Identification of Orthosteric and Allosteric Pharmacological Chaperones for Mucopolysaccharidosis Type IIIB.

Mucopolysaccharidosis type IIIB (MPS IIIB) is an autosomal inherited disease caused by mutations in gene encoding the lysosomal enzyme N-acetyl-alpha-glucosaminidase (NAGLU). These mutations result in reduced NAGLU activity, preventing it from catalyzing the hydrolysis of the glycosaminoglycan heparan sulfate (HS). There are currently no approved treatments for MPS IIIB. A novel approach in the treatment of lysosomal storage diseases is the use of pharmacological chaperones (PC). In this study, we used a drug repurposing approach to identify and characterize novel potential PCs for NAGLU enzyme. We modeled the interaction of natural and artificial substrates within the active cavity of NAGLU (orthosteric site) and predicted potential allosteric sites. We performed a virtual screening for both the orthosteric and the predicted allosteric site against a curated database of human tested molecules. Considering the binding affinity and predicted blood-brain barrier permeability and gastrointestinal absorption, we selected atovaquone and piperaquine as orthosteric and allosteric PCs. The PCs were evaluated by their capacity to bind NAGLU and the ability to restore the enzymatic activity in human MPS IIIB fibroblasts These results represent novel PCs described for MPS IIIB and demonstrate the potential to develop novel therapeutic alternatives for this and other protein deficiency diseases.

Genetic features of patients with MPS type IIIB: Description of five pathogenic gene variations.

BACKGROUND: There are four distinct forms of Sanfilippo syndrome (MPS type III), each of which is an autosomal lysosomal storage disorder. These forms are caused by abnormalities in one of four lysosomal enzymes. This study aimed to identify possible genetic variants that contribute to Sanfilippo IIIB in 14 independent families in Southwest Iran. METHODS: Patients were included if their clinical features and enzyme assay results were suggestive. The patients were subsequently subjected to Sanger Sequencing to screen for Sanfilippo-related genes. Additional investigations have been conducted using various computational analyses to determine the probable functional effects of diagnosed variants. RESULTS: Five distinct variations were identified in the NAGLU gene. This included two novel variants in two distinct families and three previously reported variants in 12 distinct families. All of these variations were recognized as pathogenic using the MutationTaster web server. In silico analysis showed that all detected variants affected protein structural stability; four destabilized protein structures, and the fifth variation had the opposite effect. CONCLUSION: In this study, two novel variations in the NAGLU gene were identified. The results of this study positively contribute to the mutation diversity of the NAGLU gene. To identify new disease biomarkers and therapeutic targets, precision medicine must precisely characterize and account for genetic variations. New harmful gene variants are valuable for updating gene databases concerning Sanfilippo disease variations and NGS gene panels. This may also improve genetic counselling for rapid risk examinations and disease surveillance.

Clinical, biochemical, and molecular characterization of mucopolysaccharidosis type III in 34 Egyptian patients.

Mucopolysaccharidosis type III (MPS III) is a rare autosomal recessive lysosomal storage disorder characterized by progressive neurocognitive deterioration. There are four MPS III subtypes (A, B, C, and D) that are clinically indistinguishable with variable rates of progression. A retrospective analysis was carried out on 34 patients with MPS III types at Cairo University Children's Hospital. We described the clinical, biochemical, and molecular spectrum of MPS III patients. Of 34 patients, 22 patients had MPS IIIB, 7/34 had MPS IIIC, 4/34 had MPS IIIA, and only 1 had MPS IIID. All patients presented with developmental delay/intellectual disability, and speech delay. Ataxia was reported in a patient with MPS IIIC, and cerebellar atrophy in a patient with MPS IIIA. We reported 25 variants in the 4 MPS III genes, 11 of which were not previously reported. This is the first study to analyze the clinical and genetic spectrum of MPS III patients in Egypt. This study explores the genetic map of MPS III in the Egyptian population. It will pave the way for a national registry for rare diseases in Egypt, a country with a high rate of consanguineous marriage and consequently a high rate of autosomal recessive disorders.

Safety, pharmacokinetics and CNS distribution of tralesinidase alfa administered via intracerebroventricular infusion to juvenile cynomolgus monkeys.

Mucopolysaccharidosis Type IIIB (MPS IIIB) is an ultrarare, fatal pediatric disease with no approved therapy. It is caused by mutations in the gene encoding for lysosomal enzyme alpha-N-acetylglucosaminidase (NAGLU). Tralesinidase alfa (TA) is a fusion protein comprised of recombinant NAGLU and a modified human insulin-like growth factor 2 that is being developed as an enzyme replacement therapy for MPS IIIB. Since MPS IIIB is a pediatric disease the safety/toxicity, pharmacokinetics and biodistribution of TA were evaluated in juvenile non-human primates that were administered up to 5 weekly intracerebroventricular (ICV) or single intravenous (IV) infusions of TA. TA administered by ICV slow-, ICV isovolumetric bolus- or IV-infusion was well-tolerated, and no effects were observed on clinical observations, electrocardiographic or ophthalmologic parameters, or respiratory rates. The drug-related changes observed were limited to increased cell infiltrates in the CSF and along the ICV catheter track after ICV administration. These findings were not associated with functional changes and are associated with the use of ICV catheters. The CSF PK profiles were consistent across all conditions tested and TA distributed widely in the CNS after ICV administration. Anti-drug antibodies were observed but did not appear to significantly affect the exposure to TA. Correlations between TA concentrations in plasma and brain regions in direct contact with the cisterna magna suggest glymphatic drainage may be responsible for clearance of TA from the CNS. The data support the administration of TA by isovolumetric bolus ICV infusion to pediatric patients with MPS IIIB.

Mucopolysaccharidosis type III (subtype IIIB) diagnosis as a spectrum disorder: A case report from Kosovo.

Mucopolysaccharidosis type IIIB (MPS IIIB), also known as Sanfilippo syndrome type B, is a metabolic disease caused by mutations in both alleles of the NAGLU gene encoding for the enzyme α-N-acetylglucosaminidase. A malfunction of this enzyme causes  inability to degrade heparan sulfate, which leads to accumulation of glycosaminoglycans in the cells. MPS IIIB is associated with different symptoms such as neurodegeneration, extreme hyperactivity, sleeping problems, aggressive behavior, reduced fear, and cognitive  deterioration. The condition is by now not curable. Here we describe a patient with MPS IIIB diagnosed at the age of 5 presenting with communication problems, motor dysfunctions, and speech and sleeping problems.Standard biochemical tests for neurodegenerative disorders and DNA analyses including NAGLU mutation screening were performed. We also did some psychological tests assessing the patient's communication skills and behavior. The patient was heterozygote for two mutations in the gene NAGLU (Y140C and Ser169fs). Thus, he suffered from MPS IIIB due to two mutations in the disease-causing gene.The patient presented with clear signs and symptoms of MPS IIIB with at least one of the two mutations affecting the α-N-acetylglucosaminidase protein function severely. Here we report the combination of a well-known and previously unreported mutation in the NAGLU gene; this could be dependent on geographical origin of the patient, which needs to be clarified by molecular studies of more MPS IIIB patients from Southeast Europe.

Early-onset motor polyneuropathy associated with a novel dominant NAGLU mutation.

INTRODUCTION: NAGLU encodes N-acetyl-alpha-glucosaminidase, an enzyme that degrades heparan sulfate. Biallelic NAGLU mutations cause mucopolysaccharidosis IIIB, a severe childhood-onset neurodegenerative disease, while monoallelic mutations are associated to late-onset, dominantly inherited painful sensory neuropathy. However, to date, only one family with a dominant NAGLU-related neuropathy has been described. CASE REPORT: Here we describe a patient with early-onset motor polyneuropathy harboring a novel monoallelic NAGLU mutation. We found reduced NAGLU enzymatic activity thus corroborating the pathogenic role of the new variant. DISCUSSION: Our report represents the second ever described case with dominant NAGLU-related neuropathy and the first case with early-onset motor symptoms. We underlie the importance of a thorough clinical description of this probably underestimated new clinical entity.

dNAGLU Extends Life Span and Promotes Fitness and Stress Resistance in Drosophila.

To identify new factors that promote longevity and healthy aging, we studied Drosophila CG13397, an ortholog of the human NAGLU gene, a lysosomal enzyme overexpressed in centenarians. We found that the overexpression of CG13397 (dNAGLU) ubiquitously, or tissue specifically, in the nervous system or fat body could extend fly life span. It also extended the life span of flies overexpressing human Aß42, in a Drosophila Alzheimer's disease (AD) model. To investigate whether dNAGLU could influence health span, we analyzed the effect of its overexpression on AD flies and found that it improved the climbing ability and stress resistance, including desiccation and hunger, suggesting that dNAGLU improved fly health span. We found that the deposition of Aß42 in the mushroom body, which is the fly central nervous system, was reduced, and the lysosomal activity in the intestine was increased in dNAGLU over-expressing flies. When NAGLU was overexpressed in human U251-APP cells, which expresses a mutant form of the Aß-precursor protein (APP), APP-p.M671L, these cells exhibited stronger lysosomal activity and and enhanced expression of lysosomal pathway genes. The concentration of Aß42 in the cell supernatant was reduced, and the growth arrest caused by APP expression was reversed, suggesting that NAGLU could play a wider role beyond its catalytic activity to enhance lysosomal activity. These results also suggest that NAGLU overexpression could be explored to promote healthy aging and to prevent the onset of neurodegenerative diseases, including AD.

Sanfilippo syndrome type B: Analysis of patients diagnosed by the MPS Brazil Network.

Mucopolysaccharidosis type IIIB is a rare autosomal recessive disorder characterized by deficiency of the enzyme N-acetyl-alpha-d-glucosaminidase (NAGLU), caused by biallelic pathogenic variants in the NAGLU gene, which leads to storage of heparan sulfate and a series of clinical consequences which hallmark is neurodegeneration. In this study clinical, epidemiological, and biochemical data were obtained from MPS IIIB patients diagnosed from 2004-2019 by the MPS Brazil Network ("Rede MPS Brasil"), which was created with the goal to provide an easily accessible and comprehensive investigation of all MPS types. One hundred and ten MPS IIIB patients were diagnosed during this period. Mean age at diagnosis was 10.9 years. Patients were from all over Brazil, with a few from abroad, with a possible cluster of MPS IIIB identified in Ecuador. All patients had increased urinary levels of glycosaminoglycans and low NAGLU activity in blood. Main clinical symptoms reported at diagnosis were coarse facies and neurocognitive regression. The most common variant was p.Leu496Pro (30% of alleles). MPS IIIB seems to be relatively frequent in Brazil, but patients are diagnosed later than in other countries, and reasons for that probably include the limited awareness about the disease by health professionals and the difficulties to access diagnostic tests, factors that the MPS Brazil Network is trying to mitigate.

Competitive binding of extracellular accumulated heparan sulfate reduces lysosomal storage defects and triggers neuronal differentiation in a model of Mucopolysaccharidosis IIIB.

Mucopolysaccharidoses (MPSs) are a group of inherited lysosomal storage disorders associated with the deficiency of lysosomal enzymes involved in glycosaminoglycan (GAG) degradation. The resulting cellular accumulation of GAGs is responsible for widespread tissue and organ dysfunctions. The MPS III, caused by mutations in the genes responsible for the degradation of heparan sulfate (HS), includes four subtypes (A, B, C, and D) that present significant neurological manifestations such as progressive cognitive decline and behavioral disorders. The established treatments for the MPS III do not cure the disease but only ameliorate non-neurological clinical symptoms. We previously demonstrated that the natural variant of the hepatocyte growth factor NK1 reduces the lysosomal pathology and reactivates impaired growth factor signaling in fibroblasts from MPS IIIB patients. Here, we show that the recombinant NK1 is effective in rescuing the morphological and functional dysfunctions of lysosomes in a neuronal cellular model of the MPS IIIB. More importantly, NK1 treatment is able to stimulate neuronal differentiation of neuroblastoma SK-NBE cells stable silenced for the NAGLU gene causative of the MPS IIIB. These results provide the basis for the development of a novel approach to possibly correct the neurological phenotypes of the MPS IIIB as well as of other MPSs characterized by the accumulation of HS and progressive neurodegeneration.

A GH89 human α-N-acetylglucosaminidase (hNAGLU) homologue from gut microbe Bacteroides thetaiotaomicron capable of hydrolyzing heparosan oligosaccharides.

Carbohydrate-Active enZYme (CAZY) GH89 family enzymes catalyze the cleavage of terminal α-N-acetylglucosamine from glycans and glycoconjugates. Although structurally and mechanistically similar to the human lysosomal α-N-acetylglucosaminidase (hNAGLU) in GH89 which is involved in the degradation of heparan sulfate in the lysosome, the reported bacterial GH89 enzymes characterized so far have no or low activity toward α-N-acetylglucosamine-terminated heparosan oligosaccharides, the preferred substrates of hNAGLU. We cloned and expressed several soluble and active recombinant bacterial GH89 enzymes in Escherichia coli. Among these enzymes, a truncated recombinant α-N-acetylglucosaminidase from gut symbiotic bacterium Bacteroides thetaiotaomicron ∆22Bt3590 was found to catalyze the cleavage of the terminal α1-4-linked N-acetylglucosamine (GlcNAc) from a heparosan disaccharide with high efficiency. Heparosan oligosaccharides with lengths up to decasaccharide were also suitable substrates. This bacterial α-N-acetylglucosaminidase could be a useful catalyst for heparan sulfate analysis.

Cell-Mediated Immunity to NAGLU Transgene Following Intracerebral Gene Therapy in Children With Mucopolysaccharidosis Type IIIB Syndrome.

Mucopolysaccharidosis type IIIB syndrome (Sanfilippo disease) is a rare autosomic recessif disorder caused by mutations in the α-N-acetylglucosaminidase (NAGLU) gene coding for a lysosomal enzyme, leading to neurodegeneration and progressive deterioration of cognitive abilities in affected children. To supply the missing enzyme, several recent human gene therapy trials relied on the deposit of adeno-associated virus (AAV) vectors directly into the brain. We reported safety and efficacy of an intracerebral therapy in a phase 1/2 clinical trial (https://clinicaltrials.gov/ct2/show/NCT03300453), with a recombinant AAV serotype 2/5 (rAAV2/5) coding human NAGLU in four children with MPS IIIB syndrome receiving immunosuppression. It was reported that AAV-mediated gene therapies might elicit a strong host immune response resulting in decreased transgene expression. To address this issue, we performed a comprehensive analysis of cellular immunity and cytokine patterns generated against the therapeutic enzyme in the four treated children over 5.5 years of follow-up. We report the emergence of memory and polyfunctional CD4+ and CD8+ T lymphocytes sensitized to the transgene soon after the start of therapy, and appearing in peripheral blood in waves throughout the follow-up. However, this response had no apparent impact on CNS transgene expression, which remained stable 66 months after surgery, possibly a consequence of the long-term immunosuppressive treatment. We also report that gene therapy did not trigger neuroinflammation, evaluated through the expression of cytokines and chemokines in patients' CSF. Milder disease progression in the youngest patient was found associated with low level and less differentiated circulating NAGLU-specific T cells, together with the lack of proinflammatory cytokines in the CSF. Findings in this study support a systematic and comprehensive immunomonitoring approach for understanding the impact immune reactions might have on treatment safety and efficacy of gene therapies.

Evaluation of artificial signal peptides for secretion of two lysosomal enzymes in CHO cells.

Enzyme replacement therapy (ERT) is a scientifically rational and clinically proven treatment for lysosomal storage diseases. Most enzymes used for ERT are purified from the culture supernatant of mammalian cells. However, it is challenging to purify lysosomal enzymes with sufficient quality and quantity for clinical use due to their low secretion levels in mammalian cell systems. To improve the secretion efficiency of recombinant lysosomal enzymes, we evaluated the impact of artificial signal peptides on the production of recombinant lysosomal enzymes in Chinese hamster ovary (CHO) cell lines. We engineered two recombinant human lysosomal enzymes, N-acetyl-α-glucosaminidase (rhNAGLU) and glucosamine (N-acetyl)-6-sulfatase (rhGNS), by replacing their native signal peptides with nine different signal peptides derived from highly secretory proteins and expressed them in CHO K1 cells. When comparing the native signal peptides, we found that rhGNS was secreted into media at higher levels than rhNAGLU. The secretion of rhNAGLU and rhGNS can, however, be carefully controlled by altering signal peptides. The secretion of rhNAGLU was relatively higher with murine Igκ light chain and human chymotrypsinogen B1 signal peptides, whereas Igκ light chain signal peptide 1 and human chymotrypsinogen B1 signal peptides were more effective for rhGNS secretion, suggesting that human chymotrypsinogen B1 signal peptide is the most appropriate for increasing lysosomal enzyme secretion. Collectively, our results indicate that altering signal peptide can modulate the secretion of recombinant lysosome enzymes and will enable lysosomal enzyme production for clinical use.

Mutación novel en el Síndrome Sanfilippo B. Presentación de un caso / A novel mutation in Sanfilippo Syndrome type B. Case report

Introducción: El Síndrome Sanfilippo B es un error innato en el metabolismo lisosomal, con herencia autosómica recesiva. Se caracteriza por facie ligeramente tosca, deterioro neurológico progresivo y poca repercusión somática, provocado por mutaciones en el gen NAGLU, cuyo locus es 17q21.2. La incidencia internacionalmente es muy baja y en Cuba solo se han diagnosticado siete pacientes desde 1985. Objetivo: Describir las manifestaciones clínicas, bioquímicas y moleculares de un paciente cubano diagnosticado con Síndrome Sanfilippo B. Presentación de Caso: Se describió un paciente de 13 años, cuyas principales manifestaciones clínicas fueron: facie ligeramente tosca, sinofris, alteraciones de conducta y deterioro neurológico progresivo. El trastorno del sueño fue ocasional y frecuente las infecciones respiratorias. Se demostró la presencia de colitis ulcerativa y pólipo intestinal. Se confirmó excreción aumentada de heparán sulfato y disminución de la actividad enzimática N-acetil αD-glucosaminidasa. Se identificó la mutación c.640dupC en el gen NAGLU en homocigosis en el paciente y ambos padres resultaron ser portadores. Conclusiones: Predominaron las alteraciones de conducta, deterioro neurológico progresivo e infecciones respiratorias en el caso reportado; siendo la colitis ulcerativa y el pólipo intestinal un hallazgo no descrito anteriormente para esta enfermedad. Los estudios cromatográficos y enzimáticos resultaron positivos para Sanfilippo B. El genotipo de este paciente resultó ser homocigótico para una nueva variante alélica patogénica en el gen NAGLU. Se demostró la segregación mendeliana de la mutación en la familia(AU)

Introduction: Sanfilippo syndrome type B is an autosomal recessive lysosomal storage disease. The frequent clinical manifestations include slightly coarse facial features, progressive neurodegeneration and mild somatic repercussion caused by mutations in the NAGLU gene, whose locus is 17q21.2. The worldwide incidence is very low and only seven patients have been diagnosed in Cuba since 1985. Objective: To describe clinical, biochemical and molecular characteristics of a Cuban patient with the diagnosis of Sanfilippo Syndrome type B. Case presentation: A 13 years old patient was described. The main clinical manifestations included mild coarse facie, synophrys, behavior disturbances, and progressive neurologic deterioration. Intermittent sleep disturbance and frequent upper respiratory infections were identified. Ulcerative colitis and intestinal polyp were demonstrated. Increased excretion of heparan sulfate and very low N-acetyl α-Dglucosaminidase activity were confirmed. In addition, the presence of mutation c.640dupC in NAGLU gene was identified. The patient had homozygous genotype and both parents were heterozygous. Conclusions: Behavioral alterations, progressive neurological deterioration and respiratory infections predominated in the reported case. Other findings such as ulcerative colitis and intestinal polyps were not previously described in this disease. The chromatographic and enzymatic studies were positive for Sanfilippo type B. This patient's genotype was found to be homozygous for a novel pathogenic allelic variant in the NAGLU gene. Mendelian segregation of the mutation in the family was demonstrated(AU)

Clinical and genetic features of 13 patients with mucopolysaccarhidosis type IIIB: Description of two novel NAGLU gene mutations.

AIM: Mucopolysaccharidosis type III B (MPS IIIB) is an autosomal recessive lysosomal storage disease caused by mutations in the NAGLU gene which codes the lysosomal enzyme alpha-N-acetylglucosaminidase. The major symptoms of the disease are cognitive and neurological defects. In this study, the molecular spectrums of 13 MPS IIIB patients were evaluated. MATERIAL AND METHODS: Thirteen MPS IIIB patients from 11 families were included in this study. All patients were both clinically and molecularly diagnosed. NAGLU gene sequencing was performed using a next generation sequencing platform (Illumina MiSeq). Demographic, clinical and laboratory findings of the patients were obtained via the hospital records. RESULTS: Ten different mutations from the 13 MPS IIIB patients were identified. Eight of the 10 mutations were missense, one was splice site, and one large deletion was also observed. Two mutations c.509G>T (p.Gly170Val) and c.700C>G (p.Arg234Gly) have been defined for the first time in this study. CONCLUSION: Our study expanded the mutation spectrum of the NAGLU gene thereby contributing to the improved genetic counselling of MPS IIIB patients. Confirming the literature, missense mutations were also found to be the most common NAGLU mutations in our study.

Downregulation of NAGLU in VEC Increases Abnormal Accumulation of Lysosomes and Represents a Predictive Biomarker in Early Atherosclerosis.

Cardiovascular diseases (CVDs), predominantly caused by atherosclerosis (AS), are the leading cause of mortality worldwide. Although a great number of previous studies have attempted to reveal the molecular mechanism of AS, the underlying mechanism has not been fully elucidated. The aberrant expression profiling of vascular endothelial cells (VECs) gene in early atherosclerosis (EAS) was analyzed according to the dataset (GSE132651) downloaded from the Gene Expression Omnibus (GEO) database. We primarily performed functional annotation analysis on the downregulated genes (DRGs). We further identified that α-N-acetylglucosaminidase (NAGLU), one of the DRGs, played a critical role in the progression of EAS. NAGLU is a key enzyme for the degradation of heparan sulfate (HS), and its deficiency could cause lysosomal accumulation and lead to dysfunctions of VECs. We found that siRNA knockdown of NAGLU in human umbilical vein endothelial cell (HUVEC) aggravated the abnormal accumulation of lysosomes and HS. In addition, the expression of NAGLU was reduced in the EAS model constructed by ApoE -/- mice. Furthermore, we also showed that heparin-binding EGF-like growth factor (HB-EGF) protein was upregulated while NAGLU knockdown in HUVEC could specifically bind to vascular endothelial growth factor receptor 2 (VEGFR2) and promote its phosphorylation, ultimately activating the phosphorylation levels of extracellular signal-regulated kinases (ERKs). However, the application of selective VEGFR2 and ERKs inhibitors, SU5614 and PD98059, respectively, could reverse the abnormal lysosomal storage caused by NAGLU knockdown. These results indicated that downregulation of NAGLU in HUVEC increases the abnormal accumulation of lysosomes and may be a potential biomarker for the diagnosis of EAS.

Determination of Reference Values for Alpha-N-Acetylglucosaminidase Activities in Patients with Sanfilippo Type B Disease and Control Population in Colombia

Abstract Sanfilippo B is a lysosomal disorder characterized by the pathological accumulation of heparan sulfate. It is caused by mutations in the NAGLU gene that codes for the alpha-N-acetylglucosaminidase enzyme. The objective of this study was to determine the reference values and frequency of Sanfilippo B in Colombia through an enzyme analysis of leukocytes extracts. We aim to inform the community and the health system so that they can work in a preventive way, providing an early diagnosis of patients and thus providing an appropriate management of the symptoms. We carried out an endpoint assay that indirectly quantifies NAGLU activity through the cleavage of 4-methylumbelliferone from the 4-methylumbelliferyl-2-acetamido-2-deoxy-α-D-glucopyranoside substrate. The activity of 463 healthy volunteers (Range: 0.6 - 4 nmol/mg/h, Median: 1.69 +/- 0.73) as well as 462 patients referred for clinical suspicion, was calculated. From the last group, 7 cases turned out to be positive (Range: 0 - 0.24 nmol/mg/h, Median: 0.13 +/- 0.09). The cut-off point according to ROC analysis between affected patients and controls was 0.42 nmol/mg/h. To our knowledge, this study is the first in Colombia where an estimated frequency of Sanfilippo type B is calculated by providing enzyme activity ranges and a cut-off point.

A Novel Mutation (p.Met1?) of a Cuban Patient in the NAGLU Gene with Mucopolysaccharidosis IIIB

Abstract Mucopolysaccharidosis III (MPSIII) or Sanfilippo syndrome is an autosomal recessive disorder of lysosomal metabolism. MPS III is caused by mutations in genes that encode for the enzymes involved in the degradation of heparan sulfate. It is classified into 4 subtypes (MPSIII A-D). MPS IIIB is induced by mutations in the gene encoding the alpha-N-acetylglucosaminidase enzyme. We report a 6-year-old boy with phenotypic findings of Sanfilippo syndrome type B, such as mild coarse facie, clear corneas, hirsutism, hepatomegaly, mild joint stiffness and mild dysostosis multiplex. He also presents frequent upper respiratory infections, bilateral hearing loss, sleep disturbances, progressive neurologic deterioration and behavioral problems. He is compound heterozygous for the NAGLU gene (c.503G˃A; p.Trp168Ter/ c.3G˃A; p.met1?). One of the mutation was described in two patients before. A novel pathogenic variant was detected.

Coinheritance of novel mutations in NAGLU causing mucopolysaccharidosis type IIIB and in DDHD2 causing spastic paraplegia54 in a Turkish family.

Mucopolysaccharidosis type IIIB (MPSIIIB) is one of the lysosomal storage diseases, clinically related to developmental delay in the early phase and loss of skills in the late phases of the disease. The disease is caused by homozygous mutations in the NAGLU gene. Spastic paraplegia54 (SPG54) is a neurodegenerative disorder caused by homozygous mutations in the DDHD2 gene. Clinical features are progressive spasticity and weakness in the lower limbs and corpus callosum agenesis. We report on two siblings in a consanguineous family, presenting both the clinical and molecular diagnoses of MPSIIIB and SPG54 with novel mutations by using whole exome sequencing (WES). This interesting finding shows that we should be aware of the importance of using WES for diagnosing rare diseases in consanguineous families.

Proteomic Analysis of Mucopolysaccharidosis IIIB Mouse Brain.

Mucopolysaccharidosis IIIB (MPS IIIB) is an inherited metabolic disease due to deficiency of α-N-Acetylglucosaminidase (NAGLU) enzyme with subsequent storage of undegraded heparan sulfate (HS). The main clinical manifestations of the disease are profound intellectual disability and neurodegeneration. A label-free quantitative proteomic approach was applied to compare the proteome profile of brains from MPS IIIB and control mice to identify altered neuropathological pathways of MPS IIIB. Proteins were identified through a bottom up analysis and 130 were significantly under-represented and 74 over-represented in MPS IIIB mouse brains compared to wild type (WT). Multiple bioinformatic analyses allowed to identify three major clusters of the differentially abundant proteins: proteins involved in cytoskeletal regulation, synaptic vesicle trafficking, and energy metabolism. The proteome profile of NAGLU-/- mouse brain could pave the way for further studies aimed at identifying novel therapeutic targets for the MPS IIIB. Data are available via ProteomeXchange with the identifier PXD017363.

A Novel Pathogenic Variant in NAGLU (N-Acetyl-Alpha-Glucosaminidase) gene Identified by Targeted Next-Generation Sequencing Followed by in Silico Analysis.

BACKGROUND: Mucopolysaccharidosis IIIB (MPS IIIB) (Sanfilippo Syndrome Type B; OMIM 252920) is an autosomal recessive metabolic disorder caused by mutations in the NAGLU gene which encode lysosomal enzyme N-acetyl-glucosaminidase, involved in degradation of complex polysaccharide, heparan sulfate. The disease is characterized by progressive cognitive decline and behavioral difficulties and motor function retardation. MATERIALS & METHODS: In this study, targeted exome sequencing was used in consanguineous parent (mother) of a deceased child with clinical diagnosis of mucopolysaccharidosis. Sanger sequencing was performed to confirm the candidate pathogenic variants in extended family members and segregation analysis. In silico pathogenicity assessment of detected variant using multiple computational predictive tools were performed. Computational docking using the Molegro Virtual Docker (MVD) 6.0.1 software applied to evaluate affinity binding of altered protein for its ligand, N-Acetyl-D-Glucosamine. Moreover, with I-TASSER software functional alterations between wild and mutant proteins evaluated. RESULTS: We identified a novel heterozygote deletion variant (c.1294-1304 del CTCTTCCCCAA, p.432LeufsX25) in the NAGLU gene. The variant was classified as pathogenic based on the American College of Medical Genetics and Genomics guideline. Computational docking with the Molegro Virtual Docker (MVD) 6.0.1 software confirmed different affinity binding of truncated protein for its ligand. Moreover, I-TASSER software revealed structural and functional alterations of mutant proteins. CONCLUSION: This study expands the spectrum of NAGLU pathogenic variants and confirms the utility of targeted NGS sequencing in genetic diagnosis and also the utility and power of additional family information.