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Project Title:
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STRUCTURE/FUNCTION RELATIONSHIPS IN THE MUNC13-4 PROTEIN: MOLECULAR CONSEQUENCES OF FAMILIAL HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS ASSOCIATED MUTATIONS.
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Investigator:
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Janos Sumegi MD
Cincinnati Children's Hospital Medical Center - Cincinnati, Ohio
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Amount:
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$40,500
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Length:
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One year
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Summary
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| Hemophagocytic lymphohistiocytosis is a rare life-threatening immune disorder, often referred to as "orphan", characterized by fever, hepatosplenomegaly, and pancytopenia, hypertriglyceridemia, hypofibrinogenemia and, frequently seizures. HLH has been categorized, clinically as primary or secondary HLH. The primary form, known as familial HLH (FHLH), an autosomal recessive disease invariably fatal when untreated, can result from a variety of pathologic gene mutations that affect critical pathways, which regulate the function of cytotoxic T cells and NK cells. Genetic defects in NK cell function permit excessive expansion of antigen presenting cells (histiocytes) and T cells in response to immune activation, and the consequent prolonged secretion of proinflammatory cytokines. The failure of NK cell regulation of activated populations of immune cells results in the clinical manifestations of HLH. Mutations in the MUNC13-4 gene encoding a protein which primes the function of the downstream protein receptors required for exocytosis of the lytic granules. Exocytosis of the lytic granules is a key event in the apoptotic killing of target cells suggesting that the underlying defect in FHLH is the lack of apoptosis triggering within the immune system. This proposal offers: to study the consequences of naturally occuring pathologic mutations of MUNC13-4 on the structure and function of the protein. |
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Project Title:
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THE MECHANISM OF REGULATION OF DENDRITIC CELL DEFFERENTIATION BY ADENOSINE
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Investigator:
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Mikhail M. Dikov Ph.D.
Vanderbilt University Medical Center - Nashville, Tennessee
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Amount:
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$45,000
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Length:
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One year
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Summary
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The optimal therapeutic strategies for multisystem Langerhans cell histiocytosis (LCH) remain to be defined. Advances in LCH immunology suggest that aberrant differentiation of dendritic cells (DCs) might underlie the defective interaction of immune cells, leading to a unique pathological picture within LCH lesions, which combines features of both carcinogenesis and chronic inflammation.
Recent works including ours have identified adenosine as an important factor responsible for altered differentiation of DCs and Langerhans cells (LCs). We have preliminary data demonstrating that these cells express a variety of proinflammatory and angiogenic cytokines and factors, the hallmarks of histiocytotic lesions, and thus can contribute to the development of the disease.
In the current project, we propose to determine the molecular mechanisms of adenosine effects on DC and LC differentiation and to provide rationale for therapeutic interventions aimed on disruption of adenosine signaling. We also intend to test in vitro the efficiency of adenosine receptor subtype-specific antagonist treatment in preventing accumulation of intermediate DCs and rescuing normal DC/LC differentiation. The goal of the study is to improve our understanding of the role of adenosine receptors in the differentiation of DC/LCs and pathophysiology of LCH and to validate the use of anti-adenosine receptor subtype-specific therapy in LCH. The generated data will lay basis for future clinical trials of adenosine receptor antagonists in treatment of LCH. |
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Project Title:
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HOMOZYGOZITY MAPPING FOR IDENTIFICATION OF A NOVEL GENETIC DEFECT IN PATIENTS WITH FAMILIAL HEMPHAGOCYTIC LYMPHOHISTIOCYTOSIS
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Investigator:
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Udo zur Stadt MD
University Medical Center Hamburg Eppendorf - Hamburg, GERMANY
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Amount:
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$44,100
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Length:
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One year
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Summary
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Despite the fact that FHL is a very rare disease, the identification of underlying mutations is highly relevant since affected persons have a usually fatal outcome if the disease is not correctly diagnosed and if appropriate therapy is not initiated rapidly. Therefore, the identification of several disease causing mutations has substantially improved the exact diagnosis and treatment modalities such as stem cell transplantation in individual patients. Starting with the identification of mutations in perforin 1, as a key player in cytotoxic death, in 1999, it has become clear that causative FHL genes are involved in pathways affecting cell types that directly take part in the defense against pathogens. Although the cell biological picture of activated macrophages and a reduced cytotoxic answer to infections could contribute to understanding these defects, first evidence was given only after underlying genetic defects have been clarified in different subsets of the disease. The fact that perforin 1 is directly involved in the killing machinery of cytotoxic cells, and the fact that hMunc13-4 delivers such molecules through cytotoxic granules to the plasma membrane of immune cells, allowed the identification of exact mechanisms of viral defense.
The identification of (a) novel genetic subtype(s) will further improve our understanding of these pathways, thus allowing to clarify the etiology of the disease, and make it clear how the respective genetic basis of this heterogeneous disease establishes the clinical phenotype. Furthermore, identification of a gene defect will differentiate the more lethal familial (primary) form from secondary cases that often present with a milder clinical phenotype. Whereas in familial cases a peripheral blood stem cell transplantation is absolutely required, the latter one can be cured wtih standard chemo-immunotherapy protocols. |
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Project Title:
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ANALYSIS OF T CELL FUNCTION IN LANGERHANS CELL HISTIOCYTOSIS
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Investigators:
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Kenneth L. McClain MD, PhD
Carl E. Allen MD, PhD
Texas Children's Hospital, Baylor College of Medicine - Houston, Texas
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Amount:
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$45,000
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Length:
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One year
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Summary
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| Langerhans Cell Histiocytosis is a potentially fatal disease characterized by uncontrolled proliferation of a type of cell called a Langerhans Cell. Typically, Langerhans cells are located in the skin. The job of this cell is to recognize signals from the environment and to activate the immune system to respond to "danger" signals. When the Langerhans cell is activated it migrates to lymph nodes where it communicates with T cells. The T cells then become activated and create an inflammatory response that fights cells with "foreign" material such as viral proteins. In LCH, the pathologic Langerhans cells can be found in skin and lymph nodes, but also in virtually any other organ system. The LCH lesions are a mix of Langerhans cells, T cells, and other cells of the immune system. In the LCH lesions, proteins that induce inflammation are found at high levels. It is not clear which cells, the Langerhans cells, the T cells, or other cells, are producing these pro-inflammatory signals or why. In this study, we hypothesize that the T cells are, at least in part, responsible for the uncontrolled inflammation that leads to proliferation of LCH lesions and to morbidity and mortality in patients with LCH. We propose to study this hypothesis first by characterizing the types of T cells within the LCH lesions. We then propose to characterize the status major pro- and anti-inflammatory pathways in T cells. Fianlly, we propose to compare the sets of genes expressed by T cells within the LCH lesions in patients with varying levels of disease using microarray technology. With this technology we can evaluate the level of expression of over 30,000 genes in each group of T cells we study. Our expectation is that this research will lead to improved understanding of the casue of LCH, and may also point to future studies that will allow us to individualize treatment for patients based on T cell activity. |
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Project Title:
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LANGERHANS CELL AND DENDRITIC CELL HOMEOSTASIS IN HUMAN SKIN
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Investigator:
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Matthew Collin, MD
Newcastle University - Newcastle upon Tyne, UNITED KINGDOM
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Amount:
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$44,960
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Length:
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One year
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Summary
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Langerhans cell histiocytosis (LCH) is a rare but sometimes fatal disorder of unknown cause. The Langerhans cell (LC) normally inhabits the skin and respiratory system in low numbers and is part of the immune system. Patients with LCH may have abnormal collections of LC in the bone, skin, lungs, brain and other organs which cause deformity, metabolic problems and life-threatening complications.
We are trying to find out why LC accumulate in histiocytosis by studying the normal development of these cells. In the past, it has been assumed that LC come from a bone marrow derived cell that enters the tissues at a steady rate from the bloodstream. However, recent evidence from mice suggests that LC are dividing, self-renewing cells, that may grow continuously in the tissues, independently of the bone marrow. Whether this is also true in humans is not known but it is vital for understanding the cause of LCH: it means that LCH may be directly related to abnormal regulation of LC proliferation in the tissues rather than a problem with cells originating in the bone marrow. This concept might explain why many patients with LCH have single or focal lesions rather than widespread disease.
We are testing the idea that LC normally replicate in the tissues by measuring the proliferation of human LC in skin cultured in vitro. We will then use this to test growth factors and inhibitors that influence the rate of proliferation, allowing us to demonstrate which biochemical pathways are functioning in LC and to identify potential drug targets for LCH.
To prove that the replication of LC is important in vivo in humans we will examine the LC of a number of patients with bone marrow failure (myelodysplasia) or genetically abnormal bone marrow (chronic myelpgenous leukemia). In bone marrow failure, the blood precursor cells are lost and LC will become depleted unless they can replenish themselves. In genetically abnormal bone marrow, the LC will become marked with abnormal genes only if they are continuosly replaced by cells from the blood. Although these studies do not involve LCH patients they are 'experiments of nature' that will tell us about the way LC are controlled in the body and how loss of control might lead to LCH. |
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Project Title:
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GENOMIC ARRAY AND EXPRESSION ANALYSIS OF FREQUENT AMPLIFICATIONS IN CD1a+LCH CELLS
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Investigator:
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Nicola Elizabeth Annels, MD
Leiden University Medical Center - Leiden, NETHERLANDS
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Amount:
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$45,000
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Length:
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One year
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Summary
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| Langerhans cell histiocytosis is not only a disease of unknown cause but also there is still a lot of controversy about whether this is a malignant disease. Defining what is a tumour is not easy. However, in LCH there is clearly a dividing clonal cell population, a feature widely considered to be characteristic of a malignant tumour. Further evidence that a cell is a tumour cell is that it has genetic abnormalities. Genetic studies carried out to date in LCH have given conflicting or inconsistent results. Thus, this project aims to provide a comprehensive study that will show whether there is indeed a genetic cause behind LCH. Due to the fact that the disease process underlying LCH has so far remained unknown, many current treatments for LCH are empirical. Thus, only once the nature of the disease has been resolved can a rational approach to treatment be achieved. |
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Project Title:
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CIGARETTE SMOKING PROMOTES DENDRITIC CELL SURVIVAL AND RESISTANCE TO APOPTOSIS THROUGH HEME-OXYGENASE-1 DEPENDENT MECHANISMS
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Investigator:
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Robert Vassallo, MD
Mayo Clinic - Rochester, Minnesota
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Amount:
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$45,000
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Length:
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One year
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Summary
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Pulmonary LCH is an unusual lung disease that occurs almost exclusively in cigarette smokers. Although the link between smoking and pulmonary LCH is strong, the mechanisms by which smoking may precipitate LCH in susceptible individuals are unknown. In this application, we propose that smoking inappropriately promotes the survival of dendritic cells in the lungs. We provide preliminary evidence that smoking does prolong the life-span of dendritic cells by switching on certain molecular pathways inside the dendritic cell, particularly the heme-oxygenase-1 (HO-1) protein. We also show in preliminary studies that smoking activates Bcl-xL, a cellular protein that confers resistance to cell death. These observations are important because a recent study showed that the pathologic cells in pulmonary LCH frequently expressed the Bcl-xL protein, suggesting that in the lungs of LCH patients, cells may be accumulating because they resist normal mechanisms that would otherwise cause their clearance.
This application will explore specific mechanisms by which smoking may confer enhanced survival of dendritic cells, the key cellular participant in the LCH lesion. We will also define which component in cigarette smoke (nicotine, carbon monoxide, or oxidative stress) is most likely to be causing this effect. We will also study the expression of HO-1 and Bcl-xL in lung biopsy specimens of patients with pulmonary LCH and determine if the intensity of expression is a marker of disease severity.
Through the studies propose in this application we will have greater insight into the mechanisms that lead to pulmonary LCH. We will also identify new targets for the treatment of this disease. For example, the use of potent anti-oxidant strategies may be helpful to reverse the abnormalities in cell regulation induced by oxidative stress from cigarette smoke. Understanding mechanisms causing pulmonary LCH is absolutely critical in the quest for new treatment approaches for this frustrating disease. |
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Project Title:
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GENETIC STUDIES ON THE PATHOGENESIS OF FHL
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Investigator:
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Magnus Nordenskjöld, M.D., Ph.D.
Karolinska University Hospital - Stockholm, SWEDEN
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Amount:
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$45.000
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Length:
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One year
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Summary
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Familial hemophagocytic lymphohistiocytosis (FHL) is a devastating disease affecting mainly infants and young children. It causes fever, enlarged liver and spleen, as well as depleted blood counts. Without treatment, FHL is invariably fatal, with a median survival after onset of 1-2 months. With the treatment protocol HLH-94, which is based on chemotherapy and immunotherapy followed by bone marrow transplantation, the survival has increased to around 50%.
FHL is considered to be an inherited disease, with an autosomal recessive pattern. This means that each parent to an affected child carries one healthy and one defect gene (i.e. the specific gene causing the disease). The affected child has been unfortunate to inherit the defect gene from both of the parents. The risk for this to happen is 25% for each child (50% risk to inherit the defect gene from the father times 50% from the mother). The disease will not affect an individual with at least one healthy gene.
The symptoms in FHL are caused by an uncontrolled accumulation and activation of immunological cells (white blood cells, mainly T-lymphocytes and macrophages). The underlying reason for this accumulation has until recently been unknown but in early 1999 we published data suggesting that the accumulation of lymphocytes in FHL was due to decreased programmed cell death (cell suicide). This made sense, since if the cells will not enter path leading to programmed cell death they will instead accumulate, and that is found in these children. We suggested that this might be due to a deficient triggering of this mechanism. Later 1999 a group of researchers (from France, Texas, and our group) were able to confirm this suggestion, when a gene (perforin, chromosome 10) was shown to be deficient in a subset of FHL children. Perforin has its effect by opening (perforating) the cell membrane to allow the influx of toxic substances which initiate the cell death program. Thus, lack of normal perforin will decrease the programmed cell death and cause cell accumulation.
Subsequently it was shown that mutations in the gene Munc 13-4 may also cause FHL and recently our group was involved in identifying a third genetic cause of FHL (the gene syntaxin-11). Nevertheless, our data suggest that for many FHL families the genes casing the disease have not yet been revealed. We have reported that only a minority (20-40%) of the FHL families have perforin defects and in individuals with FHL ethnically originating from Western Europe only 30% have a known genetic defect. Consequently, the major aim with the proposed research project is to find additional genes that may cause FHL. There are a number of substances involved in the regulation of programmed cell death that we intend to study. Moreover, we have DNA from a large number of families without mutations in the genes perforin, Munc 13-4 and syntaxin-11, and we have therefore the potential to make the proposed research project fruitful.
In summary:Our purpose is to find additional gene(s) and mutations that are defect in FHL. This would be of importance for:
1. Diagnosis. The differential diagnosis between FHL (primary HLH) and secondary HLH is at present sometimes very difficult. Since treatment of FHL includes bone marrow transplant, which is not the case in secondary HLH, better diagnostic tools are highly desired, such as a distinct genetic analysis.
2. Genetic counseling: If a child dies of HLH, a distinct information to the family regarding whether the disease is inherited or not is also very valuable.
3. Prenatal diagnosis: Many families who have had a child who suffered from FHL would appreciate the opportunity of early prenatal diagnosis, which could provide them with an option of an early interruption of the pregnancy (at the choice of the family).
4. Therapy: Increased understanding of the cause of the disease might improve treatment further. In addition, with improved genetic diagnosis thrapeutic decisions are facilitated and improved, not least the decision on whether to perform a BMT or not in an affected child, and we believe that such knowledge would improve survival further. |
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Project Title:
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HOMOZYGOSITY MAPPING TO REVEAL NOVEL GENETIC DEFECTS IN PATIENTS WITH FAMILIAL HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS
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Investigator:
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Maurizio Aricò MD
Osoedale dei Bambini "G. Di Cristina" - Palermo, ITALY
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Amount:
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$45,000
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Length:
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One year
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Summary
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FHL is rare, familial disease which is usually fatal in early infancy. Delayed onset is being increasingly recognized and the disease has been recently diagnosed also in youg adults. It may be not easy to differentiate the true genetic form of HLH, called FHL, from some cases which are extremely similar on the clinical ground but are not due to a constitutional defect, and rather depend on curcumstances triggered by common pathogens. Chemo-immunotherapy is helpful for all cases of HLH. Yet, patients with FHL are candidate to disease recurrence and fatal outcome unless they undergo hematopoietic stem cell transplant (HSCT), which is instead contraindicated in patients with the transient, non genetic form ("secondary HLH", VAHS, IAHS).
During the last few years genetic markers for this disease -- PFR1, MUNC13-4 and STX11 -- became available, allowing a more precise diagnosis and treatment. In those families in which a genetic marker is not available, the disease may be uneasy to confirm, especially if the child has atypical clinical manifestations, leading to a delay in the specific treatment; furthermore, genetic counseling is not allowed, including identification of healthy carriers and also of patients who are at present not ill, but may develop the disease later on, if they are genetically affected. In rare cases, such patients have been selected as HSCT donor for an affected sibling, resulting in dramatic events of both kids getting ill after transplantation. Finally, prenatal diagnosis may be available only for families with a genetic marker. Refining our knowledge of genes involved in FHL may allow the clinicians to expand the number of families in which a genetic diagnosis can be done.
Furthermore, identification of novel genes responsible for FHL may allow better knowledge of the mechanisms underlying the defensive mechanisms played by the cytotoxic lymphocytes against infections and cancer. Thus, information raised for care of a very small number of children with a fatal, familial disease may result into a progress of our knowledge of the mechanisms producing the immune system shielding of the human being against the most common cause of fatal disease.
To achieve this goal we plan to apply to this disease a well-known genetic method, i.e. the “homozygosity mapping”. This is based on the assumption that since FHL is an autosomal recessive disorder, the disease-related gene has to be contained in a chromosomal region in which both copies of the information (gene) are abnormal and identical. In the case of related parents one can assume that the same error is inherited by the patient and thus can be recognized by the investigators. According to study design, we have selected a group of families with FHL and related parents. By using a set of predefined genetic markers we will be able to identify the chromosomal regions in which the child has two identical copies of the same message. Among them, by definition, the disease-related gene could be hidden. By screening the functions of the genes contained in such chromosomal regions, we plan to identify a list of genes involved in the mechanisms of cellular cytotoxicity, which is known to be impaired in FHL an also in these group of families. The genes which appear to be most likely involved will be studied by two methods, the analysis of the proteins and the direct sequence analysis. Identification of defective protein(s) or mutations in the genes will provide the basis for building a definite correlation between these gene(s) and novel subset(s) of the disease. It is important to remark that, given the heterogeneity of the disease, we will not assume that two or more families of the group will share the by definition the novel genetic defect. Thus any information achieved will be tested on the entire group and possibly also on additional, non consanguineous families with FHL.
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