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Intravenous SBC-103, a recombinant human alpha-N-
acetylglucosaminidase reduces CNS heparan sulfate content
in a mucopolysaccharidosis type IIIB mouse model
Joseph V. Rutkowskia, Kari Harbertb, Hao Xua, Wei Hua, Matthew
Renningerc, Mark C.C. Leavitta, Qinming Chena, Sandra Rojas-Caroa,
Anthony G. Quinna, aSynageva BioPharma, Lexington, MA, USA, bBRM, Inc,
Worcester, MA, USA, cCovance, Inc, Greenfield, IN, USA
Mucopolysaccharidosis (Sanfilippo syndrome type B) is a lysosomal
disorder caused by deficiency in the enzyme alpha-N-acetylglu-
cosaminidase (NAGLU). At present, there is no treatment for this disease.
We previously reported initial findings from a mucopolysaccharidosis
type IIIB mouse model that both intravenously (IV) and intrathecally (IT)
administered SBC-103 decreased brain heparan sulfate disaccharide
with a concomitant increase in brain NAGLU enzyme activity. NAGLU
activity and heparan sulfate disaccharide (HSD) levels were measured in
tissue from NAGLU (-/-) mice in separate studies following (i) IV
administration once weekly for 3 to 6 weeks; (ii) IV administration once
weekly for 4 weeks; (iii) IT administration once every other week for
12 weeks. The IV doses of 5, 10, and 20 mg/kg body weight corresponded
to dose levels of 0.42, 0.83, and 1.67 mg/g brain weight. The IT doses of
1.7, 5 and 10 mg/kg body weight corresponded to dose levels of 0.14,
0.42 and 0.83 mg/g brain weight. Whole blood and tissues were collected
at the scheduled sacrifice and analyzed. Dose dependent reductions in
heparan sulfate in NAGLU (-/-) mice were seen with IV dosing of SBC-
103. Reductions were also seen with IT dosing but a minimally effective
dose was not defined across the doses tested. Decreases in heparan
sulfate were accompanied by increases in perfused brain enzyme activity
levels. These data confirm our initial findings of the effects of IV SBC-103
on CNS substrate accumulation in this disease model, and support
further investigation of IV dosing of SBC-103 as an approach for patients
with MPS IIIB.
doi:10.1016/j.ymgme.2013.12.222
211
Morquio syndrome type A locus-specific database: a framework
for a curated database to identify and characterize pathogenic
variants in the GALNS gene
April B. Rylesa, Chaoyi Dub, Tal Oronb, Robert Atwoodb, Sean D.
Mooneyb, Patricia Francis-Lyona, aUniversity of San Francisco, San
Francisco, CA, USA, bThe Buck Institute for Research on Aging, Novato,
CA, USA
Mutations in the N-acetylgalactosamine-6-sulfatase (GALNS) gene
cause the genetic disease mucopolysaccharidosis type IV A (Morquio
syndrome type A, MPS IV A). MPS IV A is an autosomal recessive
lysosomal disorder. Currently there are more than 100 known missense
and other mutations in the GALNS gene that are causative. MPS IV A is
characterized by short stature due to skeletal dysplasia, as well as
pulmonary and cardiovascular disease, all which may contribute to
limited walking ability and endurance (OMIM #253000). The gold
standard for diagnosis of MPS IV A is a documented enzyme defect in the
enzyme GALNS. Novel disease causing mutations in GALNS continue to
be reported and several polymorphisms are present in the population.
Novel GALNS mutant alleles provide useful information for genetic
counseling but often these alleles are not publicly available or made open
access. Genotype to phenotype correlations of disease severity and the
basis for phenotypic expressivity and penetrance of mutations remain
elusive. In order to address these challenges, we have created a web-
based resource for curating and coalescing mutations discovered in the
Abstracts
GALNS gene. The website is based on the Python Django framework and
deployed on the cloud using Heroku. In this poster, we will present our
initial database schema, currently annotated variants and our workflow
for recruiting and training curators of novel variants discovered in this
gene. We will also present our ideas for expanding the resource to
include information about complex heterozygotes and secure patient
level data.
doi:10.1016/j.ymgme.2013.12.223
212
Development of enzyme replacement therapy for Fabry disease
with a modified α-N-acetylgalactosaminidase
Hitoshi Sakuraba, Meiji Pharmaceutical University, Kiyose, Japan
In this study, we tried to develop a stable enzyme which is expected
to be well incorporated into the target organs and escape from immune
reaction on enzyme replacement therapy (ERT) for Fabry disease. A
modified alpha-N-acetylgalactosaminidase (NAGA) with alpha-galacto-
sidase A (GLA)-like substrate specificity was designed on the basis of
structural study and was produced in Chinese hamster ovary cells. The
modified NAGA purified by column chromatography was characterized
biochemically and immunologically. Then the enzyme was injected into
tail veins of GLA knock-out mice and human NAGA transgenic/GLA
knock-out mice, and the effects on cleavage of the accumulated
glycolipids and immune reaction were examined. The modified NAGA
retained the original NAGA’s stability in plasma and N-glycans
containing mannose 6-phosphate (M6P) residues, which are advanta-
geous for uptake by cells via M6P receptors. The results of the surface
plasmon resonance analysis and the enzyme-linked immunosorbent
assay revealed that there was no immunological cross-reactivity
between the modified NAGA and GLA. The modified NAGA did not react
to serum from a Fabry patient recurrently treated with a recombinant
GLA. The modified NAGA intravenously injected into GLA knock-out
mice prevented globotriaosylceramide storage in the liver, kidneys, and
heart and improved the pathological changes in the organs. The long-
term injection of the modified NAGA into human NAGA transgenic/GLA
knock-out mice did not show any injurious accidents and it did not
produce any anti-modified NAGA IgG1. The modified NAGA is highly
promising as a new safe enzyme for ERT for Fabry disease.
doi:10.1016/j.ymgme.2013.12.224
213
Using a computational human metabolic reconstruction on the
study of mucopolysaccharidosis
Diego Salazar, Alexander Rodríguez-López, George E. Barreto, Janeth
González, Carlos J. Almeciga-Diaz, Pontificia Universidad Javeriana,
Bogota, Colombia
Glycosaminoglycans (GAG) are the main biomarker used in the
diagnosis of mucopolysaccharidosis (MPS). However, during the last
years there have been growing interests in the identification of new
biomarkers that allow to overcome some of the limitations observed
with GAG. In this study we used a computational human metabolic
reconstruction (Recon2) to model the metabolic changes observed after
silencing of each one of the MPS-related genes, as well as to predict new
biomarkers for MPS. Models were done and analyzed using COBRA
toolbox through flux balance and variability analysis. Genes associated
with reactions that changed after gene silencing were subjected to
 Abstracts
enrichment analysis. Overall, 895 reactions out of 7441 were commonly
impaired in MPS models, which are mainly involved in cellular
respiration, mitochondrial process, amino acid and lipid metabolism,
and ion exchange processes. Metabolic changes were similar for MPS I
and II, and for MPS III A, B and C; while the remaining MPS showed
unique metabolic profiles. These results agree with previous reports
showing autophagy, cellular and mitochondrial stress on MPS, and
improved the prediction previously done using the same stratetgy for
GAG degradation pathway. Finally, models were used to predict
biomarkers for MPS. Although in-vivo validation of these biomarkers is
still needed, these results confirm the potential of the computational
human metabolic reconstruction to understand cellular alterations and
the prediction of biomarkers for MPS.
doi:10.1016/j.ymgme.2013.12.225
214
Phenotypic characterization of the spinal muscular atrophy with
progressive myoclonus epilepsy syndrome caused by
ASAH1 mutations
Swati Sathea, Toni Pearsonb, aSt. Joseph's Regional Medical Center,
Paterson, NJ, USA, bColumbia University Medical Center, New York City,
NY, USA
Objective: Phenotypic characterization of the newly described
spinal muscular atrophy - progressive myoclonus epilepsy syndrome
caused by mutations in acid ceramidase ASAH1.
Background: Spinal muscular atrophy - progressive myoclonus
epilepsy is a very rare autosomal recessive disorder. Recently,
mutations in ASAH1 were described as pathogenic in this entity.
Case report: We describe a 15-year-old girl with decline in
cognitive and motor domains starting at age six. Initial symptom was
progressive cognitive decline, requiring transfer from a regular
school to special school by third grade. Seizures started around age
seven shortly followed by motor weakness. Seizures were charac-
terized as drop attacks and staring spells; there were no generalized
tonic clonic seizures. Several medications were tried for seizure
control; a combination of leveteracitam and clonazepam offered the
best benefit. Myoclonus was present in the form of a jerky irregular
tremor, as well as more generalized frequent myoclonic jerks. Motor
weakness presented as difficulty climbing stairs which progressed to
difficulty walking over next three years. Parkinsonism in the form of
cog wheeling, bradyphrenia, hypomimia and diminished arm swing
as well as cervical dystonia developed at age of nine to ten years.
There was no benefit from a trial of levodopa/carbidopa. By age
twelve, pulmonary function test showed decreased FVC with reduced
inspiratory and expiratory muscle pressures compatible with
neuromuscular function. Cognitive decline was relentless, eventually
causing an home bound state, hypophonia and inability to carry out
activities of daily living. EMG showed fibrillations and positive sharp
waves in multiple muscles with no fasciculation suggestive of
anterior horn cell involvement. Brain MRI was normal. Extensive
work up for Wilson’s, lysosomal disorder, mitochondrial disease and
progressive myoclonus epilepsy was negative. Ultimately, whole
genome sequencing showed heterozygous mutations in ASAH1,
536C N T and c. 124A N G, carrier state confirmed in each parent.
Conclusion/Relevance: Phenotype of spinal muscular atrophy with
progressive myoclonus epilepsy syndrome caused by ASAH1 muta-
tions is yet not completely delineated. Parkinsonism and dystonia,
not previously described, were predominant features in this patient.
doi:10.1016/j.ymgme.2013.12.226
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Farber disease: characterization of behavior and brain pathology
in a new murine model
Tomo Sawadaa, Shaalee Dworskib, Jakub Sikoraa, Matthew Micsenyia,
Jeffrey A. Medinb, Steven U. Walkleya, David R. Hampsonb, aAlbert
Einstein College of Medicine, Bronx, NY, USA, bUniversity of Toronto,
Toronto, ON, Canada
Farber disease is caused by deficiency of acid ceramidase leading
to accumulation of ceramide in various organs. Characteristic
features of this disorder are painful swelling of joints, respiratory
difficulties, and severe impairment of psychomotor development.
Here we report CNS phenotyping of a mouse model of Farber disease
that we recently generated. Asah1P361R/P361R homozygous Farber
mice are normal at birth, exhibit growth retardation, and die
between 7-13 weeks of age. Behaviorally, Farber mice exhibited
(1) compromised rotarod performance; (2) weakness in inverted
grip tests; (3) decreased locomotion, increased thigmotaxis behavior,
and decreased rearing; (4) increased stereotypic behavior in the
open-field test; (5) increased time chewing compared to WT
littermates; and (6) decreased stereotype behavior in marble
burying tests. For brain pathology analysis, hydrocephaly was
observed by MRI in Farber mice at 7 and 9 weeks, but not in
5 week-old animals or WT littermates. Cerebral blood flow was
analyzed in 10 week-old homozygous mice and found to be reduced
by 50% in the cerebral cortex. GM2 ganglioside labeling of neurons in
the CA4 region of hippocampus, the granule cell layer of the
cerebellum, and the septal nucleus was observed. Filipin labeling
for unesterified cholesterol also showed minor accumulation in
neurons of the septal nucleus and in cerebellar Purkinje cells.
Interestingly, immunolabeling for the macroautophagy adapter
protein p62/SQSTM1 revealed numerous aggregates in these same
neurons. Western blot analysis revealed increases in LC3-II (vs. LC3-
I) and p62 protein consistent with autophagy impairment. With
electron microscopy of cerebral cortex from 9 week-old homozygous
Farber mice we observed electron lucent membrane-bound vacuoles
in the cytoplasm of cortical neurons that also contained zebra-like
profiles. Full characterization of this new Farber disease model will
allow greater understanding of the importance of ceramide degra-
dation in different types of cells, reveal insights into disease
pathogenesis, and provide an opportunity to effectively test thera-
peutic strategies.
doi:10.1016/j.ymgme.2013.12.227
216
Is mucolipidosis type IV a neurodegenerative disorder?
Raphael Schiffmanna, Joan Mayfieldb, Caren Swifta, Igor Nestrasilc,
a
Baylor Research Institute, Dallas, TX, USA, bOur Children’s House at
Baylor, Dallas, TX, USA, cUniversity of Minnesota, Minneapolis, MN, USA
Mucolipidosis type IV (MLIV) is an autosomal recessive disorder
resulting from mutations in the MCOLN1 gene. This gene encodes
the endosomal/lysosomal transient receptor potential channel pro-
tein mucolipin-1 (TRPML1). Affected patients suffer from neuro-
developmental abnormalities and progressive retinal dystrophy. In a
prospective natural history study we hypothesized the presence of
an additional slow cerebral neurodegenerative process. We have
thus far recruited 5 patients, tested their neurodevelopmental status
and measured cerebral volumes and diffusion tensor imaging using
MRI yearly. Over a period of up to 3 years, MLIV patients remained
neurologically stable. There was a trend for increased cortical and