127. Comparative proteomics and lysosomal disease

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Abstracts/Molecular Genetics and Metabolism 99 (2010) S8–S41

125. The natural history of mucolipidosis type IV Raphael Schiffman, Baylor Research Institute Mucolipidosis type IV (MLIV) is a rare autosomal recessive neurodegenerative disease caused by mutations of MCOLN1, which codes of mucolipin-1 a putative cation channel located in lysosomes. This research proposal aims to study the natural history of neurological and retinal manifestations of MLIV. It also aims to increase awareness and improve diagnosis of MLIV among patients with cerebral palsy and those with retinal dystrophy of unknown cause. Over the past decade we have been studying prospectively various clinical and genetic aspects of MLIV in the only study of its kind. We hypothesize that MLIV is a combination of mostly developmental abnormality in the CMS and a degenerative retinal process. In this application we propose to (1) recruit known and newly diagnosed patients with MLIV and systematically study the neurological and retinal function over time. (2) Contact practicing physicians that take care of cerebral palsy patients and those with retinal dystrophy to increased awareness and improve diagnosis of MLIV. Based on our experience, we believe that many MLIV patients go undiagnosed. This project will help characterize and quantify the clinical abnormalities of MLIV and thus help define a patient population and outcome measures for future clinical trials. doi:10.1016/j.ymgme.2009.10.142

126. Longitudinal studies of brain structure and function in MPS disorders: A study of the Lysosomal Disease Network

Robert Wood Johnson Medical School, Piscataway, USA, bDepartment of Pharmacology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, USA, cMcGill University, Montreal, Canada, dNeurology Department, Massachusetts General Hospital, Boston, USA, eDepartment of Biostatistics, University of Medicine and Dentistry of New Jersey, School of Public Health, Piscataway, USA Diagnosis of lysosomal storage diseases (LSD) is typically straightforward with well established clinical criteria that are validated by biochemical and genetic tests. However, there is a significant population of individuals that are diagnosed with apparent LSD where the gene defect remains elusive. In some, obtaining a definitive diagnosis is problematic because atypical clinical phenotypes can resemble other, geneticallydistinct disorders. In addition, LSD can result from mutations in genes that are not currently implicated with these diseases. Here, we describe comparative proteomic approaches that can provide useful insights into such cases. The majority of LSD arise from mutations in genes encoding lysosomal proteins that contain mannose 6-phosphate, a carbohydrate modification that acts as a signal for intracellular targeting to the lysosome. We purify mannose 6-phosphorylated proteins by affinity chromatography and estimate the relative abundance of individual proteins in the mixture detected by tandem mass spectrometry. Our rationale is that proteins that are decreased or absent in patients could represent candidates for the primary defect, directing biochemical or genetic studies. We demonstrate that this proteomic approach is useful for identifying defects in unsolved lysosomal disease. For example, this approach has been instrumental in the identification or validation of mutations in two lysosomal proteins, CLN5 and sulfamidase, in the adult form of neuronal ceroid lipofuscinosis. We will describe our analyses of brain autopsy samples (Sleat etal., Molecular and Cellular Proteomics, 2009) and ongoing studies of cultured cells as more accessible patient samples. doi:10.1016/j.ymgme.2009.10.144

Elsa Shapiro, University of Minnesota, Minneapolis, MN, USA The mucopolysaccharidoses are lysosomal disorders that progressively affect most organ systems in the body usually beginning in childhood. Recent treatment advances have produced amelioration of some of these malfunctions, but notably brain and bone have been difficult to treat. The objectives of this research are to identify abnormalities of central nervous system (CNS) structure and function and to measure quality-of-life (QOL) in both treated and untreated patients with MPS disorders over time. We will accomplish this through longitudinal studies of enrolled patients in core centers in North America that constitute the Lysosomal Disease Network (LDN). We hypothesize that specific and localized neuroimaging and neuropsychological findings and their relationship will be distinct for each MPS disorder. Further, without treatment, functions and structure will change over time in a predictable fashion, related to locus of abnormality and stage of disease. Specific goals: To develop standard quantitative measurements of CNS structure and function to assess stage of disease and treatment outcomes and to examine the degree to which independent variables (age, mutation, severity, medical history and other variables) as well as various treatments impact CNS structure and function and quality-of-life. Methods: (1) Standardized 3T acquisition with Volumetrics and DTI; analysis at the University of Minnesota using Brains2, FreeSurfer, and DTI Studio as well as the clinical method of Matheus etal. (2) Neuropsychological standardized measurement of IQ, attention, memory, spatial, adaptive, QOL and behavior. (3) Biomarker HCII-T analyzed by Lorne Clarke at UBC. Subjects will be recruited from patients seen at the eight centers for yearly follow-up for at least 3 years. At least 50 participants will be accrued in the first year. Case Report Forms will be available for on-line submission of de-identified data. The following are co-investigators and will provide data from their institution: Julian Raiman, Hospital for Sick Children Toronto; Gerry Berry, Childrens Hospital, Boston; Greg Pastores, New York University; Paul Fernhoff, Emory University; Paul Harmatz, Oakland Childrens Hospital; Robert Steiner, Oregon Health Sciences University; and David Viskochil, University of Utah. Current status and anticipated progress:

128. Identifying the pathogenic mechanisms associated with ML II using zebrafish and feline models Richard Steeta,b, Aaron Petreya, Heather Flanagan-Steeta, Mark Haskinsb, aUniversity of Georgia, Athens, GA, USA, bUniversity of Pennsylvania School of Veterinary Medicine, Philadelphia, PA, USA The severe pediatric disorder, mucolipidosis II (ML II; I-cell disease), is caused by defects in mannose 6-phosphate (M6P) biosynthesis. Patients with ML II exhibit multiple developmental defects including prominent skeletal, craniofacial and joint abnormalities. To date, the pathogenic mechanisms that underlie these clinical features are poorly understood. Taking advantage of a zebrafish model for ML II, we have previously shown that the cartilage morphogenesis defects in this model are associated with altered chondrocyte differentiation and abnormal deposition and/or turnover of type II collagen. More recently, our work has also demonstrated changes in the gene expression profile of sorted chondrocytes from ML II zebrafish embryos. In particular, upregulation of cathepsin and matrix metalloproteinase transcripts in the ML II chondrocyte precursors was observed. We now demonstrate that increased MMP activity is a general feature of this disease using fluorescence-based enzyme measurements in both ML II zebrafish embryos and ML II feline chondrocytes and fibroblastlike synoviocytes. Importantly, the increased MMP activity in the ML II zebrafish could not be further enhanced with APMA treatment, indicating that these proteases may undergo inappropriate activation. In addition, we have begun to investigate the enzymatic properties and mannose phosphorylation state of several zebrafish cathepsins in an attempt to identify potential initiators of the pathogenic cascade in this system. Together, our findings not only yield new cellular and molecular insight into ML II disease pathogenesis but also provide a novel perspective on the evolution of the mannose 6-phosphate targeting pathway. doi:10.1016/j.ymgme.2009.10.145

(1) University of Minnesota has recruited six MPS I and seven MPS II patients as of September 25, 2009. (2) At least 20 patients will be recruited from other centers this year and 17 additional patients at Minnesota. (3) A baseline cross sectional manuscript will be prepared at the end of the first year after initial visits of all accrued patients. doi:10.1016/j.ymgme.2009.10.143

127. Comparative proteomics and lysosomal disease David Sleata,b,c,d,e, Lin Dinga, Shudan Wangc, Caifeng Zhaoa, Yanhong Wanga, Winnie Xind, Haiyan Zhenga, Dirk Mooree, Katherine Simsd, Peter Lobelb, aCenter for Advanced Biotechnology and Medicine and University of Medicine and Dentistry of New Jersey,

129. Raising medical awareness of a childhood neurological disease Frank Stehr, NCL-Foundation The eye doctor is usually the first one to see a Batten disease child. Due to the rarity of this deadly lysosomal storage disorder it is often not recognized, and is initially misdiagnosed as Retinitis pigmentosa or Stargardt disease. Usually many years later after progression of the disease, and after running from doctor to doctor, the correct diagnosis is made. Due to the autosomal recessive inheritance, early diagnosis is critical. Families need to be aware that their children have a chance of inheriting this genetic mutation. Appropriate counseling needs to be offered. In order to reduce the number of false diagnoses, our German based non-profit organization, the NCL-Foundation, has developed various strategies to raise awareness of Batten disease in relevant medical groups. After addressing all German eye