Molecular analysis of treatment predictors in ITP
The ITP Support Association awarded a research grant to Dr. Drew Provan and Professor Newland at the Royal London Hospital in March 2001.
|Dr. Provan said: "Professor Newland and I were delighted to hear that we have been awarded a research grant from The ITP Support Association to allow us to carry out some basic studies into ITP. Having completed many clinical studies in this area at The Royal London Hospital we have now extended our studies to include DNA-based molecular studies. Our aim is to look at some immune response genes in order to predict which patients will respond to particular therapies and also see whether we can match the DNA profiles of patients with their indium scan data. In the text that follows we have attempted to put forward our thinking in this complex area. It must be remembered that genetic analysis such as this is a new area for all diseases, not least for ITP.|
We are confident, from studies carried out by other groups, that we will get some interesting answers from our patients who have kindly donated DNA samples for this study. Without volunteers such as these we would not be able to carry out any of these studies. Similarly, without the support of The ITP Support Association we would not be in a position financially to carry out these experiments. We extend our warm thanks to all who worked so hard to raise this impressive sum.
As readers of The Platelet will be well aware, ITP is a disorder affecting both adults and children. From previous studies it is obvious that childhood ITP is different to adults, inasmuch as the bruising in children may be dramatic, but they rarely require much in the way of investigations, and usually get better by themselves, the ITP resolving within a few weeks. The important point to note about childhood ITP is that it is not usually a chronic ailment, unlike the adult type, although in about 20% of cases the child will have ITP similar to that in adults. The hallmark of the adult type ITP is the long-standing relapsing and remitting low platelet count, requiring intermittent courses of steroids, intravenous immunoglobulins IVIg) and many other treatments.
We all have patients who attend our clinics who respond beautifully to steroids and have ITP which is easy to control. At the same time, we have many patients who relapse after we tail off the steroids and who require further therapy, most often with dubious benefit. Although we tend to group all of our ITP patients together, it is clear from the way patients respond to different treatments, that ITP in adults does not represent one single disorder - but rather several different types, each presumably with a different underlying cause. Unfortunately, we have no way of sub-categorising patients at present. As a result we lump all people together and label them as having 'idiopathic ITP', as if this were a single disease, expecting them to respond well to treatment equally. If only we could sub-divide patients into different groups (e.g. ITP Type 1, 2, 3, etc.), then we would be able to offer specific therapies, rather than generalised global immune suppressing drugs such as prednisolone, azathioprine and many others. In effect, we would be in a position to offer patients therapy which we knew would work.
ITP is one of many autoimmune diseases known in humans. These diseases are caused by antibodies directed against the patient's own cells or tissues. Several published studies have shown that we all produce antibodies against our own tissues at variable rates and at different times (these are the autoantibodies). In fact, we can detect T-cells and B-cells, both of which are involved in the immune response, which target 'self' tissues. Thankfully, for most of us, these self-reacting cells are removed and prevented from causing any harm, but it is likely that patients with ITP have self-reactive immune cells that somehow escape removal and cause their damage through coating cells such as platelets with antibody. Once the platelets have been coated with antibody, the spleen and other organs have no choice but to remove them since the spleen believes the platelets are 'foreign'. The platelet count then falls.
The study which we are shortly to start is a molecular study of immune response genes, in an attempt to see how they cause harm and to determine whether particular molecular 'variants' determine how a patient will respond to steroids, IVIg or splenectomy.
The immune system is an immensely complex network of interacting genes and proteins, which coordinates the destruction of invading pathogenic bacteria and viruses. The whole function of our immune system is to keep us healthy by destroying any invading micro-organism quickly and effectively. However, sometimes the immune system response overreacts by producing too much or too little of specific immune response proteins. These proteins include cytokines, and the genes for many of these have now been cloned by molecular labs and their precise DNA sequence worked out.
This provides us with sophisticated molecular methods for looking at mutations or variations (we call these polymorphisms - poly = many, morphism = form) in cytokine genes amongst patients with specific diseases, such as ITP. This has been carried out for other disorders such as juvenile chronic arthritis, which is a condition similar to adult rheumatoid arthritis, but affecting younger patients.
In 1998 a group of research workers published a detailed study showing that a single DNA base change in the interleukin-6 (IL-6) gene was strongly associated with development of juvenile chronic arthritis, and correlated with its severity. The IL-6 protein is one of the anti-inflammatory response proteins, and from studies of inflammatory versus anti-inflammatory response proteins it looks as though autoimmune diseases such as ITP and many others are caused by an imbalance between pro- and anti-inflammatory response genes. In health the body has these finely tuned with a perfect balance of pro- and anti-inflammatory proteins, but tipping the balance either way leads to a variety of disorders, of which ITP may be one example. Other factors doubtless play a role, such as virus infections and other environmental agents encountered during life.
We are also very keen to look at the effect of steroids on ITP. We know from our clinical records how patients have responded to prednisolone when this has been used as treatment for ITP. It appears that mutation within the IL-6 gene may alter responsiveness to steroids, so we are hopeful that the presence of a specific DNA sequence in patients with ITP will highlight those patients who are likely to respond to steroids, and those who are not.
A second group of genes that we wish to look at in this project is involved in the uptake of antibody-coated platelets by the spleen. There are many variants of these genes that have been described in both mice and men, and such variants are associated with differing ability of the spleen to destroy antibody-coated cells, such as platelets. It may well be that those patients who have difficult, refractory and troublesome ITP have specific variations in their antibody receptor genes, compared to other patients who have simple ITP (that responds rapidly to treatment and causes few problems). In addition, because these antibody receptors interact with IVIg the presence of specific variations may correlate with a patient's response to IVIg therapy.
The media is full of reports concerning the Human Genome Project, and you will all be aware that scientists have almost completely worked out the sequence of the entire human genome, an incredible achievement in such a short time! The increasing availability of genetic sequences for genes such as those of the immune system make it easier to design experiments to look at malfunctions of these genes, and to examine the role of such genetic variations in normal populations, and also those with diseases such as ITP.
The Royal London Hospital has been scanning patients with ITP, to determine whether the spleen is the primary site of the destruction, for some years now. These so-called indium scans are highly predictive in terms of a patient's response to splenectomy. Patients whose indium scan shows that the spleen is the main site of platelet destruction have an excellent response following removal of the spleen; however, those patients who have a pattern of platelet destruction involving the liver and spleen do much less well after splenectomy.
We have been very lucky to have the co-operation of patients who have undergone indium scanning, since they have allowed us to take an extra blood sample so that we can look at their DNA for the presence or absence of specific variations in genes, such as those described above. We intend to look at the IL-6 and antibody receptor genes in all patients undergoing indium scanning, to see if the presence of particular variations correlate with the indium scan results. We are hopeful that the presence of specific gene variants will correlate with a specific indium pattern. If we find this is the case, then we will hopefully be able to avoid indium scanning altogether and look at those specific genes, instead of having to subject patients to the indium scanning which is time-consuming and highly expensive. Looking at the genes is much simpler and cheaper - the time from the initial blood sampling to the final results will be around one day or less.
To summarise the above: we have the resources and precious analytical material in the form of ITP patient DNA for this study. We intend to look at a few critical immune response genes in order to determine whether these predict treatment to therapy and also to determine whether these correlate with the indium spleen scan data. If so, these tests will help us predict who will have easy/difficult ITP, who will respond to specific therapies and may also help us predict those who will respond to splenectomy. Like all molecular projects this is ambitious, but with your help, continuing patient support and a strong molecular genetics lab at The Royal London Hospital we are confident that we will provide some answers to this puzzling and complex disorder."
We received a grant from The ITP Support Association to examine the DNA from a number of patients looking for changes within specific genes to see if there were any obvious changes which might be linked to ITP. These results showed that within the TNF-α gene there was a definite poly- morphism linked to ITP. The paper was published in the journal Hematology, volume 16, page 243 (2011).
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