The team looking after PNH patients in Leeds has recently submitted several review articles to medical journals looking at various aspects of the disease. This document is an attempt to summarize the up-to-date understanding of PNH as outlined in these articles (but with less medical/scientific jargon). There is a glossary at the end to help explain any scientific terms. The opinions outlined in this paper are those of the team at Leeds including Dr Peter Hillmen, Dr Claire Hall and Dr Steve Richards.
PNH is caused by a change (mutation) in a gene that develops during life (ie the patient is not born with the defect and the disorder is not inherited). The mutation affects the very earliest kind of blood cell in the bone marrow (where blood is produced). This cell is known as the haematopoietic stem cell ('seed cell' of the marrow) because it has the ability to mature into all three kinds of blood cell: white blood cells, platelets and red blood cells. The PNH abnormality is therefore found in all three cell types.
The three major clinical problems associated with PNH are:
PNH is uncommon, with an incidence of 1 per 100, 000 to 1 per million. It can occur at any age and affects men and women equally.
PNH was one of the first haematological conditions to be defined clearly. Its classical presentation with dark urine in the early morning must have fascinated nineteenth century doctors.
In 1866, Sir William Gull, from Guy's Hospital in London, described an anaemic young tanner with 'haematinuria' (presumably meaning blood in the urine in the morning).
In 1882, Strübing, a German physician, recognized that it was haemoglobin (the iron-carrying pigment in blood) causing the discolouration of the urine.
Enneking, in 1928 gave the disease its picturesque title: 'Paroxysmal Nocturnal Haemoglobinuria' (haemoglobin in the urine at night and occurring in attacks rather than continuously).
| Haemoglobinuria: Urine passed 7am is dark containing a large amount of free haemoglobin which leaks out of PNH red-cells as they burst. The urine generally clears during the day. |
In the 1940s, Ham realized that the red blood cells in PNH are particularly sensitive to attack by a molecule in the blood called complement. He developed a laboratory test for PNH (Ham test) that has survived to the present day.
In the 1970s to the 1990s, work was done in various laboratories throughout the world to find out what the actual abnormalities present on the surface of blood cells are that makes them vulnerable to destruction by complement.
In 1993, Taroh Kinoshita's group in Japan isolated the gene, called pig-a, that undergoes a change (mutation) which is responsible for the cellular abnormalities and therefore the clinical picture of PNH.
The GPI (glycosylphosphatidylinositol) anchor is a molecule that attaches proteins to the surface of cells. The significance of GPI-anchoring is incompletely understood but it might have a role in immunity (fighting off infection and other foreign insults) and in cell signalling (passing messages between cells). GPI-anchored molecules are found in all types of organisms, from yeasts through birds and reptiles to mammals.
Proteins anchored to cells by the GPI system can be removed easily by the action of specific enzymes - phospholipases. Shedding of surface proteins by this method might allow the cell to avoid immune attack. In mammals, this remains theoretical but in the trypanosome (a single-cell organism which can cause diseases, such as sleeping sickness) the GPI-anchor is the means by which the organism avoids destruction by its host (the infected mammal). The trypanosome is coated in GPI-linked protein called VSG, against which the host mounts an immune reaction in an attempt to clear the organism. In return, the trypanosome activates a specific phospholipase enzyme to remove the GPI-anchor and so shed the VSG coat. The organism has the ability to produce at least 200 variations in the VSG molecule and so can continue to produce new coats and therefore avoid immune attack.
Further understanding of GPI function comes from studies involving the parasite, Schistosoma mansoni. The schistosome causes the tropical disease, Schistosomiasis, and is able to avoid destruction by the host's immune system by inserting host GPI-linked proteins into its own cell membrane possibly by means of its own GPI anchor system.
It is suggested that, where GPI-anchored proteins are found clustered with other molecules in specialized areas of the cell membrane (caveolae), they may represent 'rafts'. It is proposed that these rafts have a role in cell signalling.
It has also been found in mammalian studies that GPI-anchored proteins can transfer between cells. This is referred to as 'painting'.
The significance of all these recent findings to PNH remains unclear.
| Structure of GPI anchor: The biochemical defect in PNH occurs at the first step in the production of the GPI anchor: at the transfer of the glucosamine to the phosphatidylinositol. |
The pig-a (phosphatidylinositol glycan complementation class A) gene is found on the X chromosome and the protein it produces is responsible for the first step in the production of the GPI anchor. Over 20 other genes involved in GPI production have now been described but these are not involved in PNH. In all reported cases of PNH there is one or more abnormality of the pig-a gene. The abnormalities are extremely diverse and result in blood cells with either total (Type III cells) or partial (Type II cells) deficiency of GPI-linked proteins.
All proteins attached to the cell membrane via the GPI anchor have been found to be missing from PNH blood cells. The two missing proteins thought to cause the clinical manifestations of the disease are:
Both proteins are involved in the protection of cells from the action of complement (a protein involved in the immune system that acts to break cells down). In the absence of CD55 and CD59, blood cells are vulnerable to attack from complement; red blood cells are destroyed prematurely and platelets undergo changes that increase the risk of blood clotting.
While we understand the genetic mutations in the disease, we have little idea why the abnormal cells become predominant. Recent findings have supported the 'dual pathogenesis' theory proposed by Dacie in 1980 and developed by Rottoli and Luzzato in 1989:
Mutations of the pig-a gene have been found at very low levels in some normal individuals and are probably common events in early blood cells (stem cells). For this abnormal group of cells to increase in size, their growth must be favoured compared to the remaining normal stem cells. This might occur if the immune system attacks normal stem cells, at least in part, through GPI-linked proteins on the cell surface. The PNH cells (pig-a mutated, GPI-deficient), protected from attack, would therefore have a growth advantage.
The growth advantage of PNH cells over normal cells could explain why PNH is related to a condition called Aplastic Anaemia in which the bone marrow fails to produce blood cells. Its cause is unknown, but it is likely that the marrow stem cells are altered by an unknown factor (perhaps a virus or chemical). As a result these early blood cells are recognized as foreign by the immune system. The subsequent immune attack is probably mediated via GPI-linked proteins. This situation would favour the growth of GPI-deficient cells (which would avoid immune attack) and the emergence of PNH.
The tendency for blood clots (thrombosis) to occur in PNH was first described by Crosby in 1953. Thrombosis occurs at an increased rate in the veins (taking blood from the body to the heart) rather than the arteries (taking blood from the heart to the body). There is a particular tendency for involvement of liver, abdominal and cerebral (around the brain) veins. Thrombosis in PNH is likely to be related to the increased vulnerability of GPI-deficient platelets (cells that clot the blood) to attack by complement. The platelet surface is thus damaged, increasing the risk of thrombosis.
In a recent study of 167 patients known to our laboratory (HMDS) in Leeds, those with large PNH clones (a clone is a family of cells derived from a single stem cell) have a particular risk of thrombosis. If blood PNH cells are greater than 50% (62 patients), the risk of thrombosis was found to be 42% at 10 years. This compares with a risk of 6.4% at 10 years in those (47 patients) with PNH cells less than 50%.
In order to prevent further clots, warfarin, a blood thinning drug, is prescribed for patients who have suffered thrombosis. At Leeds, we now recommend warfarin for selected patients who have large PNH clones even if they have not suffered blood clots. Patients at risk of bleeding, either due to a low platelet count or other causes, are excluded however. In our study, 38 patients who had not had a previous thrombosis took warfarin for an average of 23 months. There were no episodes of thrombosis in this group. This compares with a group of 50 patients, not on warfarin (usually their own or their physician's choice) with large clone sizes, who had a 10 year risk of thrombosis of 34% (this excluded those individuals who had blood clots when they presented with PNH). However, two of the patients on warfarin had serious bleeding episodes and therefore the risks and benefits of taking the drug need to be carefully considered.
For many years, the Ham test was the gold standard. In this test, a blood sample is taken and the patient's red blood cells are mixed with acidified serum from a normal donor (the serum is a source of complement which is activated by the acidification). PNH blood cells are destroyed by complement whereas normal cells are more robust.
Flow cytometry is now the laboratory investigation of choice. This method measures GPI-anchored proteins directly on blood cells but requires expertise for interpretation of results. At least two types of cells are studied, usually white cells and red cells, and the percentage of GPI-deficient cells are reported.
| Granulocyte flow cytometry in PNH. Granulocytes are electronically selected (upper left plot: red R1 region), and analysed for expression of CD16, CD55 and CD66 cell-membrane proteins (lower dot-plots). Two cell populations are visible, a residual normal and the GPI-deficient PNH clone. |
| Red-cell flow cytometry in PNH. Red-cells are analysed for expression of CD55, CD59 and Glycophorin-A (CD235a; red-cell marker). The normal and GPI-deficient PNH red-cell populations (defined by CD55 and CD59) are visible in the histogram overlay plot. The lower right histogram shows three CD59-defined red-cell populations, Types I (normal), II (partial deficiency) and III (complete deficiency). |
Patients in whom Aplastic Anaemia is the predominant disease, generally have small PNH clone sizes. Those with 'haemolytic' PNH, (haemoglobin in the urine, anaemia), usually have large clones, often near to 100% affected white blood cells.
The standard treatment of haemolytic PNH involves the use of folic acid (usually 5mg per day) which the bone marrow needs to make red blood cells. Patients who have low blood iron levels (usually assessed by the ferritin level), because they pass a lot of iron out into their urine, may require iron tablets. Patients who have severe anaemia with symptoms may need occasional or regular blood transfusions.
Corticosteroids, such as prednisolone, are recommended by some doctors who treat PNH as a possible way of reducing haemolysis. There is little or no good evidence that corticosteroids are effective in PNH and they are certainly associated with some potentially serious long-term complications. We do not recommend corticosteroids for our patients with PNH but accept that not all haematologists with an interest in PNH agree with this policy.
Patients who develop blood clots need urgent medical treatment and are usually treated with medicines to thin the blood such as warfarin. There is some evidence that warfarin prior to a patient's first thrombosis may be beneficial but this requires careful assessment on an individual patient basis. Other risk factors for thrombosis, such as surgical operations, long-haul flights, hormone replacement therapy or pregnancy, can increase the risk of thrombosis and should be discussed with the patient's specialist, if at all possible.
Bone marrow transplantation (BMT) from a brother or sister carries a high risk and should only be recommended after very careful consideration by the patient and his or her haematologist. Immunosuppressive therapy, such as cyclosporin A, may be useful in patients in whom cytopenias (low blood counts) are the major problem.
Finally there are new drugs which are currently being developed that may be useful in PNH. There is reasonable hope that there will be better treatments for PNH available in the foreseeable future.
Although PNH is a rare disease, it has always aroused interest in the medical profession. In its early days this was because of its dramatic symptoms. More recently, it is hoped that growing understanding of the molecular defects in PNH might cast light on the formation of blood cells and a wider canvas of diseases affecting the blood. Some patients affected by PNH lead relatively normal lives but for many patients the disease has profound effects on their general well being. Some patients develop complications, such as thrombosis, and these complications can be life threatening. It is hoped that new effective therapies will soon be developed for PNH to reduce the symptoms and complications of the disease.
Antigen: a particle, often on the coating of cells, which the body can recognize as 'self' or 'non-self'. A molecule that can be recognised by the adaptive immune system (B or T cells).
Chromosome: a thread-shaped structure, consisting of DNA in the form of a helix, which is contained within living cells and carries genes.
Clone: the cells produced by division from a single cell.
Enzyme: a complex protein that promotes (or catalyzes) biochemical changes without being used up in the reaction. Enzymes are produced in living cells and are usually highly specific.
Gene: a unit of the material by which living things inherit their characteristics. In humans this is coded in DNA, carried on chromosomes.
Glycosylphosphatidylinositol (GPI) anchor: the structure by which some antigens are attached to the cell surface. It is this structure that is disrupted in PNH cells which leads to the loss of GPI-linked antigens and thus to the symptoms of PNH.
Haematopoiesis: the production of blood cells.
Haemoglobin: a red pigment, partly formed from protein, which is present in red blood cells. It links oxygen with iron and transports it round the body.
Host: an organism infected by a parasite.
Immunity: the ability of an organism to resist infection by the recognition of 'self' and 'non-self'. The immune system involves cells (mainly lymphocytes), antibodies and antigens.
Membrane: the coating of a cell, consisting of a double layer of proteins and lipids. This complex surface regulates transport in and out of the cell. The specificity and configuration of its proteins are involved in immunity, cell recognition and signalling.
Molecule: a specific combination of atoms, the building units of organic chemistry.
Mutation: a change in chromosomal DNA, causing a transformation in individual genes.
Protein: an organic compound consisting of a chain of amino acids folded into a complex structure.
Stem cells: cells with the potential to differentiate into a diversity of cell types.
Thrombosis: a clot, or the physiological process by which clotting occurs.
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