Neuropathology of murine Sanfilippo D syndrome.

Sanfilippo D syndrome (mucopolysaccharidosis type IIID) is a lysosomal storage disorder caused by the deficiency of N-acetylglucosamine-6-sulfatase (GNS). A mouse model was generated by constitutive knockout of the Gns gene. We studied affected mice and controls at 12, 24, 36, and 48 weeks of age for neuropathological markers of disease in the somatosensory cortex, primary motor cortex, ventral posterior nuclei of the thalamus, striatum, hippocampus, and lateral and medial entorhinal cortex. We found significantly increased immunostaining for glial fibrillary associated protein (GFAP), CD68 (a marker of activated microglia), and lysosomal-associated membrane protein-1 (LAMP-1) in Sanfilippo D mice compared to controls at 12 weeks of age in all brain regions. Intergroup differences were marked for GFAP and CD68 staining, with levels in Sanfilippo D mice consistently above controls at all age groups. Intergroup differences in LAMP-1 staining were more pronounced in 12- and 24-week age groups compared to 36- and 48-week groups, as control animals showed some LAMP-1 staining at later timepoints in some brain regions. We also evaluated the somatosensory cortex, medial entorhinal cortex, reticular nucleus of the thalamus, medial amygdala, and hippocampal hilus for subunit c of mitochondrial ATP synthase (SCMAS). We found a progressive accumulation of SCMAS in most brain regions of Sanfilippo D mice compared to controls by 24 weeks of age. Cataloging the regional neuropathology of Sanfilippo D mice may aid in understanding the disease pathogenesis and designing preclinical studies to test brain-directed treatments.

Sanfilippo Syndrome: Molecular Basis, Disease Models and Therapeutic Approaches.

Sanfilippo syndrome or mucopolysaccharidosis III is a lysosomal storage disorder caused by mutations in genes responsible for the degradation of heparan sulfate, a glycosaminoglycan located in the extracellular membrane. Undegraded heparan sulfate molecules accumulate within lysosomes leading to cellular dysfunction and pathology in several organs, with severe central nervous system degeneration as the main phenotypical feature. The exact molecular and cellular mechanisms by which impaired degradation and storage lead to cellular dysfunction and neuronal degeneration are still not fully understood. Here, we compile the knowledge on this issue and review all available animal and cellular models that can be used to contribute to increase our understanding of Sanfilippo syndrome disease mechanisms. Moreover, we provide an update in advances regarding the different and most successful therapeutic approaches that are currently under study to treat Sanfilippo syndrome patients and discuss the potential of new tools such as induced pluripotent stem cells to be used for disease modeling and therapy development.

Mucopolysaccharidosis III in Taiwan: Natural history, clinical and molecular characteristics of 28 patients diagnosed during a 21-year period.

Mucopolysaccharidosis type III (MPS III, Sanfilippo syndrome) has a variable age of onset and variable rate of progression. However, information regarding the natural history of this disorder in Asian populations is limited. A retrospective analysis was carried out for 28 patients with MPS III (types IIIA [n = 3], IIIB [n = 23], and IIIC [n = 2]; 15 males and 13 females; median age, 8.2 years; age range, 2.7-26.5 years) seen in six medical centers in Taiwan from January 1996 through October 2017. The median age at confirmed diagnosis was 4.6 years. The most common initial symptom was speech delay (75%), followed by hirsutism (64%) and hyperactivity (54%). Both z scores for height and weight were negatively correlated with age (r = -.693 and -0.718, respectively; p < .01). The most prevalent clinical manifestations were speech delay (100%) and intellectual disability (100%), followed by hirsutism (93%), hyperactivity (79%), coarse facial features (68%), sleep disorders (61%), and hepatosplenomegaly (61%). Ten patients (36%) had epilepsy, and the median age at the first seizure was 11 years. Thirteen patients (46%) experienced at least one surgical procedure. At the time of the present study, 7 of the 28 patients had passed away at the median age of 13.0 years. Molecular studies showed an allelic heterogeneity without clear genotype and phenotype correlations. MPS IIIB is the most frequent subtype among MPS III in the Taiwanese population. An understanding of the natural history of MPS III may allow early diagnosis and timely management of the disease facilitating better treatment outcomes.

Genomic basis of mucopolysaccharidosis type IIID (MIM 252940) revealed by sequencing of GNS encoding N-acetylglucosamine-6-sulfatase.

Mucopolysaccharidosis type IIID (MPS IIID; Sanfilippo syndrome type D; MIM 252940) is caused by deficiency of the activity of N-acetylglucosamine-6-sulfatase (GNS), which is normally required for degradation of heparan sulfate. The clinical features of MPS IIID include progressive neurodegeneration, with relatively mild somatic symptoms. Biochemical features include accumulation of heparan sulfate and N-acetylglucosamine-6-sulfate in the brain and viscera. To date, diagnosis required a specific lysosomal enzyme assay for GNS activity. From genomic DNA of a subject with MPS IIID, we amplified and sequenced the promoter and 14 exons of GNS. We found a homozygous nonsense mutation in exon 9 (1063C --> T), which predicted premature termination of translation (R355X). We also identified two common synonymous coding single-nucleotide polymorphisms and genotyped these in samples from four ethnic groups. This first report of a mutation in GNS resulting in MPS IIID indicates the potential utility of molecular diagnosis for this rare condition.

Mouse model of Sanfilippo syndrome type B produced by targeted disruption of the gene encoding alpha-N-acetylglucosaminidase.

The Sanfilippo syndrome type B is an autosomal recessive disorder caused by mutation in the gene (NAGLU) encoding alpha-N-acetylglucosaminidase, a lysosomal enzyme required for the stepwise degradation of heparan sulfate. The most serious manifestations are profound mental retardation, intractable behavior problems, and death in the second decade. To generate a model for studies of pathophysiology and of potential therapy, we disrupted exon 6 of Naglu, the homologous mouse gene. Naglu-/- mice were healthy and fertile while young and could survive for 8-12 mo. They were totally deficient in alpha-N-acetylglucosaminidase and had massive accumulation of heparan sulfate in liver and kidney as well as secondary changes in activity of several other lysosomal enzymes in liver and brain and elevation of gangliosides G(M2) and G(M3) in brain. Vacuolation was seen in many cells, including macrophages, epithelial cells, and neurons, and became more prominent with age. Although most vacuoles contained finely granular material characteristic of glycosaminoglycan accumulation, large pleiomorphic inclusions were seen in some neurons and pericytes in the brain. Abnormal hypoactive behavior was manifested by 4.5-mo-old Naglu-/- mice in an open field test; the hyperactivity that is characteristic of affected children was not observed even in younger mice. In a Pavlovian fear conditioning test, the 4.5-mo-old mutant mice showed normal response to context, indicating intact hippocampal-dependent learning, but reduced response to a conditioning tone, perhaps attributable to hearing impairment. The phenotype of the alpha-N-acetylglucosaminidase-deficient mice is sufficiently similar to that of patients with the Sanfilippo syndrome type B to make these mice a good model for study of pathophysiology and for development of therapy.

Expression and characterization of wild type and mutant recombinant human sulfamidase. Implications for Sanfilippo (Mucopolysaccharidosis IIIA) syndrome.

Mucopolysaccharidosis IIIA (MPS-IIIA) is an autosomal recessive lysosomal storage disorder caused by the deficiency of sulfamidase (NS; EC 3.10.1.1), resulting in defective degradation and storage of heparan sulfate. This paper reports the production and characterization of monoclonal and polyclonal antibodies against recombinant human sulfamidase (rhNS) to quantitate and characterize normal and mutant sulfamidase produced from the wild type NS expression vector. Glycosylation and phosphorylation studies of immunoprecipitated rhNS show that all five potential glycosylation sites are utilized, with three high mannose/hybrid oligosaccharides and two simpler chains, with at least one functional mannose 6-phosphate group. An NS quantification system was developed to determine the effect of the three most common and severe patient mutations: S66W (Italy), R74C (Poland), and R245H (The Netherlands). The quantity and specific activity of expressed mutant rhNS was significantly lower than expressed normal rhNS, with 0.3, 0.2, and 0.05% of normal rhNS produced and 15, 17, and 83% of normal specific activity for S66W, R74C, and R245H observed, respectively. The recent structural elucidation of N-acetylgalactosamine-4-sulfatase was utilized to postulate the effect on the structure-function relationship of NS. The characterization of normal and mutated rhNS has relevance for efficient diagnosis and therapeutic developments for MPS-IIIA patients.