Small molecule therapies for juvenile neuronal ceroid lipofuscinosis

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Abstracts / Molecular Genetics and Metabolism 120 (2016) S17–S145

Guimarães, Portugal, cDr. Joaquim Chaves, Laboratório de Análises Clínicas, Miraflores, Portugal, dRoyal Free London NHS Foundation Trust and University College London, London, United Kingdom Cardiovascular disease is the main cause of death in Fabry disease (FD) and a major determinant of overall disease prognosis, with worst outcomes in patients with myocardial fibrosis. Currently, late gadolinium enhancement (LGE) is the gold standard for evaluation of replacement myocardial fibrosis. However, this is an end stage / irreversible event, thus identification of biomarkers of earlier diffuse fibrosis or increased collagen deposition is paramount. Type I collagen synthesis and degradation biomarkers (carboxy-terminal propeptide of procollagen type I [PICP], carboxy-terminal telopeptide of type I collagen and matrix metalloproteinases [MMP] 1 and 2), as well as markers of bone synthesis and degradation were evaluated (to adjust type I collagen metabolism to bone turnover) in FD patients and controls. FD patients were grouped by cardiomyopathy severity: 1) normal echocardiogram; 2) tissue Doppler abnormalities in echocardiogram; 3) left ventricular (LV) hypertrophy in echocardiogram. 60 FD patients (20 in each group; 38,3% males, mean age 50,2±14,7years, 65,0% on ERT) and 20 controls (age- and sexmatched with group 1) were recruited. A significant increase of PICP and a significant decrease in MMPs was observed in FD patients, even in the group 1 for the former biomarker. There was a significant correlation between LV mass and PICP (β= 0.413, p=0.001) or MMP-1 (β=-0.339, p= 0.01). PICP (adjusted for bone turnover) was the better predictor of LV mass in a multivariate regression model and its diagnostic accuracy to predict LGE was also significant. Collagen type I synthesis is increased in FD cardiomyopathy (even in the earlier stages of the disease) and this profibrotic state is a good predictor and is likely to be critical to the development of overt LV hypertrophy. Moreover, inhibition of enzymes involved in collagen type I cleavage seems to be a crucial pathophysiological link in myocardial collagen deposition. doi:10.1016/j.ymgme.2016.11.012

4 Characterization of a secondary deficiency of neuraminidase activity in a unique ovine model of GM1 gangliosidosis Amelia Ahern-Rindell, Alexandra Quackenbush, Sara McCrohan, University of Portland, Portland, OR, United States Ongoing characterization of an ovine lysosomal disease with dual deficiencies of beta-galactosidase and alpha-neuraminidase activities has provided additional evidence to support the hypothesis that this sheep model is a variant of GM1 gangliosidosis (GM1). Mutations in the GLB1 gene, which codes for beta-galactosidase, have been identified including a missence mutation we reported on in this dual deficiency sheep model. The amino acid substitution is most likely the primary cause for the profound deficiency of betagalactosidase activity as a result of its altered protein structure and possible premature degradation. Our explanation for the less severe, secondary deficiency of alpha-neuraminidase activity is based on the fact that these two hydrolases interact with one another as a part of the Lysosomal Multienzyme Complex (LMC). We proposed that the structurally altered beta-galactosidase interferes with normal formation of the LMC, which indirectly affects the alpha-neuraminidase activity. The neuraminidase protein is coded for by the NEU1 gene and mutations in this gene results in another lysosomal disease known as sialidosis. We believe it is highly unlikely that our affected sheep have two separate disease-causing mutations, one in each gene. To verify that there is

no mutation in the coding region of the NEU1 gene, we isolated genomic DNA from normal and affected sheep and performed PCR amplification of the exonic regions. No significant differences were found as a result of a comparison between the affected sheep DNA sequence and that of the normal sheep, and the reference sequence of NEU1 in GenBank. We also conducted assays on fibroblasts from affected sheep cultured in the presence and absence of the protease inhibitor MG132. We found an increase in alphaneuraminidase activity in the presence of the inhibitor. We conclude that the structurally altered beta-galactosidase leads to the malformed LMC that indirectly causes premature degradation of alpha-neuraminidase.

doi:10.1016/j.ymgme.2016.11.013

5 Neurocognitive and behavioral longitudinal trajectory of Hurler-Scheie syndrome patients with L238Q mutation Alia Ahmed, Elsa Shapiro, Kyle Rudser, Chester Whitley, University of Minnesota, Minneapolis, MN, United States A cross sectional study of the medical, cognitive, and psychiatric outcomes of patients affected with Hurler-Scheie syndrome with a heterozygous mutation of L238Q were previously published [PMC3939822]. This study extends prior work to investigate how the outcomes change over time and affect their life adjustment in comparison to non-L238Q Hurler-Scheie syndrome patients. Among 6 patients with a L238Q mutation, 4 were paired with a nonsense, 1 paired with a deletion, and 1 paired with a splice site mutation. This L238Q group was compared to 6 patients with Hurler-Scheie syndrome matched in age range. For the comparison group, 3 patients had a missense paired with nonsense mutation, 2 patients with missense paired with missense, and 1 patient with nonsense paired with splice site. To measure the somatic disease burden the Physical Symptom Score (PSS) was used [PMID: 26303610]. We measured FSIQ, attention, memory, adaptive function and psychological/behavioral status from 2009 to 2014. FSIQ scores increased on average by 1.5 per year for the L238Q group and 2.0 per year in the comparison group, but the range of FSIQ scores were uniformly below average in the L238Q group and within the average range for the comparison group (p ≤ 0.05). Attention and adaptive function scores did not change significantly over time but the range was below average in the L238Q group and average in the comparison group. PSS worsened with age in both groups. For psychological/ behavioral measures, parent report of anxiety worsened but scores on depression, withdrawal, and atypicality improved, whereas selfreport of anxiety, depression and atypicality improved over time. The L238Q mutation has a significant impact on FSIQ, memory, attention, adaptive functions and psychological/behavioral status. Although their deficits appear stable in their current age range, they continue to need yearly follow up. (Support by NIH U54-NS065768, UL1TR000114, CTSI and CNBD.)

doi:10.1016/j.ymgme.2016.11.014

6 Small molecule therapies for juvenile neuronal ceroid lipofuscinosis Rebecca C Ahrens-Nicklasa, Luis Tecedora, Mark L Schultzb, Elena Lysenkoa, Shyam Ramachandrana, Eric D Marsha, Beverly L Davidsona,

Abstracts / Molecular Genetics and Metabolism 120 (2016) S17–S145 a

The Children's Hospital of Philadelphia, Philadelphia, PA, United States, University of Michigan, Ann Arbor, MI, United States

b

Juvenile neuronal ceroid lipofuscinosis (JNCL) is a childhoodonset neuro-degenerative disorder caused by mutations in CLN3. Using CLN3 knock-out mice brain endothelial cells (MBEC) as an in vitro model, we previously documented membrane instability and brain endothelial cell defects. In current investigations we found that carbenoxolone (CBX) treatment modifies plasma membrane fluidity and rescues caveolin-1 trafficking, fluid-phase endocytosis, and Cdc42 defects in CLN3-null MBEC. Moreover, mice treated orally with CBX exhibited recovery of blood-brain barrier hypotonic stress, improved astrocytic end-feet morphology and reduced autofluorescence in the brain, suggesting that carbenoxolone, or its metabolites may improve JNCL phenotypes. Additional work in our cell-based screen has revealed several other promising small molecules for JNCL therapy. Further studies will examine the effects of these treatments on our recently identified quantifiable seizure phenotypes, an important step to clinical translation.

doi:10.1016/j.ymgme.2016.11.015

7 Niemann-Pick disease type C: a diagnostic challenge Nihal M Al Menabawya, Iman G Mahmouda, Nour M EL Khateeba, Ahmed M Sobhya, Maha S Zakib, Joseph Gleesonc, Arndt Rolfsd, Laila A Selima, aCairo University Children Hospital, Cairo, Egypt, bNational Research Centre, Cairo, Egypt, cSan Diego University, California, CA, United States, dRostock University, Rostock, Germany Niemann-Pick disease type C (NP-C) is a neurovisceral lysosomal cholesterol trafficking and lipid storage disorder caused by mutations in one of the two genes, NPC1 or NPC2. Diagnosis has often been a difficult task, due to the wide range in age of onset of NP-C and clinical presentation of the disease. The main objective is to highlight the different clinical presentation of NP-C and to point the challenges in diagnosis of such heterogeneous disorder. The clinical, biochemical, radiological and neurophysiologic data as well as genetic testing of thirteen paediatric patients with a genetically confirmed diagnosis of Niemann-pick disease type C were analysed. Patients were gathered from the neurometabolic clinic at Cairo University Children Hospital. The patients were grouped into neonatal, infantile and juvenile forms according to the age of presentation. Two patients were diagnosed b3 months, with the main clinical presentation of prolonged neonatal icterus and visceral enlargement. While 7 /13 cases were diagnosed as infantile NP-C, their age ranged from 9 months to 3 years and they presented with reduction of intellectual development, psychomotor regression and organomegaly. The juvenile group included 4 cases who were all above 10 years old. They presented mainly with progressive ataxia, organic psychosis and loss of previously acquired milestones without organomegaly and this has accounted for the delay in their diagnosis. Genetic testing for NPCI, NPCII were performed for the neonatal and infantile form, while the advanced technique of whole exome sequencing was the only diagnostic clue for the juvenile group. NP-C is commonly misdiagnosed due to its highly variable clinical presentation that are not specific and wide range in age of onset. This makes NP-C diagnosis very challenging. Whole exome sequencing aids in early diagnosis of such cases which is mandatory to prevent the devastating outcome of this potentially treatable disorder.

doi:10.1016/j.ymgme.2016.11.016

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8 Urinary glycosaminoglycan levels in a mucopolysaccharidosis type II pediatric population receiving idursulfase therapy: data from the Hunter Outcome Survey (HOS) for patients aged b18 months Kyrieckos A Alecka, Anna Tylki-Szymańskab, Can Ficiciogluc, Barbara K Burtond, Virginie Jegoe, Nathalie Guffonf, aPhoenix Children's Medical Group, Phoenix, AZ, United States, bChildren’s Memorial Health Institute, Warsaw, Poland, cThe Children’s Hospital of Philadelphia, Perelman School of Medicine, University of Pennsylvania, PhoenixPhiladelphia, PA, United States, dAnn & Robert H Lurie Children’s Hospital of Chicago, Northwestern University, Chicago, IL, United States, eCytel, Inc., Geneva, Switzerland, f Hôpital Femme Mère Enfants, Bron, France Specific treatment for mucopolysaccharidosis type II (MPS II; Hunter syndrome) is available in the form of intravenous enzyme replacement therapy (ERT). Idursulfase (Shire, Lexington, MA, USA) stabilizes or improves many of the somatic features of MPS II in patients aged ≥ 5years; tolerability and initial efficacy outcomes in patients starting idursulfase therapy aged 1.4-7.5 years have been shown to be similar to those of the first clinical studies but experience in patients aged ≤ 18 months remains limited. Our ability to diagnose MPS II early in life is improving, creating a need for increased understanding of the impact of early initiation of ERT. We analyzed data from patients enrolled in the HOS global, observational registry who started idursulfase aged ≤ 18 months (n = 45 prospective patients; all male; June 2016). Patients were diagnosed at a median (P10, P90) age of 0.4 (0.0, 1.3) years; symptom onset was at age 0.4 (0.0, 1.1) years. Almost half had cognitive impairment (48.8%; 20/41). Patients had received idursulfase for 53.3 (2.1, 91.9) months, starting at age 0.8 (0.2, 1.4) years; three individuals stopped ERT and never re-started. Sixteen patients started idursulfase at b6 months (median 0.3 [0.0, 0.4] years). Urinary glycosaminoglycan (uGAG) measurements (dimethylmethylene blue spectrophotometric assay, adjusted for urine creatinine; n = 33 patients) showed a trend towards a decrease from elevated baseline levels with idursulfase therapy. Among those with data at both baseline and 1 year of idursulfase (n = 11), uGAG levels changed by − 61.6% (− 81.8, 44.1%). Between baseline and year 2 (n = 9), the change was − 73.5% (− 87.7, 115.7%). As yet, patient numbers are low, but current findings indicate decreased uGAG levels following idursulfase therapy in this group. Additional follow-up and analysis of a range of clinical outcomes will further increase our understanding of early treatment with idursulfase. (Funding information: Shire sponsors HOS and funds medical writing support.)

doi:10.1016/j.ymgme.2016.11.017

9 Preliminary validation of telecounseling for depression in patients with Fabry disease Nadia Ali, Scott Gillespie, Dawn Laney, Emory University, Decatur, GA, United States Fabry disease (FD) is a genetic X-linked, multi-systemic, progressive lysosomal storage disorder. While the majority of FD research focuses on physical aspects, psychological symptoms such as depression have emerged as a disease complication as well, with prevalence estimates ranging from 15-62%. The present study examines the effects of psychological counseling for depression