Efficient urine filter paper mass spectrometry methodology for glycosaminoglycan quantification

Abstracts / Molecular Genetics and Metabolism 102 (2011) S3–S47

group with a ≥50% reduction in the number of GL-3 inclusions per interstitial capillary is greater than that in the placebo group using the exact Cochran–Mantel–Haenszel test stratified by sex. A supportive analysis will be performed using similar methods for the per protocol population. doi:10.1016/j.ymgme.2010.11.013

Neuropsychological and neuroimaging findings in MPSVI Alia Ahmed, Julie Eisengart, Kyle Rudser, Kathleen Delaney, Igor Nestrasil, Elsa Shapiro, University of Minnesota, Minneapolis, MN, USA Background: Mucopolysaccharidosis type VI (MPSVI) or Maroteaux Lamy Syndrome is a rare inherited lysosomal disease arising from a deficiency of enzyme arylsulfatase B with a spectrum of severity from mild to severe. MPS VI is described as affecting somatic systems with little CNS involvement. However, scattered reports have indicated both cognitive impairment and abnormal appearing brain scans. A careful longitudinal study has not been done. Until the development of enzyme replacement therapy (ERT) in 2005, hematopoietic cell transplantation (HCT) was the only treatment. Goal: To characterize the CNS phenotype and the effects of treatment using quantitative MRI and neuropsychological data. Methods: As part of a longitudinal study, 8 patients with MPSVI were recruited. They all had an unsedated 3 Tesla brain MRI. Manual tracing of hippocampus was done with Brains2; automated volumes were obtained using FreeSurfer. Neuropsychological testing included IQ, attention, memory, spatial, and behavioral assessment. These results reflect first year cross-sectional data. Results: Of 8 patients, 2 had ERT only, 6 had HCT, 2 of which were unengrafted. Three were female. In the six with HCT, average IQ, below average attention, and low average reaction time were found. All 8 subjects had visual motor difficulties compared to normative data. Two patients with ERT have above average intelligence, average reaction time, but below average attention. Overall brain volume as well as corpus callosum values was smaller in patients who had HCT. Two HCT patients with small brain and hippocampal volumes demonstrated lower intelligence and attention scores. Two ERT patients will soon be added to these data. We conclude that HCT treatment has a long-term negative effect on brain volume and overall cognition in MPS VI. doi:10.1016/j.ymgme.2010.11.014

Will psychological intervention improve adaptive functioning in Fabry disease patients? Nadia Ali, Laney Dawn, Fernhoff Paul, Emory University, Decatur, GA, USA Fabry disease (FD) is an X-linked genetic lysosomal storage disorder caused by a deficiency of the lysosomal enzyme αgalactosidase A. Historically, the focus of FD treatment has been on somatic manifestations of the disease, with very little attention paid to potential psychiatric manifestations. Anecdotal evidence points towards FD patients' increased difficulties in performing daily tasks, including keeping a full time job and being medically compliant. These difficulties suggest ;that they have decreased social and occupational adaptive functioning. Adaptive functioning refers to the effectiveness with which an individual copes with the daily demands of everyday tasks and responsibilities. Two fundamental aspects of adaptive functioning are personal independence and social responsibility. Adaptive function

S5

can be measured through questionnaires and evaluations focused on an individual's relationships, job, education, substance use, psychological issues, and coping skills. A 2009 pilot study by the Emory LSDC team determined that poorer adaptive functioning correlated with significantly greater rates of depression and anxiety in their FD population. These data suggest that focused treatment of Fabry patients' psychological issues should improve adaptive functioning, increase treatment compliance, and help quality of life. Our current study thus randomizes 20 FD adults with adaptive function disorders into either a control group or individual therapy for 1 h every other week with a health psychologist, for a period of one year. Questionnaires are administered every 3 months to measure changes in adaptive function and related psychiatric conditions. We hypothesize that psychological treatment of FD patients will improve adaptive functioning. We further hypothesize that improvements in adaptive functioning will increase medical treatment compliance. doi:10.1016/j.ymgme.2010.11.015

Production and characterization of a recombinant N-acetylgalactosamine-6-sulfate sulfatase produced in E. coli BL21 Carlos Alméciga-Diaza, Angela Mosqueraa, Felice Reyesb, Carlos Sotoa, Alexander Rodrigueza, Lina Lizarasoa, Angela Espejoa, Oscar Sanchezb, Luis Barrerab, aPontificia Universidad Javeriana, Bogotá, D.C., Colombia, b Universidad de los Andes, Bogotá, D.C., Colombia Morquio A disease (MPS IVA) is an autosomal recessive disorder caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS) that leads to the accumulation of keratan sulfate and chondroitin-6-sulfate mainly in bone and cornea. Previously, we showed the feasibility for producing an active recombinant GALNS (rGALNS) enzyme in E. coli BL21 at shacked and bench scales. In this work, we report the production of a rGALNS at bench scale by using a fed-batch strategy, and the purification and characterization of the recombinant enzyme. This strategy allowed a 5-fold increment in enzyme production in the crude protein extract from cell lysate compare to those levels observed with a batch strategy (0.05 U/mg vs 0.01 U/mg). Noteworthy, recombinant enzyme was detected in culture media from fed-batch cultures; with enzyme activities of up to 2.9 U/mg. rGALNS was purified by anion exchange and gel filtration chromatography. Enzyme stability of the purified enzyme both from cell lysate and culture medium was characterized at pH from 3.0 to 8.0 at 4, 37 and 45 °C. Finally enzyme uptake was evaluated in HEK293 cells and human MPS IVA fibroblasts. In summary, these results constitute valuable evidence towards the development of an enzyme replacement therapy for MPS IVA using a prokaryotic recombinant enzyme. doi:10.1016/j.ymgme.2010.11.016

Efficient urine filter paper mass spectrometry methodology for glycosaminoglycan quantification Christiane Auray-Blaisa, Paméla Lavoiea, René Gagnona, Joe T.R. Clarkea, Yan Anb, Sarah P. Youngb, Haoyue Zhangb, David S. Millingtonb, aFaculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Quebec, Canada, bDuke University Medical Center, Durham, NC, USA Introduction: Mucopolysaccharidoses (MPSs) are complex storage disorders caused by specific lysosomal enzyme deficiencies resulting in the accumulation of glycosaminoglycans (GAGs) in urine, plasma,

Q43

S6

Abstracts / Molecular Genetics and Metabolism 102 (2011) S3–S47

and various tissues. We developed a tandem mass spectrometry methodology to quantify GAGs, dermatan sulfate (DS) and heparan sulfate (HS) in urine samples collected on filter paper. Method: A 5 cm urine filter paper disc was eluted with ammonium hydroxide and the eluate (900 L) was evaporated under nitrogen. One milliliter of methanol–HCl·3N was used for methanolysis performed by heating at 65 °C for 75 min. After evaporation under nitrogen, the residue was reconstituted in 300 L of 10 mM ammonium acetate/water containing deuterated DS and HS internal standards. Twenty microliters was injected onto an Alliance 2795XE LC coupled to a Quattro micro MS/MS (Waters). Chromatographic separation (9 min) was done on an Atlantis T3 column using solutions of 10 mM ammonium acetate in water and methanol. Data were recorded in positive electrospray ionization. Results: Linearity (r2 > 0.995), accuracy (Bias < 20%) and precision (CV < 15%) data were excellent. Levels of DS and HS in controls were below LOQs. We were able to efficiently discriminate MPS I, II and VI patients from controls. Conclusion: We devised a simple and efficient filter paper methodology to measure urinary GAGs. The method is applicable to high-risk screening as well as monitoring MPS patients on treatment. Moreover, urine filter paper specimens present major advantages for sample collection, storage and shipping. doi:10.1016/j.ymgme.2010.11.017

Clinical follow-up of Niemann–Pick disease type C patients, natural history and the importance of the disability scale assessing neurological disease progression Q2

Ramses Badilla-Porrasa,b, Maha Salehb, Don Mahuranc, Brigitte Rigatc, J.T.R. Clarkeb, Margaret Mackrellb, Julian Raimanb, aThe Hospital for Sick Children, Toronto, ON, Canada, bDepartment of Clinical and Metabolic Genetics, The Hospital for Sick Children, Toronto, ON, Canada, c Genetics and Genomic Biology, Research Institute, The Hospital for Sick Children, Toronto, ON, Canada Niemann–Pick disease type C (NP-C) is an autosomal recessive neurodegenerative disorder characterized by accumulation of unesterified cholesterol and glycosphingolipids in the endosomal/lysosomal system. It is characterized by a variety of progressive, disabling neurological symptoms including ataxia, dysarthria, dysphagia and cognitive deterioration. We present a description of the natural history and progression of the disease using a modified Disability Scale (Wraith et al., 2009). This is an observational retrospective cohort study designed to describe the natural history and progression of neurological disease. The modified scale rates from 0 (best) to 1 (worst) the severity in the four domains of: ambulation, manipulation, language and swallowing. The scores of every visit, calculated for 36 months, were on a yearly basis to assess progression. 15 patients (4 females and 11 males) were included. 3 had an identifiable NPC2 mutation and 10 a NPC1 mutation, Clinically 1 case presented as prenatal form (<3 months), 7 as early infantile (3 months to <2 years), 4 as late infantile (2 years to <6 years) and 1 as juvenile/ classical (6 years to 15 years). All NPC2 patients died before 6 years of age and reached a maximum score average of 3.875. Patients with NPC1 showed that annual rates of change were .069 in early infantile group, .076 in late infantile group and .020 in the juvenile group. NPC1 patients showed a maximum score average of 2.09. These results indicate severe phenotype in patients with NPC2 mutations and a higher progression rate in the infantile types among the NPC1 patients. doi:10.1016/j.ymgme.2010.11.018

Small molecule inhibitors of ganglioside biosynthesis as substrate reduction therapy for the gangliosidoses Xiaomei Bai, Tram Nguyen, Jillian Brown, Charles Glass, Sergio Duron, Shripad Bhagwat, Brett Crawford, Zacharon Pharmaceuticals, Inc., San Diego, CA, USA Tay–Sachs and Sandhoff diseases are lysosomal storage diseases caused by a deficiency in the lysosomal hexosaminidase required for the degradation of gangliosides. This deficiency leads to high levels of GM2 accumulation in the central nervous system which leads to progressive neurological deterioration and death. To date there is no effective therapy for these devastating neurological disorders. To address this unmet medical need, we are developing a small molecule CNS penetrant substrate reduction therapy. The goal of substrate reduction is to reduce the synthesis of the substrate (gangliosides) to alleviate the burden on the compromised lysosomal system. The therapeutic approach has been validated with known glucosylceramide synthase inhibitors. However, the efficacy of these compounds is potentially limited due low CNS penetration, limited ability to reduce ganglioside synthesis through the inhibition of upstream precursors (glucosylceramide), and doselimiting toxicity. Using a cell-based high throughput screen, we have identified a set of compounds that inhibit the synthesis of gangliosides without inhibiting the synthesis of early precursor glycolipids such as lactosylceramide. By focusing only on ganglioside inhibition, we believe this therapeutic strategy can reduce ganglioside accumulation in Tay–Sachs and Sandhoff diseases while avoiding the potential toxicity associated with inhibiting upstream precursors required for the synthesis of a broad set of glycolipids. Three unique scaffolds have been identified that i) inhibit ganglioside synthesis downstream of the lactosylceramide synthase, ii) penetrate the blood-brain barrier and iii) reduce GM2 storage in human Tay–Sachs and Sandhoff fibroblasts. At the time of abstract submission, we are currently testing a series of analogs of these three scaffolds to determine the best compound series to further develop as a novel CNS penetrant substrate reduction therapy for the gangliosidoses. doi:10.1016/j.ymgme.2010.11.019

Bortezomib: A solution to the challenge of antibodies in diseases treated with therapeutic proteins? Suhrad Banugariaa, Prater Seana, Sheela Nampoothirib, Jonathan Feldmanc, Joyce Koborid, Judeth McGannc, Dwight Koeberla, Priya Kishnania, aDuke University, Durham, NC, USA, bAmrita Institute of Medical Sciences & Research Centre, Cochin, Kerala, India, cKaiser Permanente, Santa Clara, CA, USA, dKaiser Permanente, San Jose, CA, USA Enzyme replacement therapy (ERT) is now available for a multitude of disorders. However, therapeutic enzymes are potentially immunogenic, inducing antibodies leading to decreased efficacy. Pompe disease (PD) is a lysosomal disorder caused by a deficiency of acid alphaglucosidase. Infantile PD is characterized by cardiomyopathy, hypotonia, respiratory insufficiency and death by age 1–2 years. Since the availability of ERT with alglucosidase alfa, the history of this disease has changed, with prolonged survival and enhanced quality of life. However, the immune response to ERT remains a challenge. Toleranceinducing therapies have been successful in at-risk (CRIM-negative) patients in the naïve setting or after early exposure to ERT. Once hightiter antibodies are formed, tolerization therapy has uniformly failed to lower antibodies. Notably lacking are drugs that target long-lived plasma cells, whose elimination is vital in reversing entrenched immune responses. Bortezomib, an FDA-approved therapy for multiple myeloma, is a 26S proteasome inhibitor which targets antibody-producing plasma