Genetic and clinical studies of Gaucher disease and other lysosomal disorders

National Human Genome Research Institute

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Abstract

A bench to bedside approach is used in our studies of human genetic disorders, integrating both basic and clinical research. We focus on a rare genetic disorder, Gaucher disease, and a common complex disorder, Parkinson disease. Most inherited diseases are characterized by a wide range of patient presentations, yet the factors contributing to this heterogeneity are elusive. Gaucher disease, the most common of the sphingolipidoses, is studied as a prototype because of the broad spectrum of clinical diversity resulting from this recessively inherited enzyme deficiency. Gaucher disease affects approximately 10,000 to 20,000 Americans and is more common among Ashkenazi Jews. Natural history studies, as well as molecular and biochemical evaluations in humans and animals, are used to enhance our understanding of heterogeneity in Gaucher disease and our ability to develop rational therapy for patients. In contrast, Parkinson disease is a common disorder that affects over 1.5 million Americans and over 10 million globally and the incidence is rapidly rising. The techniques, insights, and experience gained from our studies of Gaucher disease are being applied to the challenges of complex illnesses like Parkinson disease. Our research directly lead to the discovery of an association between Gaucher disease and parkinsonism. Our clinical studies of patients with both disorders, neuropathological evaluations, family studies and screening of tissues and DNA samples from subjects with Parkinson disease have all supported this link. Evaluations of patients with Gaucher disease and Parkinson disease demonstrate both classic and atypical features. Many different genotypes are encountered. Longitudinal imaging studies of these subjects and their affected or at-risk relatives using Positron Emission Tomography studies of the brain have been completed, establishing the rate of disease progression and degree of non-penetrance in our cohort. Longitudinal clinical evaluations of these at- risk individuals are continuing. Our work has led to the recognition that while mutations in glucocerebrosidase are the most frequent inherited risk factor associated with parkinsonism, most patients with Gaucher disease never develop Parkinson disease, implicating other genetic or environmental modifiers of disease prevalence. As a result of our research, investigator in the field of neurodegeneration are now focusing on the role of lysosomal pathways in disease pathogenesis. Over the past three decades under our clinical protocols, we have established a vast bank of clinical data, DNA, RNA and tissue samples enabling us to understand the natural history and genotype/phenotype correlation in Gaucher disease. Our research has shown that although Gaucher disease is classically divided into three types, there is actually a continuum of manifestations. We have uncovered several unexpected phenotypes and have solid evidence that modifiers must contribute to this phenotypic diversity. Studying specific families, we have identified some with atypical inheritance resulting from germline mosiacism and uniparental disomy. Now utilizing next-generation sequencing strategies, we can better investigate the intricate relationships between clinical phenotypes, metabolic defects and genes in patient samples. Understanding the mechanisms leading to the diverse phenotypes in Gaucher disease may help in the identification of modifier genes and will provide insights relevant to other disorders. To enhance our studies of Gaucher disease and of parkinsonism we have been working to develop new cellular models. We have successfully studied patient-derived macrophages in culture and show that they mimic the storage phenotype seen in patients. We have now also generated induced pluripotent stem cells (iPSCs) from patient fibroblasts and have differentiated these into macrophages, astrocytes and dopaminergic neurons. We find that they have an appropriate phenotype, with deficient glucocerebrosidase and increased glycolipid storage. iPSC-derived dopaminergic neurons from patients with both Gaucher disease and Parkinson disease demonstrate elevated levels of alpha-synuclein. Using new technologies we have made isogenic lines from a single patient so that differentiated cells with mutant, absent and wildtype enzyme from the same individual can be compared. These isogenic lines were engineered so that lysosomes within neurons can be easily isolated and evaluated. We have also developed lines from siblings with Gaucher disease where only one sib has Parkinson disease, and are evaluating the differentiated neurons using proteomics, lipidomics, metabolomics, Whole genome sequencing and RNA sequencing to identify the other factors that contribute to the development of Parkinson disease. We have also developed a mouse model of GBA1-associated Parkinson disease, by crossing murine Gaucher models with mutant alpha-synuclein over-expressing mice. These models can be used both to test therapeutics and to perform screens for genetic modifiers. Another important goal is to develop new treatment strategies for patients. Enzyme replacement therapy for Gaucher disease has been shown to be clinically effective, but it is extremely costly, and requires life-long intravenous infusions. A potential alternative therapy is the use of small molecules that may function as chemical chaperones that can stabilize and enhance the patient's mutant enzyme. We are now exploring whether small molecule drugs may enhance the delivery of mutant glucocerebrosidase to the lysosome. In collaboration with NCATS, we have screened large libraries of compounds using mutant forms of the enzyme, identifying exciting lead molecules that are being developed as treatments for Gaucher disease. For the first time, we identified non-inhibitory chaperone molecules that result in translocation of the enzyme to the lysosome and enhancement of activity. These new non-inhibitory leads have been validated in our macrophage models, and in iPSC-derived cellular models. The therapy resulted in enhanced enzymatic activity, decreased lipid storage and improved cellular function in these patient-derived models. Treatment of dopaminergic neurons from patients with parkinsonism also decreased the levels of alpha-synuclein. We are now developing better assays, models, tools and screens that can enhance development of new therapeutics. In collaboration with Dr. Charles Venditti , we have made gene therapy constructs to deliver GCase, and have tested them successfully in murine Gaucher models. These approaches may lead to more convenient and less costly therapy for Gaucher disease and will have important implications for the treatment of Parkinson disease.