thrombosis ).

The cause lies in the bone marrow , where blood cells are formed from blood-forming stem cells. In people with PNH there is a defect in the PIGA gene in one or more blood-forming stem cells , which means that a certain substance can no longer be formed: the glycolipid GPI. GPI is a so-called protein anchor that attaches various proteins to the cell surface. Among these proteins are two important proteins ( CD55 and CD59), which otherwise protect the blood cells from attack by the complement system. Therefore, all blood cells without GPI are attacked by the complement system, but the red blood cells are much more vulnerable than the other cells.

PNH is often an accompanying phenomenon of aplastic anemia , in which there is a general decline in the number of stem cells in the bone marrow. This is probably due to an autoimmune reaction against normal stem cells with GPI, which allows stem cells with GPI defects to spread. The processes in the bone marrow are the subject of current research.

In 1882 the Greifswald doctor Paul Strübing described the first case of a patient with paroxysmal nocturnal hemoglobinuria. Research into paroxysmal nocturnal hemoglobinuria led to the discovery of the alternative pathway for complement activation and thereby to a better understanding of the humoral immune response . At the same time, the disease is an example of how research depends on both careful experimentation and lucky coincidence .

Until 2007 the disease could only be treated supportively, e.g. B. with blood transfusions . For severe cases, the bone marrow transplant was available as a last resort . A targeted therapy of the disease has been possible with eculizumab since 2007 . The active ingredient inhibits attack by the complement system.

Clinical picture

Epidemiology

The disease is not inheritable. An accumulation among relatives or a special gender distribution has not yet been observed, but in a large observational study with predominantly white patients from western industrialized countries, women are slightly in the majority with 54%. The prevalence is estimated at around 16/1 million people and the rate of new cases is estimated at around 1.3 / 1 million per year according to figures for Great Britain and France. The disease often breaks out between the ages of 25 and 45. Worldwide, the incidence is between 1 and 1.5 new cases per 1 million population per year. In Asia, PNH occurs more frequently than in other parts of the world.

Symptoms

Main symptoms of PNH

The severity of the symptoms varies greatly from patient to patient. Often the disease is first noticed by haemolytic anemia , in many cases it is combined with aplastic anemia due to bone marrow failure. Many patients already have a general decrease in all blood cells (pancytopenia) as an expression of bone marrow failure. The eponymous paroxysmal, nocturnal excretion of hemoglobin in the urine (hemoglobinuria) with dark-colored urine only occurs in about every fourth patient. The third cardinal symptom besides anemia and pancytopenia is a pronounced tendency to clot blood (thrombophilia). Many patients suffer from severe exhaustion ( fatigue). It is not related to the anemia but to the extent of the hemolytic activity. It is often made worse by infections, physical exertion, surgery and pregnancy. Some patients complain of stomach and back pain, esophageal cramps, difficulty swallowing and erectile dysfunction . Most patients have or develop impaired kidney function and high blood pressure in the pulmonary arteries ( pulmonary hypertension ).

In addition to these symptoms, which can be traced back to hemolysis, there are also symptoms caused by anemia: dizziness, headache, shortness of breath, rapid heartbeat and paleness. Depending on how far the bone marrow failure has progressed, there are also the consequences of pancytopenia: susceptibility to infection due to the lack of immune cells and a tendency to bleeding due to the lack of blood platelets.

Pregnancy represents a considerable risk for patients with PNH. The risk of thrombosis increases again through pregnancy, which means that pregnant women have an increased mortality rate compared to other PNH patients and suffer more miscarriages than healthy women.

Course and prognosis

The natural course of PNH can drag on for years. In the 1990s, half of the sick died within 10 years of being diagnosed. In the early 2000s, that time lengthened to 20 years. Patients on long-term eculizumab therapy (see below) can have the same life expectancy as healthy people.

Before effective therapies were introduced, thrombosis was the leading cause of death in PNH patients. The thromboses mostly occur in the liver veins ( Budd-Chiari syndrome ), other frequently affected veins are the portal vein, the mesenteric veins, the splenic vein and the venous sinuses of the brain ( sinus thrombosis ). The deep veins of the legs and arms can also be affected, causing pulmonary embolismcomes. Arterial thromboses are less common and can cause heart attacks and strokes, among other things. Paroxysmal nocturnal hemoglobinuria usually occurs with bone marrow failure due to an autoimmune reaction. In patients with pronounced aplastic anemia, bone marrow failure leads to long-term death. In about one to two percent of the sick, PNH turns into acute myeloid leukemia .

Disease emergence

Pathogenesis

The complement system destroys cells by punching a hole in their cell membrane with a membrane attack complex . CD55 acts against the complement factor C3 in the alternative way of complement activation. CD59 inhibits the membrane attack complex. Without these protective proteins, the complement system reacts to the body's own blood cells. (Click to enlarge)

The complement system consists of various proteins in the blood, the task of which is to recognize invading microorganisms such as bacteria, to open up their cell membranes and thereby destroy (lyse) them. The body's blood cells are spared because certain proteins on the surface of the cells, CD55 and CD59 , prevent the complement system from attacking. However, the proteins must be anchored to the cell membrane with a glycolipid , this anchor is called glycosylphosphatidylinositol , or GPI for short. Many blood cells in PNH lack GPI, which is why they are exposed to attacks by the complement system without CD55 and CD59. The red blood cells ( erythrocytes ) are most vulnerable to destruction because they do not have a nucleus.

The cause lies in the blood-forming stem cells of the bone marrow . In PNH, an acquired defect ( somatic mutation ) in the PIGA gene occurs in one or more stem cells . PIGA is the gene for the enzyme N-acetylglucosaminyltransferase. This is responsible for the first step of the GPI synthesis and loses its function due to the mutation. Theoretically, PNH can result from the loss of each of the more than 20 genes involved in GPI production ( synthesis ). In practice there is only one known case in which a PIGA mutation was not the cause. That's because PIGAis on the X chromosome . Men have only one X chromosome, women have two, but only one of them is active and the other is "switched off". Therefore, a single mutation in PIGA is enough to prevent GPI synthesis. All other genes are duplicated, so both variants ( alleles ) of the same gene would have to be switched off by a mutation in order to impair GPI synthesis. This constellation is unlikely and therefore rare.

The PIGA mutation alone is not sufficient for the onset of the disease , since PIGA mutated stem cells can also be detected in most healthy people without PNH . So there must be factors that make the mutated stem cell predominate among the other, non-mutated, stem cells in the bone marrow. On the one hand, an autoimmune reaction against healthy, GPI-bearing stem cells is discussed here. This explains the frequently observed simultaneous occurrence of PNH and bone marrow failure ( aplastic anemia ). With the loss of healthy stem cells, the GPI-free stem cells gain space to spread. On the other hand, genome studies showed that the PIGA-mutated stem cells in PNH, other mutations occur in genes that play a role in regulating cell growth and are often also found in blood cancers.

Pathophysiology

Anemia and hemolysis

The central sign of PNH is constant hemolysis by the complement system. It is intensified by certain triggers such as infections because the complement system is then activated more intensely via antibodies, and during sleep. The hemoglobin released by hemolysis is excreted through the kidneys and causes the urine to darken. Research has long been concerned with the connection between sleep and hemolysis. The common assumption is that slight hypoventilation occurs during sleep . This means that less CO 2 is exhaled, which consequently accumulates in the blood. This lowers the pHSo the blood becomes more acidic, which increases the activity of the complement system. This hypothesis has never been proven, especially since only a minority of patients show classic “nocturnal” hemoglobinuria. Not all PNH erythrocytes are equally sensitive to attacks by the complement system. Depending on the underlying defect in PIGA , the affected cells can show GPI-anchored proteins on their cell membrane to different degrees. The severity of hemolysis then depends on how much (or how little) CD55 and CD59 remain on the cell surface.

Bone marrow failure

PNH is associated with other bone marrow diseases. Approximately 20% of PNH patients also have signs of a general decline in bone marrow blood formation due to aplastic anemia at the time of diagnosis . For a better understanding of this connection, it helps to consider the cause of aplastic anemia: the sudden onset of aplastic anemia in younger people is usually an attack by cytotoxic T cellson your own bone marrow. It is not clear what triggers this autoimmune reaction; there are likely several triggers and targets. In a little more than every tenth case, however, bone marrow cells “escape” the attack. Several properties of bone marrow cells are known that lead to their being spared from attacking T cells. So if the PNH stem cells without GPI are protected from the autoimmune reaction, the conclusion that this must have something to do with the GPI anchor - but to date, no antigen has been identified.

Smooth muscle cell dystonia and thrombosis

Some symptoms, such as pain, difficulty swallowing, impaired kidney function, and pulmonary hypertension, can be explained by tension in the smooth muscle cells found in the walls of blood vessels and digestive organs. They are a result of hemolysis. Free hemoglobin in the blood is normally eliminated through various mechanisms. In the case of hemolytic seizures, however, so much hemoglobin is produced that the protective mechanisms are overwhelmed. This free hemoglobin then binds nitric oxide(NO) in the vessels. Nitric oxide relaxes the smooth muscle cells; if it is missing, the smooth muscle cells contract. This causes blood vessels to constrict, which explains the erectile dysfunction, kidney problems and high blood pressure in the lungs. Muscle cells also contract in the walls of the digestive tract, hence abdominal cramps and swallowing disorders.

The mechanism by which the thrombosis develops is unclear and a problem for future research. One possibility is increased platelet activity due to nitric oxide deficiency, which is also associated with smooth muscle cell dystonia.

Classification

Because PNH is linked to other bone marrow diseases, there are many manifestations of the disease. There are two classifications that attempt to depict this, but none has been established so far. The older one from 2005 distinguishes three subcategories: 1. PNH with hemolysis and thrombosis. 2. PNH in the context of another bone marrow disease (aplastic anemia, myelodysplastic syndrome). 3. the subclinical PNH. Here patients have low levels of PNH cells, but no symptoms. The newer from 2008 is now more common. It also distinguishes between three sub-categories: 1. Hemolytic (classic) PNH. There is thrombosis or hemolysis. 2. aplastic anemia PNH (AA-PNH) with additional evidence of bone marrow failure. These two groups are defined by limit values ​​for the hemoglobin concentration, the number of certain immune cells and the number of blood platelets. Patients who cannot be assigned to either of the two sub-categories based on their blood values ​​therefore fall into category 3: Intermediate PNH , in German roughly "medium PNH".

diagnosis

Diagnosis

The diagnosis is largely guided by the patient's symptoms. Paroxysmal nocturnal hemoglobinuria is often suspected in patients who have been diagnosed with anemia.

A laboratory examination of the blood then shows signs of hemolytic anemia: the hemoglobin level and the number of red blood cells are decreased. At the same time, the concentration of lactate dehydrogenase (LDH) is increased because it is released from the destroyed red blood cells. Unconjugated bilirubin (a breakdown product of hemoglobin) is also increased. The haptoglobin that binds free hemoglobin in the blood is decreased or cannot be detected. Usually a blood smear is also done. This allows the appearance of the erythrocytes to be assessed. In particular, there are no fragmentocytes in PNH , as occur in other hemolytic anemias (see Sect.Differential diagnosis ).

The ham test is no longer used today. In this case, the patient's blood is slightly acidified, in PNH hemolysis occurs. Another test that is no longer used is the sucrose lysis test. Here the complement system is activated by adding sucrose . Both tests were not conclusive enough. Instead, flow cytometry is used to reliably detect the diseaseApplied to a peripheral blood sample: This test can be used to detect the lack of GPI-anchored proteins. If there are blood cells from at least two different blood cell series (e.g. erythrocytes and granulocytes) that are missing at least two such proteins, PNH is very likely. A newer method detects GPI directly: Fluorescence-labeled Aerolysin (FLAER). Aerolysin is a toxin from the bacterium Aeromonas hydrophilius that binds directly to the GPI anchor. It has a fluorescent dye that can be seen under the microscope ( fluorescence microscopy ). As a result, cells that lack GPI do not stain.

Differential diagnoses

The most important differential diagnosis to PNH is autoimmune hemolytic anemia . In this case, the immune system makes antibodies against the red blood cells. These antibodies can be detected in the Coombs test . In PNH, the Coombs test remains negative because there are no antibodies against red blood cells (Coombs-negative hemolytic anemia). Other differential diagnoses are congenital hemolytic anemias, which usually show in childhood, and other acquired hemolytic anemias:

therapy

The therapy recommendations for German-speaking countries are published in cooperation with the German , Austrian and Swiss societies for hematology on onkopedia.com (see web links). In general, the anaplastic anemia, which is usually present, is treated according to the guideline for anaplastic anemia. The therapy of hemolysis-related symptoms depends on the severity of the symptoms. In hemolytic anemia, iron and vitamin B12 can be given to support the formation of new blood. In the event of severe blood loss, blood may need to be transfused. If patients have thromboses, they need a lifelong inhibition of blood coagulation , for example with Marcumar ,Heparin or DOACs . However, this therapy does not rule out re-thrombosis in the case of PNH. Bacterial infections should be treated early with antibiotics to avoid hemolytic attacks.

For a long time the only prospect of a cure was a bone marrow transplant . However, this therapy carries a high risk of death for the patient, which is why it was only an option in severe cases as a last resort.

Eculizumab was presented in 1996 : the genetically engineered monoclonal antibody against complement factor C5 was tested in several clinical studies in the following years. Eculizumab has been in the US and the EU since 2007approved under the brand name Soliris. Study results with the active ingredient showed rapid and sustained control of chronic hemolysis. PNH patients suffer almost no more thrombosis during eculizumab therapy, which was otherwise the main cause of death. Almost half of the patients no longer required blood transfusions. Symptoms typical of PNH such as B. abdominal pain, difficulty swallowing (dysphagia) and erectile dysfunction (erectile dysfunction) could be improved with eculizumab treatment. Overall, there was a significant increase in the quality of life of the patients u. a. by improving the symptoms of fatigue. Eculizumab was well tolerated and the side effect profile was comparable to that of the placebo group. Since eculizumab blocks part of the complement cascade and thus also hinders the defense against bacteria, the risk of a meningococcal infection with the result of meningitis is increased. For this reason, all patients must be vaccinated against meningococci at least two weeks before starting therapy. Long-term survival data from patients who have been treated with eculizumab for up to 8 years show that the life expectancy of patients is approximating that of the normal population.

In December 2018, the American FDA approved ravulizumab (trade name: Ultomiris), which is also a monoclonal antibody against complement factor C5. Ravulizumab was approved in the EU on July 2, 2019.

The German guideline recommends eculizumab and ravulizumab equally for the treatment of symptomatic patients.

Research history

The American hematologist Charles Parker from Salt Lake City , Utahhas dealt intensively with the research history of paroxysmal nocturnal hemoglobinuria. In an extensive article from 2002, he divides the history of research into an "early history" and a "modern history". The first phase was characterized by the description and definition of the clinical picture. The discovery of the alternative activation pathway of the complement system marked a turning point because from now on the molecular-biological basis of the disease was uncovered. Research reached its preliminary climax in 1993 with the discovery of the genetic cause. Parker asks why such a rare disease received relatively much research attention. He believes that the "elegant complexity" of nature that can be recognized in the pathophysiology of PNH, hold a fascination for hematologists. On the one hand, PNH research drove progress in research into the immune system. On the other hand, it is an example of how incidental observations from neighboring research areas can provide crucial insights for one another.

From the first case report to the alternative path

The description of the disease

The first description of a patient with paroxysmal nocturnal hemoglobinuria is attributed to the Greifswald doctor Paul Strübing , who in 1882 was still an assistant doctor who dealt with the 29-year-old cartwrightCarl G. describes. Since 1876 he noticed a morning dark discoloration of the urine, while his skin was noticeably paler and turned gray-yellow in color. The symptoms were increased like attacks and were accompanied by exhaustion, dizziness and palpitations as well as pain in the spleen and kidney area. Strübing found that the urine contained hemoglobin, but no red blood cells. He also observed that after severe seizures, the blood plasma also turned red. From this he drew the (correct) conclusion that there must be a destruction of red blood cells in the blood vessels. He assumed that sleep played a decisive role because dark urine was excreted after the patient woke up at night, but otherwise not. Strübing was known to have erythrocyteslyse faster in an acidic environment (with incubation with CO 2 , because a higher CO 2 concentration in the blood acidifies it). He therefore hypothesized that the cause of hemolysis lies in the fact that the CO 2 concentration increases during sleep due to slower blood flow . This makes the blood more acidic, which causes hemolysis. Strübing tried to substantiate his hypothesis: he administered acid to his patient to induce a seizure, but this did not succeed.

In fact, Strübing's description is not the oldest publication describing a case of PNH. For example, an older description comes from the British doctor William Gull (1866); the oldest could be from 1793. Strübing's achievement, however, was to recognize PNH as an independent disease and to differentiate it from hemoglobinuria caused by cold ( autoimmune hemolytic anemia of the cold type) and march hemoglobinuria , which is why he is recognized as the first to describe it. Strübing later embarked on a career outside of hematology, which is why he did not pursue the disease any further.

From 1908 the disease was increasingly researched and described, a review article from 1949 names 73 case reports that were published between 1908 and 1949. From this period, the work of the Italians Ettore Marchiafava and Ferdinando Micheli, who worked on cases in 1928 and 1931 and described them as a syndrome, should be emphasized. Due to its achievements, the disease was known as Marchiafava-Micheli syndrome until the 1960s , before the term went out of fashion. The name "paroxysmal nocturnal hemoglobinuria" was first used in 1928 by the Amsterdam doctor J. Enneking and was becoming increasingly popular.

Van den Berg's experiments

An important work for the understanding of the disease was done by the Dutchman Hijmans van den Berg . In 1911 he showed that the erythrocytes of PNH patients lyse in serum in vitro (i.e. in a test tube) in a CO 2 atmosphere - in the patient's serum , but also when they were added to the blood serum of healthy people of the same blood group . At the same time, it showed that the erythrocytes of healthy test subjects survived the same treatment unscathed. This proved that the cause of the hemolysis lay in the erythrocytes and that it could not be a pathological factor in the blood serum.

Van den Berg's experiments revealed another important finding because they were the first to bring the complement system into play. At that time, three components of the humoral immune response were known: antigen , antibodyand the complement system, which complements the function of the antibodies. The complement system was known to be destroyed by heat and restored to function by giving a small amount of blood serum (containing complement factors). Van den Berg observed that there was in fact no hemolysis if the serum was heated beforehand. This suggested that complement is involved in hemolysis. But, contrary to what was expected prior to the knowledge of the time, hemolytic activity could not be restored by adding fresh serum. Van den Berg therefore rejected the assumption that the complement system could be involved in hemolysis.

Today we know that the properties of the complement system known at the time apply to the classic way of complement activation. This is the complement activation by binding an antibody to an antigen. This mechanism plays a role in autoimmune hemolytic anemia, but not in PNH. In PNH, complement factors bind directly to structures on the cell surface and thereby activate the membrane attack complex- the alternative way of activation, the existence of which was only proven in the 1960s. The classic activation via the formation of antibody-antigen complexes works even with high dilutions, which is what restores the complement activity by adding small amounts of serum. In contrast, the alternative activation is no longer possible even if the complement factors are only slightly diluted. This is why van den Berg was able to prevent hemolysis by heating the complement, but when the fresh complement factors were added, they were too diluted to be activated via the alternative route.

Thomas Hale Ham

From 1937 Thomas Hale Ham , a doctor from Cleveland , Ohio , began studying PNH. His work should point the way for research over the next decades. Like Strübing, whose publication Ham did not know, he first looked at the connection between sleep and hemoglobinuria. By shifting a study patient's sleep rhythm, he was able to show that hemoglobinuria depends on sleep and not on the time of day. He was also able to induce hemolytic seizures in study patients by giving them acid, and vice versa, he was able to prevent seizures by alkalizing their blood (by giving them bases or by giving them an iron lung while sleeping hyperventilated ). Like Strübing, Ham believed that hemolysis was caused by an increased CO 2 concentration in the blood caused by hypoventilation during sleep.

Next, Ham supplemented his experiments on humans with laboratory experiments. He repeated van den Berg's essential experiments, although he made no reference to van den Berg's work - it is not clear whether they were still unknown to him at the time. Ham also correctly concluded that the cause of the disease lies in the patient's erythrocytes, while the hemolytic factor must be a normal component of the blood. Ham explained why no antibodies are involved in the process of hemolysis, but came to the wrong conclusion that a previously unknown system different from the complement system must mediate hemolysis.

From the observation made in this series of experiments that PNH erythrocytes lyse when the blood acidifies, a diagnostic test was later developed, the Ham test . This was the standard method in diagnostics for 50 years until it was replaced by flow cytometry.

In 1939 Ham published one of the most influential papers with his colleague John Holmes Dinglein the research history of PNH, in which they explain in detail that a previously unknown mechanism lyses the erythrocytes. The finding that no antibodies are involved was completely new and did not fit into the previous picture of the humoral immune response. Although they describe several strong indications of a role for the complement system, and presumably themselves believed that it is responsible for hemolysis, they claim in the discussion part of the article that the complement system is not the lysing factor. Presumably, they were reluctant to draw their conclusions because they suspected that their results would meet with skepticism and rejection in the professional world. A secondary finding that was significant in retrospect was the observation

Discovery of the alternative pathway for complement activation

The decade after Ham and Dingle's publication saw little progress. The research continued to gather data that spoke for and against a role for the complement system. In retrospect, an important discovery was that the hemolytic process in PNH (via the then unknown alternative complement activation ) depends on magnesium ions (Mg2 +). This is where it differs from autoimmune hemolytic anemia.

Louis Pillemer provided an initial explanation of which part of the immune system mediates hemolysis in PNH . In 1954, while trying to isolate what was then known as the third complement factor (which actually consists of several complement factors ), Pillemer accidentally isolated properdin - a protein that supports the addition of complement factor C3b to factor B. Pillemer noted that properdin was only active in the presence of complement and magnesium. It worked on the destruction of bacteria , virusesand “certain red blood cells” with - PNH erythrocytes. It was found that antibodies were not required for properdin activity. Two years later, Pillemer and his colleagues Carl F. Hinz and William S. Jordan demonstrated that although properdin is absolutely necessary for hemolysis of PNH erythrocytes, it is not necessary for hemolysis in autoimmune hemolytic anemia. Pillemer's studies on properdin and PNH provided strong evidence that there is antibody-independent complement activation. However, Pillemer's hypothesis of the properdin system could not prevail among complement system researchers, especially because small amounts of antibodies were found in his samples for technical reasons. His colleagues therefore assumed that these explained complement activation. Pillemer died in 1957 of an overdose of sleeping pills after meeting other scientists in the field. By contrast, the properdin system quickly found widespread acceptance among hematologists as an explanation for the hemolysis of PNH, as it best matched the observations of the previous decades. Only after the development of better processing methods and 10 years after Pillemer's death could his assumptions be confirmed. By 1980 the components of the alternative activation path had been clarified. Only after the development of better processing methods and 10 years after Pillemer's death could his assumptions be confirmed. By 1980 the components of the alternative activation path had been clarified. Only after the development of better processing methods and 10 years after Pillemer's death could his assumptions be confirmed. By 1980 the components of the alternative activation path had been clarified.

From the alternative path to the genetic cause

PNH is a mosaic

According to Ham and Dingle, researchers observed time and again that hemolysis never affected all erythrocytes in PNH patients, which suggested that two different populations of erythrocytes are found in the blood of those affected (i.e. form a mosaic). The British hematologists Wendell F. Rosse and John Dacie examined this question in more detail in 1966. By closely observing the course of hemolysis in healthy blood and the blood of PNH patients, they demonstrated that PNH patients have a certain proportion of erythrocytes susceptible to hemolysis, while the rest of their red blood cells show normal sensitivity to the complement system. The proportion of erythrocytes susceptible to hemolysis was between 4 and 80%, which explains the different severity of the symptoms. Originally it was assumed that there are only two populations of PNH cells: one that is very susceptible to hemolysis by the complement system, and one that is not. This assessment had to be corrected by further studies in the 1970s, however, because several erythrocyte populations were found in some patients whose susceptibility to hemolysis was between the two originally postulated groups.

Also in 1969 it was shown that the blood platelets and certain immune cells of PNH patients are more susceptible to complement-mediated hemolysis, which supports the thesis that these cells have a common ancestor and that the cause of PNH is a somatic mutation. At this point in time, the existence of hematopoietic stem cells, from which all blood cell lines can arise, had not yet been proven and was highly controversial. The results of PNH research provided arguments for the proponents of this theory.

Discovery of the complement regulators

Edward M. Hoffmann, a Florida hematologist , was the first to report in 1969 on a factor in erythrocytes that can prevent complement-mediated hemolysis. He called this decay accelerating factor (DAF) because it accelerates the decline in complement activity. DAF (now known as CD55) prevents the conversion of the complement factor C3 to C3b. In doing so, it blocks complement activation via both the classic and the alternative route. In 1983 Anne Nicholson-Weller reportedand colleagues that PNH erythrocytes lack DAF. It quickly became generally accepted that this lack of DAF would play a causal role in the pathogenesis of the disease. He did not explain everything, however, because there were also indications of impaired regulation of the membrane attack complex (MAC). The regulator for MAC could only be identified in 1989. Its discoverers gave it the name membrane inhibitor of reactive lysis (MIRL, in German "Membrane inhibitor of reactive lysis"), today it is known as CD59 .

Knowing these two regulators could explain why PNH erythrocytes are differently susceptible to lysis. Erythrocytes completely lacking both regulators are the most susceptible to the complement system. There are, however, erythrocytes whose susceptibility is less pronounced because they still have less CD55 and CD59 on their surface. While the amount of CD59 is still sufficient to control the MAC, CD55 can no longer prevent the conversion of C3 to C3b.

The GPI anchor

The discovery of the GPI anchor falls between the discovery of DAF and MIRL . A few incidental observations ( serendipity ) led to this, and to the recognition of its importance for PNH . The first dates from 1951, when researchers who were actually interested in leukemia happened to also examine leukocytes from a PNH patient and noticed that they lacked alkaline phosphatase . Other researchers reported in the 1950s that PNH erythrocytes also lack acetylcholinesterase and 5′-nucleotidasecan be found. These enzymes are not causally related to the disease, but raised the question of why three unrelated enzymes are missing on PNH cells. The answer came in 1985 after a Science article reported experiments with a bacterial enzyme carried out in the 1970s. This enzyme was a phospholipase that cleaves phosphatidylinsositols . The article reports that the use of phospholipase leads to the release of alkaline phosphatase, acetylcholinesterase and 5'-nucleotidase, as well as Thy1 ( CD90 ), from cell membranes. Scientist from the University of New Yorkrecognized the connection to the PNH. They obtained the phospholipase and were able to show that DAF (CD55) is also released from the cell membrane by it. It was clear to the researchers that every protein that is missing on the surface of PNH erythrocytes has the same anchor, and that, conversely, every protein that is normally connected to the cell membrane via this anchor must be missing on PNH cells.

Deciphering the genetic cause

By 1987 the structure of this anchor, glycosylphosphatidylinositol, had been elucidated. It has been assumed since the 1960s that the cause of PNH could be a somatic mutation. When the defect in the GPI anchor became known to be central to the development of PNH, research was faced with the problem of identifying the causal genetic defect. In theory, damage to any of the countless genes involved in GPI synthesis and anchoring the proteins in the cell membrane was conceivable. Initial experiments also indicated that the disturbance could be in many different places.

Once again, a lucky coincidence came to the rescue. In the 1970s a line of lymphoma cells was grown to study the genetic basis of cancer development . Coincidentally, these cells also lacked CD90, which was irrelevant in the context of lymphoma research. Knowing about the anchoring of CD90 by GPI, Japanese researchers working with Taroh Kinoshita used this well-researched cell series to decipher the genetic cause of PNH in 1993.

In 1993, Kinoshita and his colleagues demonstrated in a series of challenging experiments that the genetic defect that leads to the lack of the GPI anchor in lymphoma cells must be the same as that in the PNH cells they examined. They concluded this from the fact that the cells without a GPI anchor could not produce N-acetylglucosamine phosphatidylinositol - which is the first step in GPI synthesis. They were able to identify a gene that appeared to be responsible for this step. They copied this from a healthy cell and transferred it to lymphoma cells without GPI. After transfer, the cells synthesized N-acetylglucosamine phosphatidylinositol. The researchers named the gene PIGA(Phosphatidylinosytol Glycan Class A). They compared the code of the gene in GPI-bearing and GPI-less cells and found differences in the GPI-less cells. The scientists proved that they were acquired mutations by comparing healthy and diseased cells from the same patient. The PIGA mutation was only found in the PNH cells. If the PIGA mutation were a germline mutation , it would be found in every cell. The working group provided strong evidence that this mutation occurs in a hematopoietic stem cell with the detection of the same mutation in B lymphocytes and neutrophils of the same patient. In the end she was able to PIGA locate on the X chromosome.

So far around 150 different mutations have been described in PIGA , the effects of which on GPI synthesis range from functional impairment to complete loss. This diversity in the genotype of PNH stem cells explains why PNH cells differ in their susceptibility to hemolysis.

Web links and literature

Individual evidence

  1. ↑ a b c d A Roth, U. Duhrsen: Paroxysmal nocturnal hemoglobinuria In: Dtsch Arztebl , 2007, 104, pp. 192–197
  2. ↑ a b G. Socié, H. Schrezenmeier, P. Muus, I. Lisukov, A. Röth: Changing prognosis in paroxysmal nocturnal haemoglobinuria disease subcategories: an analysis of the International PNH Registry . In: Internal Medicine Journal . tape 46 , no. 9 , 2016, ISSN  1445-5994 , p. 1044-1053 , doi : 10.1111 / imj.13160 .
  3. ↑ a b c d e Jörg Schubert, Alexander Röth, Peter Bettelheim, Georg Stüssi, Britta Höchsmann, Jens Panse, Tim Henrik Brümmendorf, Hubert Schrezenmeier: Paroxysmal nocturnal hemoglobinuria (PNH) . DGHO guidelines on onkopedia.com, as of November 2019. Last accessed on March 20, 2020.
  4. ↑ a b c d e f g h i j k l m n o p Anita Hill, Amy E. DeZern, Taroh Kinoshita, Robert A. Brodsky: Paroxysmal nocturnal haemoglobinuria . In: Nature Reviews Disease Primers . tape 3 , no. 1 , May 18, 2017, ISSN  2056-676X , p. 1–14 , doi : 10.1038 / nrdp.2017.28 .
  5. ↑ a b c d Lucio Luzzatto: Haemolytic anemia and anemia due to acute blood loss , for the German edition by Andreas Loew and Julia Jesse. In: Harrison's Internal Medicine , 19th edition. ABW-Verlag, Berlin 2016. ISBN 978-3-88624-560-4 . P. 804
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