- Email: [email protected]
14. Polyneuropathy in Type 1 Gaucher disease: A two-year,multinational, prospective observational study
S10
Abstracts/Molecular Genetics and Metabolism 99 (2010) S8–S41
Gaucher disease, characterized by glucocerebrosidase deficiency, provokes glucosylceramide accumulation in cellular lysosomes. Current therapy uses mammalian cell production of recombinant glucocerebrosidase for enzyme replacement therapy which requires post-expression glycan remodeling for exposing mannose structures, which are required for uptake by Gaucher macrophages cells. Expressing taliglucerase alpha in specifically targeted plant cell organelles enables the cellular control of the mannose glycosylation pattern and consistency. Thus, taliglucerase has intrinsic exposed mannose residues and demonstrates high batch to batch uniformity. taliglucerase exhibits similar crystal structure and biological activity to that of the CHO expressed imiglucerase and demonstrates superior uptake to macrophage cells. Preclinical and Phase I clinical trials showed that Taliglucerase alpha was well tolerated, with no significant immune reaction or production of neutralizing antibodies. A multi-center Phase III pivotal trial performed under FDA Special Protocol Assessment has been recently concluded. The trial was a randomized, double-blind, parallel group, dose-ranging trial to assess the safety and efficacy of Taliglucerase in naive Gaucher disease patients. In the pivotal trial, patients were selected randomly for one of two dosing arms, 30 or 60 U/kg infusions every 2 weeks over 9 months. The primary endpoint of the study is the reduction in spleen volume from baseline, as measured by MRI. In addition, secondary end points such as liver volume, hemoglobin and platelet counts where followed, as well as additional biomarkers and bone parameters. Results of this pivotal study will be presented and discussed. doi:10.1016/j.ymgme.2009.10.026
10. Life expectancy and cause of death in Fabry disease: Findings from the Fabry Registry Maryam Banikazemia, Manesh Patelb, Roberta Lemayc, Stephen Waldekd, aColumbia University Medical Center, New york, NY, USA, bDivision of Cardiovascular Medicine, Duke University School of Medicine, Durham, NC, USA, cBiomedical Data Sciences and Informatics, Genzyme Corporation, Cambridge, MA, USA, dSalford Royal NHS Foundation Trust, Manchester M6 8HD, United Kingdom Clinical and investigative data from 2848 patients in the Fabry Registry were used to evaluate life expectancy and cause of death. Among this cohort, 75 of 1422 males and 12 of 1426 females were reported to have died, at a median age of 54.3 years in males and 62.0 years in females. The 87 deceased patients were diagnosed at a much older age than other patients in the Fabry Registry: median age at diagnosis was 40 years versus 24 years in males and 55 years versus 33 years in females. Life expectancies were calculated from birth date, based on data from the entire Fabry Registry population. Life expectancy of males with Fabry disease was 58.2 years, compared to 74.8 years in the general US population. Life expectancy of females with Fabry disease was 75.4 years, compared to 80.1 years in the general US population. The most common cause of death was cardiovascular disease. The deaths of 30 of 56 males with a known cause of death (54%) and 5 of 10 females with a known cause of death (50%) were classified as due to cardiovascular disease. Most Fabry patients who died of cardiovascular disease (20 of 35, 57%) had previously received renal replacement therapy. Cardiac function should be closely monitored in Fabry patients. Patients who exhibit signs or risk factors for cardiovascular, renal, or cerebrovascular disease should receive prompt and appropriate treatment for these conditions. doi:10.1016/j.ymgme.2009.10.027
11. Marked variability in urinary Gb3/creatinine excretion in healthy infants Caroline Barr, Joe T.R. Clarke, Aim Ntwari, Rgen Drouin, Christiane Auray-Blais, CHUS– Faculty of Medicine and Health Sciences, Universit de Sherbrooke, Sherbrooke, Canada Introduction: Fabry disease is a lysosomal storage disorder resulting in glycosphingolipid accumulation, mainly globotriaosylceramide (Gb3), in various tissues and physiological fluids. In order to establish reference values for infants less than 12 months of age, we measured urinary Gb3 by a liquid chromatography–tandem mass spectrometry (LC–MS/MS) methodology previously developed in our lab. Method: We analyzed 728 urine specimens collected by parents from 68 healthy infants of both sexes at intervals from birth to 6 months of age. Results: Parental compliance was good at 90%. Gb3/creatinine results were grouped into four periods according to age: <6 days; 6–29 days; 30–90 days; >90 days for statistical analyses. In both girls and boys, Gb3 excretion decreased with postnatal age. Urinary creatinine levels were increased at birth and decreased rapidly with time. There was no significant difference (p = 0.4620) between creatinine excretions for either sexes vs postnatal age. In contrast to infants >90 days of age, we found no statistically significant difference between boys and girls during the first 3 periods (p = 1.0000 for all comparisons), though the levels tend to be higher in girls. The anal-
ysis of variance of transformed Gb3/creatinine ratios for 728 samples according to sex and period showed a significant difference between boys (14.6 g/mmol creat.) and girls (42.7 g/mmol creat.) in period 4 (p = 0.0005). Conclusion: Urinary Gb3 excretion varied markedly among healthy infants of both sexes at all ages from birth to 6 months. doi:10.1016/j.ymgme.2009.10.028
12. The blood–brain barrier: A central role in the pathology and treatment of neuronopathic lysosomal storage disorders David Begley, Kings College, London, United Kingdom The central nervous system requires an extremely stable and specialised extracellular fluid environment in order to ensure correct neuronal and synaptic function. This extracellular fluid compartment of the brain is maintained separate and distinct from that of the blood and the extracellular fluid of other tissues. The concentrations of ions, metabolites, peptides, proteins and other solutes in cerebrospinal fluid and brain extracellular fluid are precisely regulated and can be very different from those in blood. It is the blood–brain barrier formed by the cerebral capillaries which enable this distinct fluid environment of the brain to be maintained. The endothelial cells forming the capillaries of the brain maintain tight junctions at the cell margins which prevent any paracellular movement of solutes. Transfers of solutes into and out of the brain thus have to be transcellular across the endothelial cells. Transport mechanisms in the cell membranes of these endothelial cells are also polarised and differ in the blood and brain facing cell membranes. The presence of the blood–brain barrier severely restricts the usefulness of many drugs and therapeutic compounds in the treatment of central nervous disease as they do not reach the brain in effective quantities. These properties of the blood–brain barrier will restrict the entry of chaperones and substrate reduction therapies into the brain and totally exclude large molecular weight enzyme replacement therapy. There is also evidence that the function of the blood–brain barrier is also disturbed in some lysosomal storage disorders and may contribute to cerebral damage and dysfunction. doi:10.1016/j.ymgme.2009.10.029
13. A pharmacogenetic approach to pharmacological chaperone therapy for Fabry disease Elfrida Benjamin, Xiaoyang Wu, Evan Katz, Kirsten Mascioli, Kate Chang, David Lockhart, Kenneth Valenzano, Amicus Therapeutics, Cranbury, USA Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the gene (GLA) that encodes galactosidase A (a-Gal A). More than 600 mutations have been reported; 60% are missense. AT 1001 (migalastat hydrochloride) is a pharmacological chaperone that selectively binds a-Gal A, increasing physical stability, lysosomal trafficking, and total cellular activity. A systematic approach to select patients that may be candidates for AT 1001 therapy was developed. Each of 415 known Fabry disease-causing missense and small in-frame insertion or deletion mutations were expressed in HEK-293 cells and tested for response to AT 1001. Increased a-Gal A levels were seen for a majority of the mutant forms. To identify the subset of mutant forms that are most likely to respond to AT 1001 invivo, criteria were developed based on the magnitude of the AT 1001 response in the HEK-293 assay. The criteria were applied to the HEK-293 results and compared to those of an AT 1001 Phase 2 trial that included male Fabry patients representing 18 different mutant forms of a-Gal A. The HEK-293 results were consistent with the patient white blood cell a-Gal A responses after oral administration of AT 1001. To date, 173 mutant forms meet these criteria. Collectively, the data suggest that a pharmacogenetic reference table comprised of the AT 1001-responsive a-Gal A mutant forms and the corresponding GLA mutations can be used to select male and female Fabry patients for pharmacological chaperone therapy with AT 1001 based on genotype. doi:10.1016/j.ymgme.2009.10.030
14. Polyneuropathy in Type 1 Gaucher disease: A two-year,multinational, prospective observational study Marieke Biegstraatena, E. Mengelb, L. Marodic, M. Petakovd, C. Niederaue, P. Giraldof, D. Hughesg, M. Mrsich, A. Mehtag, C.E.M. Hollaka, I.N. van Schaika, aAcademic Medical Center, Amsterdam, The Netherlands, bUniversitts Kinderklinik, Mainz, Germany, c University of Debrecen, Debrecen, Hungary, dClinical Center of Serbia, Belgrade, Serbia, e Klinik fr Innere Medizin, Universitt Essen, Dsseldorf, Germany, fMiguel Servet University
Abstracts/Molecular Genetics and Metabolism 99 (2010) S8–S41 Hospital and CIBERER, Zaragoza, Spain, gRoyal Free and University College Medical School, London, UK, hUniversity Hospital Centre, Department of Hematology, Zagreb, Croatia Type 1 Gaucher disease (GD1) is currently categorised as non-neuronopathic, although recent studies suggest peripheral neurological manifestations. We conducted a two-year, multicentre, prospective, observational cohort study in patients with GD1 to determine the prevalence and incidence of polyneuropathy, evaluated by standardised assessments of neurological symptoms and signs, and electrophysiological studies. Secondary outcome parameters included the prevalence and incidence of mononeuropathy, other neurological or electrophysiological abnormalities not fulfilling the criteria for a mono- or polyneuropathy, and general GD1 symptoms. Furthermore, a systematic literature search was performed to identify all studies reporting on prevalence and incidence of polyneuropathy in the population. One hundred and three patients were enrolled. At baseline, 11 patients (10.7%; 95%CI 5.5–18.3%) were diagnosed with sensory motor axonal polyneuropathy. Two (1.9%; 95%CI 0.2–6.8%) had an ulnaropathy. During two-years of follow-up six new cases of polyneuropathy were identified (5.9%; 29/1000 patient-years). Patients with polyneuropathy were older than those without. Possible associated conditions were identified in 5/17 patients only (gammopathy, vitamin B1 or B12 deficiency, diabetes mellitus, renal insufficiency and alcohol abuse). The 11 cases of polyneuropathy diagnosed at baseline remained unchanged or worsened during follow-up. According to the literature, the prevalence of polyneuropathy in the population is estimated between 0.09% and 7.5% and the annual incidence is estimated between 0.46 and 85 per 10.000 person-years. Thus, the prevalence and incidence of polyneuropathy in patients with GD1 appear to be higher than that of the general population, and may be part of the natural disease course. doi:10.1016/j.ymgme.2009.10.031
15. The effect of long-term substrate reduction therapy withgenistein in a mouse model of MPS IIIB Brian Biggera, Marcela Malinowskaa, Fiona Wilkinsona, Kia Langford-Smitha, Alexander Langford-Smitha, Rob Wynnc, Ed Wraithd, Grzegorz Wegrzynb, Brian Biggera, aUniveristy of Manchester, Manchester, UK, bUniveristy of Gdansk, Gdansk, Poland, cRoyal Manchester Childrens Hospital, Manchester, UK, dSt Marys Hospital, Manchester, UK Mucopolysaccharide (MPS) diseases IIIA,B,C,D are neuronopathic diseases which primarily accumulate the glycosaminoglycan heparan sulphate due to defects in lysosomal enzymes. Symptoms include progressive CNS degeneration characterised by mental retardation, hyperactivity and seizures, with death usually in the mid teens to early twenties. No treatments currently exist for any of the Sanfilippo diseases. We have previously shown that genistein aglycone can significantly reduce GAG and lysosomal compartment size in peripheral organs of MPS IIIB mice at several doses. In that 8 week study we did not observe drug related toxicity, neither did we observe reduction of GAG in the brain, despite evidence to suggest that 10% of the drug can cross the blood–brain barrier. Our hypothesis was that the treatment regimen was too short to observe changes in the brain. In the study presented here, we have performed a 34 week comparison of genistein (160 mg/kg/day) with placebo control in MPS IIIB and WT mice. We found improvements in the health of genistein treated MPS IIIB mice, as well as improvements or full correction of several behavioural parameters. We also present histological and biochemical findings in the brains of these mice. We have not seen toxicity from genistein despite these doses being considerably higher than those achievable with genistein in soy extract form. These data would support the development of a clinical trial of genistein aglycone in patients with MPS III and other MPS diseases. doi:10.1016/j.ymgme.2009.10.032
16. A chemical genetic approach to identifying therapeutictargets for NCL Sunita Biswasa,b, Sasja Heetvelda,b, Pavlina Wolfa,c, Yi Caoa, Stephanie Nortonb, Stephen Haggartyb, Susan Cotmana, aHarvard Medical School, Boston, USA, b Massachusetts General Hospital, Boston, USA, cBroad Institute of MIT and Harvard, Cambridge, MA, USA Neuronal Ceroid Lipofuscinosis (NCL) is a genetically heterogeneous group of disorders, characterized by overlapping clinical and pathological features, including the autophagosomal/autolysosomal accumulation of the mitochondrial ATP synthase, subunit-c protein. We have used neuronal cell culture models derived from mice harboring the most common disease mutation causing juvenile NCL (Cln3ex7/8 mice and CbCln3ex7/8 cerebellar cells) in phenotypic cell-based assays developed to screen for modifiers of NCL disease phenotypes and identify target pathways for therapeutic
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development. An autophagy GFP-LC3 based high-content screening assay, optimized to identify modulators of abnormal accumulation of autophagic vacuoles, was used in a chemical genetic screen of FDA-approved drugs and natural products. Seventeen compounds were identified as hits that altered the number of GFP-LC3 positive autophagic vacuoles in homozygous CbCln3ex7/8 cerebellar cells, compared to wild-type cells. Follow-up studies with one of the validated compounds, thapsigargin, known to induce increased cytosolic calcium levels, demonstrated a heightened sensitivity of the homozygous CbCln3ex7/8 cerebellar cells to a thapsigargin-induced autophagic response, compared to wild-type cells. Furthermore, subunit c accumulation was significantly worsened following a 24-h treatment with thapsigargin, while in wild-type cells, thapsigargin did not alter the amount of subunit c deposits. Further studies of thapsigargin in the Cln3ex7/8 genetic models will ascertain whether altered sensitivities of the wild-type and mutant cerebellar cells to thapsigargin treatment highlights differences in calcium regulation and homeostasis in the presence of NCL gene mutations. doi:10.1016/j.ymgme.2009.10.033
17. A natural history study of hexosaminidase deficiency Kendra Bjoraker, Julie Eisengart, Peter Karachunski, Gulin Oz, Chester Whitley, University of Minnesota, Minneapolis, MN, USA Hexosaminidase A deficiency results in a group of neurodegenerative disorders caused by intralysosomal storage of the specific glycosphingolipid GM2 ganglioside. Three studies are proposed: (1) focus on developmental course of infantile TSD from retrospective data and complete literature review to elucidate the clinical course of TSD; (2) collect longitudinal prospective data from patients with TSD seen at the University of Minnesota; (3) longitudinal study of LOTS to understand progression of CNS disease using MRI/MRS and neuropsychological measures. Objectives: Aim 1: Develop index of disease progression in TSD: A. First, a retrospective medical chart/record review for clinical data points. Second, caretakers of infants with TSD will be asked to complete the University of Minnesota Natural History Study Questionnaire for Infantile Tay-Sachs Disease (MNQSTD). B. Prospective: We will collect longitudinal neuropsychological (Bayley II, Vineland) and neuroimaging data in patients with infantile TSD to characterize developmental course and longitudinally examine individual growth trajectories. Aim 2: To better understand underlying CNS structure and function abnormalities in LOTS disease and to measure change in function over time through neuroimaging and neuropsychological data (WASI, Mindstreams Neurotrax, Vineland). Methods: Infantile: Recruit 5 new patients the 1st year for a total of at least 3 follow-up visits within the 5 year period. LOTS: Participants: Recruit 5 new patients 1st year. Each patient will be seen once a year. Both Infantile and LOTS: quantitative neuroimaging, biomarkers, and neuropsychological protocol will be collected as well as relevant medical history to develop quantitative markers to measure outcomes for future treatments. doi:10.1016/j.ymgme.2009.10.034
18. Axonopathy in a mouse model of Krabbe disease Ernesto Bongarzone, Ludovico Cantuti, Maria I. Givogri, Aurora Lopez-Rosas, Gustavo Pigino, Gerardo Morfini, Scott Brady, Department of Anatomy and Cell Biology, University of Illinois, Chicago, IL, USA Psychosine, a galactosyl-sphingolipid normally degraded by galactosyl-ceramidase (GALC), accumulates to toxic levels in Krabbe disease (KD), where GALC is deficient. This induces the apoptosis of myelinating glia and leads to a progressive demyelination of the nervous system and to the death in most of affected cases. While axonal degeneration has been indicated in some case reports, the cause for these pathological observations has remained associated to a secondary side effect of demyelination. Because expression of GALC is ubiquitous and because neurons are known to synthesize many of kinds of sphingolipids, we speculated that neurons in KD might also accumulate psychosine and hence, become intrinsically sensitive to this toxin. Using the Twitcher mouse, an authentic model of KD, here we report neuronal loss and axonal abnormalities before the onset of demyelination. In line with the invivo findings, psychosine was detected in Twitcher neurons, in absence of myelinating glia. We show that the abnormalities observed invivo can be replicated in cell culture by administration of psychosine to cultured cortical neurons and neuroblastoma cells, identifying psychosine as a potential mediator of axonal degeneration in KD. Of relevance, psychosine is shown to inhibit fast axonal transport invitro. These observations suggest that transport abnormalities may contribute to axonopathy in KD. Taken together, our results support the hypothesis that GALC deficiency may exert an intrinsic defect in mutant neurons and identify psychosine as a direct mediator of
Abstracts/Molecular Genetics and Metabolism 99 (2010) S8–S41
Gaucher disease, characterized by glucocerebrosidase deficiency, provokes glucosylceramide accumulation in cellular lysosomes. Current therapy uses mammalian cell production of recombinant glucocerebrosidase for enzyme replacement therapy which requires post-expression glycan remodeling for exposing mannose structures, which are required for uptake by Gaucher macrophages cells. Expressing taliglucerase alpha in specifically targeted plant cell organelles enables the cellular control of the mannose glycosylation pattern and consistency. Thus, taliglucerase has intrinsic exposed mannose residues and demonstrates high batch to batch uniformity. taliglucerase exhibits similar crystal structure and biological activity to that of the CHO expressed imiglucerase and demonstrates superior uptake to macrophage cells. Preclinical and Phase I clinical trials showed that Taliglucerase alpha was well tolerated, with no significant immune reaction or production of neutralizing antibodies. A multi-center Phase III pivotal trial performed under FDA Special Protocol Assessment has been recently concluded. The trial was a randomized, double-blind, parallel group, dose-ranging trial to assess the safety and efficacy of Taliglucerase in naive Gaucher disease patients. In the pivotal trial, patients were selected randomly for one of two dosing arms, 30 or 60 U/kg infusions every 2 weeks over 9 months. The primary endpoint of the study is the reduction in spleen volume from baseline, as measured by MRI. In addition, secondary end points such as liver volume, hemoglobin and platelet counts where followed, as well as additional biomarkers and bone parameters. Results of this pivotal study will be presented and discussed. doi:10.1016/j.ymgme.2009.10.026
10. Life expectancy and cause of death in Fabry disease: Findings from the Fabry Registry Maryam Banikazemia, Manesh Patelb, Roberta Lemayc, Stephen Waldekd, aColumbia University Medical Center, New york, NY, USA, bDivision of Cardiovascular Medicine, Duke University School of Medicine, Durham, NC, USA, cBiomedical Data Sciences and Informatics, Genzyme Corporation, Cambridge, MA, USA, dSalford Royal NHS Foundation Trust, Manchester M6 8HD, United Kingdom Clinical and investigative data from 2848 patients in the Fabry Registry were used to evaluate life expectancy and cause of death. Among this cohort, 75 of 1422 males and 12 of 1426 females were reported to have died, at a median age of 54.3 years in males and 62.0 years in females. The 87 deceased patients were diagnosed at a much older age than other patients in the Fabry Registry: median age at diagnosis was 40 years versus 24 years in males and 55 years versus 33 years in females. Life expectancies were calculated from birth date, based on data from the entire Fabry Registry population. Life expectancy of males with Fabry disease was 58.2 years, compared to 74.8 years in the general US population. Life expectancy of females with Fabry disease was 75.4 years, compared to 80.1 years in the general US population. The most common cause of death was cardiovascular disease. The deaths of 30 of 56 males with a known cause of death (54%) and 5 of 10 females with a known cause of death (50%) were classified as due to cardiovascular disease. Most Fabry patients who died of cardiovascular disease (20 of 35, 57%) had previously received renal replacement therapy. Cardiac function should be closely monitored in Fabry patients. Patients who exhibit signs or risk factors for cardiovascular, renal, or cerebrovascular disease should receive prompt and appropriate treatment for these conditions. doi:10.1016/j.ymgme.2009.10.027
11. Marked variability in urinary Gb3/creatinine excretion in healthy infants Caroline Barr, Joe T.R. Clarke, Aim Ntwari, Rgen Drouin, Christiane Auray-Blais, CHUS– Faculty of Medicine and Health Sciences, Universit de Sherbrooke, Sherbrooke, Canada Introduction: Fabry disease is a lysosomal storage disorder resulting in glycosphingolipid accumulation, mainly globotriaosylceramide (Gb3), in various tissues and physiological fluids. In order to establish reference values for infants less than 12 months of age, we measured urinary Gb3 by a liquid chromatography–tandem mass spectrometry (LC–MS/MS) methodology previously developed in our lab. Method: We analyzed 728 urine specimens collected by parents from 68 healthy infants of both sexes at intervals from birth to 6 months of age. Results: Parental compliance was good at 90%. Gb3/creatinine results were grouped into four periods according to age: <6 days; 6–29 days; 30–90 days; >90 days for statistical analyses. In both girls and boys, Gb3 excretion decreased with postnatal age. Urinary creatinine levels were increased at birth and decreased rapidly with time. There was no significant difference (p = 0.4620) between creatinine excretions for either sexes vs postnatal age. In contrast to infants >90 days of age, we found no statistically significant difference between boys and girls during the first 3 periods (p = 1.0000 for all comparisons), though the levels tend to be higher in girls. The anal-
ysis of variance of transformed Gb3/creatinine ratios for 728 samples according to sex and period showed a significant difference between boys (14.6 g/mmol creat.) and girls (42.7 g/mmol creat.) in period 4 (p = 0.0005). Conclusion: Urinary Gb3 excretion varied markedly among healthy infants of both sexes at all ages from birth to 6 months. doi:10.1016/j.ymgme.2009.10.028
12. The blood–brain barrier: A central role in the pathology and treatment of neuronopathic lysosomal storage disorders David Begley, Kings College, London, United Kingdom The central nervous system requires an extremely stable and specialised extracellular fluid environment in order to ensure correct neuronal and synaptic function. This extracellular fluid compartment of the brain is maintained separate and distinct from that of the blood and the extracellular fluid of other tissues. The concentrations of ions, metabolites, peptides, proteins and other solutes in cerebrospinal fluid and brain extracellular fluid are precisely regulated and can be very different from those in blood. It is the blood–brain barrier formed by the cerebral capillaries which enable this distinct fluid environment of the brain to be maintained. The endothelial cells forming the capillaries of the brain maintain tight junctions at the cell margins which prevent any paracellular movement of solutes. Transfers of solutes into and out of the brain thus have to be transcellular across the endothelial cells. Transport mechanisms in the cell membranes of these endothelial cells are also polarised and differ in the blood and brain facing cell membranes. The presence of the blood–brain barrier severely restricts the usefulness of many drugs and therapeutic compounds in the treatment of central nervous disease as they do not reach the brain in effective quantities. These properties of the blood–brain barrier will restrict the entry of chaperones and substrate reduction therapies into the brain and totally exclude large molecular weight enzyme replacement therapy. There is also evidence that the function of the blood–brain barrier is also disturbed in some lysosomal storage disorders and may contribute to cerebral damage and dysfunction. doi:10.1016/j.ymgme.2009.10.029
13. A pharmacogenetic approach to pharmacological chaperone therapy for Fabry disease Elfrida Benjamin, Xiaoyang Wu, Evan Katz, Kirsten Mascioli, Kate Chang, David Lockhart, Kenneth Valenzano, Amicus Therapeutics, Cranbury, USA Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the gene (GLA) that encodes galactosidase A (a-Gal A). More than 600 mutations have been reported; 60% are missense. AT 1001 (migalastat hydrochloride) is a pharmacological chaperone that selectively binds a-Gal A, increasing physical stability, lysosomal trafficking, and total cellular activity. A systematic approach to select patients that may be candidates for AT 1001 therapy was developed. Each of 415 known Fabry disease-causing missense and small in-frame insertion or deletion mutations were expressed in HEK-293 cells and tested for response to AT 1001. Increased a-Gal A levels were seen for a majority of the mutant forms. To identify the subset of mutant forms that are most likely to respond to AT 1001 invivo, criteria were developed based on the magnitude of the AT 1001 response in the HEK-293 assay. The criteria were applied to the HEK-293 results and compared to those of an AT 1001 Phase 2 trial that included male Fabry patients representing 18 different mutant forms of a-Gal A. The HEK-293 results were consistent with the patient white blood cell a-Gal A responses after oral administration of AT 1001. To date, 173 mutant forms meet these criteria. Collectively, the data suggest that a pharmacogenetic reference table comprised of the AT 1001-responsive a-Gal A mutant forms and the corresponding GLA mutations can be used to select male and female Fabry patients for pharmacological chaperone therapy with AT 1001 based on genotype. doi:10.1016/j.ymgme.2009.10.030
14. Polyneuropathy in Type 1 Gaucher disease: A two-year,multinational, prospective observational study Marieke Biegstraatena, E. Mengelb, L. Marodic, M. Petakovd, C. Niederaue, P. Giraldof, D. Hughesg, M. Mrsich, A. Mehtag, C.E.M. Hollaka, I.N. van Schaika, aAcademic Medical Center, Amsterdam, The Netherlands, bUniversitts Kinderklinik, Mainz, Germany, c University of Debrecen, Debrecen, Hungary, dClinical Center of Serbia, Belgrade, Serbia, e Klinik fr Innere Medizin, Universitt Essen, Dsseldorf, Germany, fMiguel Servet University
Abstracts/Molecular Genetics and Metabolism 99 (2010) S8–S41 Hospital and CIBERER, Zaragoza, Spain, gRoyal Free and University College Medical School, London, UK, hUniversity Hospital Centre, Department of Hematology, Zagreb, Croatia Type 1 Gaucher disease (GD1) is currently categorised as non-neuronopathic, although recent studies suggest peripheral neurological manifestations. We conducted a two-year, multicentre, prospective, observational cohort study in patients with GD1 to determine the prevalence and incidence of polyneuropathy, evaluated by standardised assessments of neurological symptoms and signs, and electrophysiological studies. Secondary outcome parameters included the prevalence and incidence of mononeuropathy, other neurological or electrophysiological abnormalities not fulfilling the criteria for a mono- or polyneuropathy, and general GD1 symptoms. Furthermore, a systematic literature search was performed to identify all studies reporting on prevalence and incidence of polyneuropathy in the population. One hundred and three patients were enrolled. At baseline, 11 patients (10.7%; 95%CI 5.5–18.3%) were diagnosed with sensory motor axonal polyneuropathy. Two (1.9%; 95%CI 0.2–6.8%) had an ulnaropathy. During two-years of follow-up six new cases of polyneuropathy were identified (5.9%; 29/1000 patient-years). Patients with polyneuropathy were older than those without. Possible associated conditions were identified in 5/17 patients only (gammopathy, vitamin B1 or B12 deficiency, diabetes mellitus, renal insufficiency and alcohol abuse). The 11 cases of polyneuropathy diagnosed at baseline remained unchanged or worsened during follow-up. According to the literature, the prevalence of polyneuropathy in the population is estimated between 0.09% and 7.5% and the annual incidence is estimated between 0.46 and 85 per 10.000 person-years. Thus, the prevalence and incidence of polyneuropathy in patients with GD1 appear to be higher than that of the general population, and may be part of the natural disease course. doi:10.1016/j.ymgme.2009.10.031
15. The effect of long-term substrate reduction therapy withgenistein in a mouse model of MPS IIIB Brian Biggera, Marcela Malinowskaa, Fiona Wilkinsona, Kia Langford-Smitha, Alexander Langford-Smitha, Rob Wynnc, Ed Wraithd, Grzegorz Wegrzynb, Brian Biggera, aUniveristy of Manchester, Manchester, UK, bUniveristy of Gdansk, Gdansk, Poland, cRoyal Manchester Childrens Hospital, Manchester, UK, dSt Marys Hospital, Manchester, UK Mucopolysaccharide (MPS) diseases IIIA,B,C,D are neuronopathic diseases which primarily accumulate the glycosaminoglycan heparan sulphate due to defects in lysosomal enzymes. Symptoms include progressive CNS degeneration characterised by mental retardation, hyperactivity and seizures, with death usually in the mid teens to early twenties. No treatments currently exist for any of the Sanfilippo diseases. We have previously shown that genistein aglycone can significantly reduce GAG and lysosomal compartment size in peripheral organs of MPS IIIB mice at several doses. In that 8 week study we did not observe drug related toxicity, neither did we observe reduction of GAG in the brain, despite evidence to suggest that 10% of the drug can cross the blood–brain barrier. Our hypothesis was that the treatment regimen was too short to observe changes in the brain. In the study presented here, we have performed a 34 week comparison of genistein (160 mg/kg/day) with placebo control in MPS IIIB and WT mice. We found improvements in the health of genistein treated MPS IIIB mice, as well as improvements or full correction of several behavioural parameters. We also present histological and biochemical findings in the brains of these mice. We have not seen toxicity from genistein despite these doses being considerably higher than those achievable with genistein in soy extract form. These data would support the development of a clinical trial of genistein aglycone in patients with MPS III and other MPS diseases. doi:10.1016/j.ymgme.2009.10.032
16. A chemical genetic approach to identifying therapeutictargets for NCL Sunita Biswasa,b, Sasja Heetvelda,b, Pavlina Wolfa,c, Yi Caoa, Stephanie Nortonb, Stephen Haggartyb, Susan Cotmana, aHarvard Medical School, Boston, USA, b Massachusetts General Hospital, Boston, USA, cBroad Institute of MIT and Harvard, Cambridge, MA, USA Neuronal Ceroid Lipofuscinosis (NCL) is a genetically heterogeneous group of disorders, characterized by overlapping clinical and pathological features, including the autophagosomal/autolysosomal accumulation of the mitochondrial ATP synthase, subunit-c protein. We have used neuronal cell culture models derived from mice harboring the most common disease mutation causing juvenile NCL (Cln3ex7/8 mice and CbCln3ex7/8 cerebellar cells) in phenotypic cell-based assays developed to screen for modifiers of NCL disease phenotypes and identify target pathways for therapeutic
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development. An autophagy GFP-LC3 based high-content screening assay, optimized to identify modulators of abnormal accumulation of autophagic vacuoles, was used in a chemical genetic screen of FDA-approved drugs and natural products. Seventeen compounds were identified as hits that altered the number of GFP-LC3 positive autophagic vacuoles in homozygous CbCln3ex7/8 cerebellar cells, compared to wild-type cells. Follow-up studies with one of the validated compounds, thapsigargin, known to induce increased cytosolic calcium levels, demonstrated a heightened sensitivity of the homozygous CbCln3ex7/8 cerebellar cells to a thapsigargin-induced autophagic response, compared to wild-type cells. Furthermore, subunit c accumulation was significantly worsened following a 24-h treatment with thapsigargin, while in wild-type cells, thapsigargin did not alter the amount of subunit c deposits. Further studies of thapsigargin in the Cln3ex7/8 genetic models will ascertain whether altered sensitivities of the wild-type and mutant cerebellar cells to thapsigargin treatment highlights differences in calcium regulation and homeostasis in the presence of NCL gene mutations. doi:10.1016/j.ymgme.2009.10.033
17. A natural history study of hexosaminidase deficiency Kendra Bjoraker, Julie Eisengart, Peter Karachunski, Gulin Oz, Chester Whitley, University of Minnesota, Minneapolis, MN, USA Hexosaminidase A deficiency results in a group of neurodegenerative disorders caused by intralysosomal storage of the specific glycosphingolipid GM2 ganglioside. Three studies are proposed: (1) focus on developmental course of infantile TSD from retrospective data and complete literature review to elucidate the clinical course of TSD; (2) collect longitudinal prospective data from patients with TSD seen at the University of Minnesota; (3) longitudinal study of LOTS to understand progression of CNS disease using MRI/MRS and neuropsychological measures. Objectives: Aim 1: Develop index of disease progression in TSD: A. First, a retrospective medical chart/record review for clinical data points. Second, caretakers of infants with TSD will be asked to complete the University of Minnesota Natural History Study Questionnaire for Infantile Tay-Sachs Disease (MNQSTD). B. Prospective: We will collect longitudinal neuropsychological (Bayley II, Vineland) and neuroimaging data in patients with infantile TSD to characterize developmental course and longitudinally examine individual growth trajectories. Aim 2: To better understand underlying CNS structure and function abnormalities in LOTS disease and to measure change in function over time through neuroimaging and neuropsychological data (WASI, Mindstreams Neurotrax, Vineland). Methods: Infantile: Recruit 5 new patients the 1st year for a total of at least 3 follow-up visits within the 5 year period. LOTS: Participants: Recruit 5 new patients 1st year. Each patient will be seen once a year. Both Infantile and LOTS: quantitative neuroimaging, biomarkers, and neuropsychological protocol will be collected as well as relevant medical history to develop quantitative markers to measure outcomes for future treatments. doi:10.1016/j.ymgme.2009.10.034
18. Axonopathy in a mouse model of Krabbe disease Ernesto Bongarzone, Ludovico Cantuti, Maria I. Givogri, Aurora Lopez-Rosas, Gustavo Pigino, Gerardo Morfini, Scott Brady, Department of Anatomy and Cell Biology, University of Illinois, Chicago, IL, USA Psychosine, a galactosyl-sphingolipid normally degraded by galactosyl-ceramidase (GALC), accumulates to toxic levels in Krabbe disease (KD), where GALC is deficient. This induces the apoptosis of myelinating glia and leads to a progressive demyelination of the nervous system and to the death in most of affected cases. While axonal degeneration has been indicated in some case reports, the cause for these pathological observations has remained associated to a secondary side effect of demyelination. Because expression of GALC is ubiquitous and because neurons are known to synthesize many of kinds of sphingolipids, we speculated that neurons in KD might also accumulate psychosine and hence, become intrinsically sensitive to this toxin. Using the Twitcher mouse, an authentic model of KD, here we report neuronal loss and axonal abnormalities before the onset of demyelination. In line with the invivo findings, psychosine was detected in Twitcher neurons, in absence of myelinating glia. We show that the abnormalities observed invivo can be replicated in cell culture by administration of psychosine to cultured cortical neurons and neuroblastoma cells, identifying psychosine as a potential mediator of axonal degeneration in KD. Of relevance, psychosine is shown to inhibit fast axonal transport invitro. These observations suggest that transport abnormalities may contribute to axonopathy in KD. Taken together, our results support the hypothesis that GALC deficiency may exert an intrinsic defect in mutant neurons and identify psychosine as a direct mediator of