1. Quantification of urinary oxidized lipids, heptanone-etheno-2-deoxyguanosine, 8-hydroxyguanine, and 8-hydroxy-2-deoxyguanosine in Friedreich ataxia patients undergoing idebenone treatment in a phase II double-blind placebo-controlled study.

Abstract

Friedreich ataxia (FRDA) is a rare, inherited neuro- and cardio-degenerative disorder. The signs and symptoms derive from decreased expression of the protein frataxin, which is involved in iron metabolism. Frataxin chaperones iron for iron-sulfur-cluster biogenesis and detoxifies iron in the mitochondrial matrix. Decreased expression of frataxin is associated with impairments of iron-sulfur-cluster biogenesis, mitochondrial dysfunction, mitochondrial iron accumulation, and oxidative stress. Compounds currently in clinical trials are directed toward improving mitochondrial function and lessening oxidative stress. A critical aspect of facilitating the development and testing of these compounds is the identification of appropriate biomarkers of oxidative stress in FRDA. The phase II double-blind placebo-controlled study of the antioxidant idebenone for FRDA being conducted by the Neurogenetics Branch of NINDS provides an unprecedented opportunity to test the most advanced methodologies for the quantification of oxidative-stress analytes and thereby identify the best approach for following disease progression and response to therapy, especially as phase I trials of additional antioxidant compounds are completed. Utilizing this opportunity, the Neurogenetics Branch is currently collaborating with the Laboratory of Molecular Genetics in NIEHS and has uncovered unique genes altered in FRDA with a subset that correlate to response with drug treatment. We now wish to extend this research into the issue of oxidative stress. This intramural-extramural collaborative project between a clinical laboratory at the NINDS and basic research laboratories at the University of Pennsylvania seeks to test the hypothesis that FRDA patients 1) accumulate urinary oxidized lipids, heptanone-etheno-2’-deoxyguanosine (H?dGuo), 8-hydroxyguanine (8-oxo-Gua), and 8-hydroxy-2’-deoxyguanosine (8-oxo-dGuo), 2) decrease urinary oxidized lipids, H?dGuo, 8-oxo-Gua, and 8-oxo-dGuo with idebenone treatment, and 3) increase urinary oxidized lipids, H?dGuo, 8-oxo-Gua, and 8-oxo-dGuo after an increase in ATP demand during a stationary bicycle exercise protocol. The proposed studies will utilize highly specific and sensitive quantitative methodologies based on stable isotope dilution immunoaffinity purification and liquid chromatography/tandem mass spectrometry (LC/MS/MS), which were specifically developed to quantify relevant biomarkers of oxidative stress. These assays have been (or are currently being) translated into urinary analyses for monitoring human populations at risk for oxidative stress. In addition to identifying biomarkers of disease progression and response to therapy for patients with FRDA, the proposed studies should elucidate further the role of oxidative stress in FRDA and the biochemical abnormalities underlying the signs and symptoms of the disorder.

2. Role of pathogen-specific IgE and histamine release in the hyper-IgE syndrome

Abstract

Hyper IgE syndrome (HIES) is a rare primary immunodeficiency characterized by eczema, recurrent skin and lung infections, elevated serum IgE and multiple connective tissue and skeletal abnormalities. The genetic etiology and exact immunologic defect remain unknown. Patients with HIES produce IgE antibodies specific for Candida albicans and Staphylococcus aureus, two of the common pathogens in this population. We hypothesize that the presence of pathogen-specific IgE combined with continuous exposure to these ubiquitous agents leads to chronic IgE mediated histamine release from basophils and mast cells, with subsequent pathogen-specific immune tolerance and an increase in pathogen specific T regulatory cells. We plan to test this hypothesis through clinical and immunologic evaluation of HIES patients before, during and after H2 blocker therapy with ranitidine. We chose this therapy because histamine has been shown to stimulate IL-10, a major down-regulatory cytokine, through the H2 receptor and clinical improvement has been observed in several patients treated with H2 blockers. Laboratory studies will include evaluation of pathogen-specific IgG4:IgE ratios, basophil activation, IL-10 producing regulatory T-cells, cellular proliferative responses to Candida and Staphylococcal antigens, and functional testing of regulatory T-cells. Clinical evaluation will include comprehensive history and physical examination, dermatologic evaluation, genetic evaluation for clinical severity scoring, pulmonary function tests, and chest CT examination. Through this study we will further our understanding of the immunologic abnormalities of HIES and determine whether a prospective, double-blind trial of H2 blockade as adjunctive therapy for HIES is indicated.

3. Life-threatening pulmonary complications of organ transplantation: An investigation of the pathogenesis of bronchiolitis obliterans and its novel treatment with aerosolized liposomal cyclosporine A

Abstract

Bronchiolitis obliterans is an obstructive disease of the small airways of the lung that may develop after either hematopoietic stem cell or lung transplantation. It is associated with considerable morbidity and mortality in long-term transplant survivors and thus represents a major impediment to the success of these therapies Mortality due to bronchiolitis obliterans-associated respiratory failure may occur in more than 55% of patients developing this complication typically within 3 to 5 years following transplantation. Treatment is based primarily on increased systemic immunosuppression which results in attendant increased risk of infection and morbidity. As a consequence, conventional therapy for bronchiolitis obliterans has not improved long-term outcome of this disorder. Recently, inhaled cyclosporine in propylene glycol improved survival of chronic bronchiolitis obliterans in lung transplant recipients. While promising as a new organ-targeted approach for the prevention and treatment of bronchiolitis obliterans, the propylene glycol vehicle had significant toxicities precluding further development of this drug preparation. To study the pathogenesis and treatment of bronchiolitis obliterans, we propose to perform a comprehensive assessment of lung function and structure in hematopoietic stem cell and lung transplant recipients. The interventional arm of the study will use a novel inhaled liposomal cyclosporine preparation in transplant recipients who have developed or who are at high risk for this potentially lethal complication. These data will provide proof of principle and set the stage for larger prophylactic interventional trials directed at recipients of both hematopoietic stem cell and lung transplants.

4. Sensitivity and resistance to Rituximab therapy in SLL/CLL: The role of antigenic modulation, immune effector mechanisms and direct pro-apoptotic signaling.

Abstract

Immunotherapies of cancer that are based on targeting with mAbs have the potential for “real time” analyses in which the malignant cell phenotype and genetic profile of both the targeted cells and surrounding tissue can be evaluated before, during and after mAb treatment. Such measurements have the potential to allow investigators to determine, on a cellular and molecular basis, how effectively the therapies are working at targeting and killing the cells, and this information can be readily used in the design of the next generation of therapies. Our recent studies in chronic lymphocytic leukemia (CLL), in which circulating cells were examined, illustrate the power of this concept, and have led us to reevaluate and design new dosing paradigms for Rituximab (RTX) in the treatment of CLL. We propose to extend this work to small lymphocytic lymphoma (SLL), where we will determine in detail how RTX treatment affects malignant cells and normal tissue in lymph nodes of patients. We will obtain lymph node biopsies before and after rituximab infusion to analyze tissue penetration of the monoclonal antibody, effects on immune effector mechanisms and on gene expression. The necessity to obtain two matched lymph node biopsies, one of which is time sensitive, is a challenging undertaking, but one for which the NIH clinical program is ideally suited. The comprehensive studies proposed here will make use of the complementary expertise of two laboratories and aim to concomitantly address the impact of RTX on all three proposed mechanisms of action: antibody-dependent cell-mediated cytotoxicity, complement mediated cell lysis, and intracellular signaling. These studies have the potential to provide new and valuable information which may allow for more effective targeting of malignant cells growing in lymph nodes. By providing guidance for optimized dosing of RTX or rational combination therapies that increase synergy between different agents, insights from our studies are expected to allow for the development of new and better treatment options for patients with SLL/CLL.

5. Genetics of inherited paragangliomas and gastric stromal tumors associated with adrenal and other tumors

Abstract

The aims of this study are (i) to describe accurately the clinical components and natural history of a rare genetic disorder, a newly identified syndrome of familial paragangliomas and gastric stromal tumors (GST) and a related condition that is characterized by paragangliomas, GST, pulmonary chondroma and other tumors, and (ii) identify the genetic defect(s) in these disorders by state-of-the-art approaches of genetic linkage methods. More specifically, in 1977, a new genetic condition was reported (1): the association of gastric leiomyosarcoma, functioning extra-adrenal paraganglioma, and pulmonary chondroma in 2 patients and 2 of the three tumors in 5 other patients, all unrelated young females. The pattern and age of tumor occurrence-multifocal lesions in multiple organs in young patients-suggested a heritable disorder (OMIM # 604287). More patients were reported later: 67 female and 12 male patients. One-fifth of the patients had the 3 tumors; the remainder had 2 of the 3, usually the gastric and pulmonary lesions. Adrenocortical adenoma was identified as a new component of the disorder, and esophageal leiomyoma as a probable one. Because of the rarity of the 3 original components, the presence of any 2 of them was considered sufficient for diagnosis of the association, particularly if the tumors were multifocal and the patient a young female. Intriguingly, 2 of the 79 patients, a young woman and a young man, each with 2 elements of the triad (gastric sarcoma and paraganglioma), had a sibling with 1 element (paraganglioma), raising the possibility that the triad might be a familial condition. The inheritance of the disorder remained unclear because none of 355 primary relatives (parents, siblings, and children) of the remaining 77 patients had manifested any of the components. A recent article described the findings in the 2 sibships and those in 3 other kindreds with apparent familial occurrence of paraganglioma and gastric stromal sarcoma (GST). From these observations, we identified a familial syndrome of paraganglioma and GST that appears to be distinct from the previously reported one (2). We now want to study these patients clinically and investigate the molecular genetics of these and related disorders.

1. Carney JA, Sheps SG, Go VL, Gordon H. The triad of gastric leiomyosarcoma, functioning extra-adrenal paraganglioma and pulmonary chondroma. N Engl J Med. 1977;296(26):1517-8.
2. Carney JA, Stratakis CA. Familial paraganglioma and gastric stromal sarcoma: a new syndrome distinct from the Carney triad. Am J Med Genet 2002;108(2):132-9.

6. Translational studies of hereditary spastic paraplegias types SPG4 and SPG20

Abstract

In this proposal, the laboratories of Drs. Craig Blackstone and James H. Hurley propose to investigate clinical, translational, and basic science aspects of a class of disorders known as hereditary spastic paraplegias (HSPs). The HSPs have as their cardinal feature lower extremity weakness and spasticity, characterized pathologically by distal, dying-back axonal degeneration of corticospinal upper motor neurons. Greater than 30 genetic loci (SPG1-33) and 13 gene products have been identified for the HSPs to date. Most disease proteins appear involved in membrane or protein trafficking, though in most cases even the basic protein functions and interactions are not yet clear. Dr. Blackstone’s laboratory has been characterizing the protein, spartin, which is mutated in autosomal recessive Troyer syndrome (SPG20). They have found that this protein is involved in EGF receptor internalization and degradation and, interestingly, that it is modified by mono-ubiquitination and also binds ubiquitin through a novel domain. The spartin protein also shares another motif, the MIT domain (for present in microtubule-interacting and trafficking proteins), with the spastin protein that is mutated in the most common cause of autosomal dominant HSP, SPG4. However, though they are closely related, our preliminary studies have shown that the MIT domains of these two proteins have significant differences in binding specificities. Here we propose a detailed investigation of the structure and function of these MIT domains and their interactions as well as of the novel ubiquitin-binding domain of spartin. If successful, the structural and functional analyses will allow us to pinpoint the mechanism of the MIT domain binding selectivity in atomic detail. We will also perform endocytosis studies in SPG4-lacking cells to see how the spastin protein functions in endocytosis. Findings from structural and in vitro studies will be correlated with cellular analysis at each stage, and we will use insights from the structural studies to generate dominant-negative constructs to probe the role of distinct interactions on EGF receptor internalization and degradation. Lastly, we will compile an extensive clinical analysis of only the second large kindred to be identified with Troyer syndrome. An appreciation of common mechanisms in protein and membrane trafficking and how they relate to the clinical phenotypes have implications not only for the relatively rare HSPs but also other dying-back axonal neuropathies.

7. WAGR syndrome: Clinical characterization and correlation with genotype

Abstract

WAGR Syndrome is a rare genetic disorder characterized by Wilms’ tumor, Aniridia, Genitourinary abnormalities, and mental Retardation. WAGR Syndrome is caused by contiguous gene deletions that produce haploinsufficiency for gene products in the 11p13 chromosomal region. Hyperphagia and early onset obesity have been reported in a subset of patients with WAGR Syndrome. We hypothesize that haploinsufficiency for the gene encoding brain-derived neurotrophic factor (BDNF, 11p14.1), which has an important role in nervous system development and also appears to serve as a downstream effector of leptin action in appetite regulation, may be important for the development of hyperphagia and more severe neurologic impairment in patients with 11p deletions. We propose a new study to investigate the correlation between phenotype and genotype of individuals with WAGR Syndrome.
Prior Investigations: In a cohort of 328 children and adolescents, enriched for subjects who severely overweight, we recently reported that serum BDNF concentrations were negatively associated with body mass index (BMI) and body fat, suggesting that insufficient BDNF may adversely affect children’s body weight regulation. We also described an 8-year-old girl, with hyperphagia, severe overweight, and developmental delay, who had a heterozygous paracentric inversion within 11p resulting in functional haploinsufficiency of BDNF, with a 15-fold lower serum BDNF concentration compared with age-matched, unaffected, overweight girls. We subsequently investigated 10 subjects with WAGR Syndrome who mailed blood samples to us, and found that those who had become overweight during childhood had significantly lower serum BDNF concentrations than those who had not. We further studied 2 of these subjects using high resolution comparative genomic hybridization (CGH) microarray to determine the extent of 11p deletion. The first subject had early onset overweight, significant neurocognitive defects, low serum BDNF, and heterozygous deletion of BDNF, while the second subject had normal weight until adulthood, higher functional IQ, normal serum BDNF, and no deletion of BDNF.
Bench Proposal: Genotyping will be carried out in 50 subjects with WAGR Syndrome. Genetic analyses will include CGH for high-resolution mapping of the deleted region and assessment of polymorphic microsatellite markers to identify maternal or paternal allelic loss. The impact of BDNF haploinsufficiency on variant BDNF mRNA transcript expression and the balance between pro-BDNF and mature-BDNF levels will also be assessed using RT-PCR and specific antibody-based assays, respectively.
Bedside Proposal: Using the multi-institute capabilities of the Hatfield CRC, we will perform a comprehensive phenotypic evaluation of patients with WAGR Syndrome. Metabolic studies of body composition and energy balance along with neurodevelopmental assessments will allow us to test critically whether BDNF haploinsufficiency is associated with obesity and neurocognitive defects in patients with WAGR. If BDNF haploinsufficiency and low serum BDNF concentrations are demonstrated to be associated with early onset obesity and more severe neurologic deficits, we will then proceed with a clinical trial to determine the effects of recombinant methionyl human BDNF on body composition and neurocognitive function. Because there has been no prior systematic, large-scale evaluation of patients with WAGR syndrome, phenotypic characterization of known aspects (renal, ophthalmologic) and evaluations intended to find as yet unidentified components of the syndrome (craniofacial, cardiac, gastrointestinal, and endocrine) will also be undertaken.
Bench-to-Bedside: Integrating the bench and bedside research, this project will address these questions:
1) What are the clinical consequences of BDNF haploinsufficiency for patients with WAGR syndrome?
2) Is low serum BDNF concentration a sensitive marker for BDNF haploinsufficiency, and associated with hyperphagia, obesity, and/or greater neurocognitive impairment?
3) Can hyperphagia and/or neurocognitive impairment associated with BDNF haploinsufficiency be ameliorated by exogenous BDNF administration?

8. Anti-proliferative therapy for severe pulmonary arterial hypertension

Abstract

Pulmonary Arterial Hypertension (PAH) is a rare disease that is characterized histologically by proliferation of smooth muscle cells with medial hypertrophy and arteriolar muscularization, in situ thrombosis, and proliferation of endothelial cells with neointimal formation. These changes lead to a rise in mean pulmonary artery pressures, severe functional limitation and early death. Traditionally, medications used to treat this illness focus mainly on inducing vasodilation of the pulmonary vasculature to relieve the increased pressures. Similar proliferative pathologic changes are seen in the coronary vasculature following vascular injury with balloon angioplasty. Successful treatment of this disease has focused on antiproliferative therapy; namely with sirolimus and paclitaxel coated intravascular stents. We believe Pulmonary Arterial Hypertension is a vascular proliferative disease and focusing on antiproliferative therapy will lead to improved outcomes for patients with PAH. We plan to explore the underlying mechanisms that drive this abnormal cell growth with a novel murine model of pulmonary hypertension, a chronic hypoxia model, and gene knockout mice that allow us to look at the role of cell cycle regulators. We believe that by testing antiproliferative drugs proven to work in coronary artery disease after vascular injury we will gain better insight into new therapeutic pathways. With a better understanding of the underlying causes that drive pulmonary hypertension we will be poised to introduce new options to patients suffering from this disease.

9. Characterization of glycosphingolipid accumulation in Smith-Lemli-Opitz syndrome and treatment with N-butyldeoxynojirimycin

Abstract

Smith-Lemli-Opitz syndrome (SLOS) is a genetic syndrome caused by an inborn error of cholesterol synthesis. Specifically, in SLOS there is a deficiency in the synthesis of cholesterol from 7-dehydrocholesterol due to mutation of the 7-dehydrocholesterol reductase gene. This results in decreased cholesterol and increased 7-dehydrocholesterol levels. The incidence of SLOS is on the order of 1/20,000 to 1/50,000. Previously we have shown that SLOS fibroblasts have a secondary defect in intracellular cholesterol transport causing an accumulation of unesterified cholesterol similar to that observed in Niemann-Pick disease, type C (NPC). NPC cells have a defect in endocytic transport that results in intracellular accumulation of both unesterified cholesterol and glycosphingolipids (GSL). GSL accumulation appears to be the initial biochemical defect that causes impaired cholesterol transport. Preliminary experiments have shown that SLOS fibroblasts also have a defect in GSL transport. GSL accumulation may have several clinical consequences with respect to SLOS pathophysiology and treatment. First, the secondary defect in intracellular cholesterol transport may limit the efficacy of dietary cholesterol supplementation. Second, bioavailability of endogenously synthesized cholesterol could be decreased. Examples of this would be impaired transport of cholesterol within neurons or transfer of cholesterol from glial cells to neurons in the central nervous system. Third, GSL accumulation likely contributes directly to the neurological dysfunction in NPC and other disorders of GSL metabolism. Thus GSL accumulation could contribute directly to neuronal dysfunction in SLOS. N-butyldeoxynojirimycin (NB-DNJ, miglustat, Zavesca) is an iminosugar that inhibits GSL synthesis. Preliminary data shows that the GSL storage defect in SLOS fibroblasts can be reversed by treatment with NB-DNJ. The basic science goals of this proposal are to characterize the GSL biochemical defect in both SLOS fibroblasts and SLOS mice, and to test the efficacy of NB-DNJ therapy in our SLOS mouse models. The clinical science goals of this proposal are to determine if a GSL abnormality exists in SLOS patients, and, if an abnormality of GSL metabolism is present, determine the safety and efficacy of NB-DNJ therapy in SLOS patients.