Disease correction in mucopolysaccharidosis type IIIB mice by intraparenchymal or cisternal delivery of a capsid modified AAV8 codon-optimized NAGLU vector.

Mucopolysaccharidosis type IIIB (MPS IIIB) is an autosomal recessive lysosomal storage disease caused by mutations in the gene that encodes the protein N-acetyl-glucosaminidase (NAGLU). Defective NAGLU activity results in aberrant retention of heparan sulfate within lysosomes leading to progressive central nervous system (CNS) degeneration. Intravenous treatment options are limited by the need to overcome the blood-brain barrier and gain successful entry into the CNS. Additionally, we have demonstrated that AAV8 provides a broader transduction area in the MPS IIIB mouse brain compared with AAV5, 9 or rh10. A triple-capsid mutant (tcm) modification of AAV8 further enhanced GFP reporter expression and distribution. Using the MPS IIIB mouse model, we performed a study using either intracranial six site or intracisterna magna injection of AAVtcm8-codon-optimized (co)-NAGLU using untreated MPS IIIB mice as controls to assess disease correction. Disease correction was evaluated based on enzyme activity, heparan sulfate storage levels, CNS lysosomal signal intensity, coordination, activity level, hearing and survival. Both histologic and enzymatic assessments show that each injection method results in supranormal levels of NAGLU expression in the brain. In this study, we have shown correction of lifespan and auditory deficits, increased CNS NAGLU activity and reduced lysosomal storage levels of heparan sulfate following AAVtcm8-coNAGLU administration and partial correction of NAGLU activity in several peripheral organs in the murine model of MPS IIIB.

A three-step "ping-pong" mechanism of a GH20 ß-N-acetylglucosaminidase from Vibrio campbellii belonging to a major Clade A-I of the phylogenetic tree of the enzyme superfamily.

ß-N-acetylglucosaminidase (GlcNAcase) is an essential biocatalyst in chitin assimilation by marine Vibrio species, which rely on chitin as their main carbon source. Structure-based phylogenetic analysis of the GlcNAcase superfamily revealed that a GlcNAcase from Vibrio campbellii, formerly named V. harveyi, (VhGlcNAcase) belongs to a major clade, Clade A-I, of the phylogenetic tree. Pre-steady-state and steady-state kinetic analysis of the reaction catalysed by VhGlcNAcase with the fluorogenic substrate 4-methylumbelliferyl N-acetyl-ß-D-glucosaminide suggested the following mechanism: (1) the Michaelis-Menten complex is formed in a rapid enzyme-substrate equilibrium with a Kd of 99.1 ± 1 µM. (2) The glycosidic bond is cleaved by the action of the catalytic residue Glu438, followed by the rapid release of the aglycone product with a rate constant (k2) of 53.3 ± 1 s-1. (3) After the formation of an oxazolinium ion intermediate with the assistance of Asp437, the anomeric carbon of the transition state is attacked by a catalytic water, followed by release of the glycone product with a rate constant (k3) of 14.6 s-1, which is rate-limiting. The result clearly indicated a three-step "ping-pong" mechanism for VhGlcNAcase.

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.

A novel endo-type chitinase possessing chitobiase activity derived from the chitinolytic bacterium, Chitiniphilus shinanonensis SAY3T.

One of the chitinases (ChiG) derived from the chitinolytic bacterium Chitiniphilus shinanonensis SAY3T exhibited chitobiase activity cleaving dimers of N-acetyl-D-glucosamine (GlcNAc) into monomers, which is not detected in typical endo-type chitinases. Analysis of the reaction products for GlcNAc hexamers revealed that all the five internal glycosidic bonds were cleaved at the initial stage. The overall reaction catalyzed by chitobiases toward GlcNAc dimers was similar to that catalyzed by N-acetyl-D-glucosaminidases (NAGs). SAY3 possesses two NAGs (ChiI and ChiT) that are thought to be important in chitin catabolism. Unexpectedly, a triple gene-disrupted mutant (ΔchiIΔchiTΔchiG) was still able to grow on synthetic medium containing GlcNAc dimers or powdered chitin, similar to the wild-type SAY3, although it exhibited only 3% of total cellular NAG activity compared to the wild-type. This indicates the presence of unidentified enzyme(s) capable of supporting normal bacterial growth on the chitin medium by NAG activity compensation.

The beta-1, 4-N-acetylglucosaminidase 1 gene, selected by domestication and breeding, is involved in cocoon construction of Bombyx mori.

Holometabolous insects have distinct larval, pupal, and adult stages. The pupal stage is typically immobile and can be subject to predation, but cocoon offers pupal protection for many insect species. The cocoon provides a space in which the pupa to adult metamorphosis occurs. It also protects the pupa from weather, predators and parasitoids. Silk protein is a precursor of the silk used in cocoon construction. We used the silkworm as a model species to identify genes affecting silk protein synthesis and cocoon construction. We used quantitative genetic analysis to demonstrate that ß-1,4-N-acetylglucosaminidase 1 (BmGlcNase1) is associated with synthesis of sericin, the main composite of cocoon. BmGlcNase1 has an expression pattern coupled with silk gland development and cocoon shell weight (CSW) variation, and CSW is an index of the ability to synthesize silk protein. Up-regulated expression of BmGlcNase1 increased sericin content by 13.9% and 22.5% while down-regulation reduced sericin content by 41.2% and 27.3% in the cocoons of females and males, respectively. Genomic sequencing revealed that sequence variation upstream of the BmGlcNase1 transcriptional start site (TSS) is associated with the expression of BmGlcNase1 and CSW. Selective pressure analysis showed that GlcNase1 was differentially selected in insects with and without cocoons (ω1 = 0.044 vs. ω2 = 0.154). This indicates that this gene has a conserved function in the cocooning process of insects. BmGlcNase1 appears to be involved in sericin synthesis and silkworm cocooning.

Disease modeling for Mucopolysaccharidosis type IIIB using patient derived induced pluripotent stem cells.

Mucopolysaccharidosis type IIIB (MPS IIIB) is a lysosomal disease caused by mutations in the NAGLU gene encoding α-N-acetylglucosaminidase (NAGLU) which degrades heparan sulfate in lysosomes. Deficiency in NAGLU results in lysosomal accumulation of glycosaminoglycans (GAGs) and neurological symptoms. Currently, there is no effective treatment or cure for this disease. In this study, induced pluripotent stem cell lines were established from two MPS IIIB patient fibroblast lines and differentiated into neural stem cells and neurons. MPS IIIB neural stem cells exhibited NAGLU deficiency accompanied with GAG accumulation, as well as lysosomal enlargement and secondary lipid accumulation. Treatments with recombinant NAGLU, δ-tocopherol, and 2-hydroxypropyl-b-cyclodextrin significantly reduced the disease phenotypes in these cells. These results indicate the MPS IIIB neural stem cells and neurons have the disease relevant phenotype and can be used as a cell-based disease model system for evaluation of drug efficacy and compound screening for drug development.

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.

Biochemical evaluation of intracerebroventricular rhNAGLU-IGF2 enzyme replacement therapy in neonatal mice with Sanfilippo B syndrome.

Mucopolysaccharidosis IIIB (MPS IIIB, Sanfilippo syndrome type B) is caused by a deficiency in α-N-acetylglucosaminidase (NAGLU) activity, which leads to the accumulation of heparan sulfate (HS). MPS IIIB causes progressive neurological decline, with affected patients having an expected lifespan of approximately 20 years. No effective treatment is available. Recent pre-clinical studies have shown that intracerebroventricular (ICV) ERT with a fusion protein of rhNAGLU-IGF2 is a feasible treatment for MPS IIIB in both canine and mouse models. In this study, we evaluated the biochemical efficacy of a single dose of rhNAGLU-IGF2 via ICV-ERT in brain and liver tissue from Naglu-/- neonatal mice. Twelve weeks after treatment, NAGLU activity levels in brain were 0.75-fold those of controls. HS and ß-hexosaminidase activity, which are elevated in MPS IIIB, decreased to normal levels. This effect persisted for at least 4 weeks after treatment. Elevated NAGLU and reduced ß-hexosaminidase activity levels were detected in liver; these effects persisted for up to 4 weeks after treatment. The overall therapeutic effects of single dose ICV-ERT with rhNAGLU-IGF2 in Naglu-/- neonatal mice were long-lasting. These results suggest a potential benefit of early treatment, followed by less-frequent ICV-ERT dosing, in patients diagnosed with MPS IIIB.

Significant neuropsychiatric symptoms: three mucopolysaccharidosis type IIIB cases, two of whom were siblings with a novel NAGLU gene mutation.

Mucopolysaccharidosis (MPS) type IIIB patients present with marked neurodevelopmental and neuropsychiatric problems and not with typical MPS symptoms such as coarse facial features, organomegaly, or short body height, especially at the first presentation. We present three pediatric cases, two of which are sisters with novel NAGLU gene mutations, to emphasize that diagnosis of MPS type IIIB should be remembered in patients presenting with neurodevelopmental and neuropsychiatric problems such as delayed speech, autistic-like symptoms, severe behavioral and sleep problems, motor deterioration or idiopathic intellectual disability with or without refractory epilepsy, especially if there is aconsanguineous marriage.

Transglycosylation toward naringenin-7-O-glucoside using an N180H mutant of Coprinopsis cinerea endo-ß-N-acetylglucosaminidase.

Flavonoids are generally glycosylated, and the glycan moieties of flavonoid glycosides are known to greatly affect their physicochemical and biological properties. Thus, the development of a variety of tools for glycan remodeling of flavonoid glycosides is highly desired. An endo-ß-N-acetylglucosaminidase mutant Endo-CC N180H, which is developed as an excellent chemoenzymatic tool for creating sialylglycoproteins, was employed for the glycosylation of flavonoids. Endo-CC N180H transferred the sialyl biantennary glycans from the sialylglyco peptide to pNP-GlcNAc and narigenin-7-O-glucoside. The kinetic parameters of Endo-CC N180H towards SGP and pNP-GlcNAc were determined. Flavonoid glucosides harboring a 1,3-diol structure in the glucose moieties acted as substrates of Endo-CC N180H. We proposed that the sialyl biantennary glycan transfer to the flavonoid by Endo-CC N180H could pave the way for the improvement of the inherent biological functions of the flavonoids and creation of novel flavonoid glycoside derivatives for future human health benefits including foods and drugs.

The N-Acetylglucosaminidase LytB of Streptococcus pneumoniae Is Involved in the Structure and Formation of Biofilms.

The N-acetylglucosaminidase LytB of Streptococcus pneumoniae is involved in nasopharyngeal colonization and is responsible for cell separation at the end of cell division; thus, ΔlytB mutants form long chains of cells. This paper reports the construction and properties of a defective pneumococcal mutant producing an inactive LytB protein (LytBE585A). It is shown that an enzymatically active LytB is required for in vitro biofilm formation, as lytB mutants (either ΔlytB or producing the inactive LytBE585A) are incapable of forming substantial biofilms, despite that extracellular DNA is present in the biofilm matrix. Adding small amounts (0.5 to 2.0 µg/ml) of exogenous LytB or some LytB constructs restored the biofilm-forming capacity of lytB mutants to wild-type levels. The LytBE585A mutant formed biofilm more rapidly than ΔlytB mutants in the presence of LytB. This suggests that the mutant protein acted in a structural role, likely through the formation of complexes with extracellular DNA. The chain-dispersing capacity of LytB allowed the separation of daughter cells, presumably facilitating the formation of microcolonies and, finally, of biofilms. A role for the possible involvement of LytB in the synthesis of the extracellular polysaccharide component of the biofilm matrix is also discussed.IMPORTANCE It has been previously accepted that biofilm formation in S. pneumoniae must be a multigenic trait because the mutation of a single gene has led to only to partial inhibition of biofilm production. In the present study, however, evidence that the N-acetylglucosaminidase LytB is crucial in biofilm formation is provided. Despite the presence of extracellular DNA, strains either deficient in LytB or producing a defective LytB enzyme formed only shallow biofilms.

Identification of the putative N-acetylglucosaminidase CseA associated with daughter cell separation in Tetragenococcus halophilus.

The lactic acid bacterium Tetragenococcus halophilus, which is used as a starter to brew soy sauce, comprises both cluster-forming strains and dispersed strains. The cluster-forming strains are industrially useful for obtaining clear soy sauce, because the cell clusters are trapped by filter cloth when the soy sauce mash is pressed. However, the molecular mechanism underlying cell cluster formation is unknown. Whole genome sequence analysis and subsequent target sequence analysis revealed that the cluster-forming strains commonly have functional defects in N-acetylglucosaminidase CseA, a peptidoglycan hydrolase. CseA is a multimodular protein that harbors a GH73 domain and six peptidoglycan-binding LysM domains. Recombinant CseA hydrolyzed peptidoglycan and promoted cell separation. Functional analysis of truncated CseA derivatives revealed that the LysM domains play an important role in efficient peptidoglycan degradation and cell separation. Taken together, the results of this study identify CseA as a factor that greatly affects the cluster formation in T. halophilus.

Assessment of predicted enzymatic activity of α-N-acetylglucosaminidase variants of unknown significance for CAGI 2016.

The NAGLU challenge of the fourth edition of the Critical Assessment of Genome Interpretation experiment (CAGI4) in 2016, invited participants to predict the impact of variants of unknown significance (VUS) on the enzymatic activity of the lysosomal hydrolase α-N-acetylglucosaminidase (NAGLU). Deficiencies in NAGLU activity lead to a rare, monogenic, recessive lysosomal storage disorder, Sanfilippo syndrome type B (MPS type IIIB). This challenge attracted 17 submissions from 10 groups. We observed that top models were able to predict the impact of missense mutations on enzymatic activity with Pearson's correlation coefficients of up to .61. We also observed that top methods were significantly more correlated with each other than they were with observed enzymatic activity values, which we believe speaks to the importance of sequence conservation across the different methods. Improved functional predictions on the VUS will help population-scale analysis of disease epidemiology and rare variant association analysis.

Structural characterization of the α-N-acetylglucosaminidase, a key enzyme in the pathogenesis of Sanfilippo syndrome B.

Mucopolysaccharidosis III B (MPS III-B) is a rare lysosomal storage disorder caused by deficiencies in Alpha-N-acetylglucosaminidase (NAGLU) for which there is currently no cure, and present treatment is largely supportive. Understanding the structure of NAGLU may allow for identification of novel therapeutic targets for MPS III-B. Here we describe the first crystal structure of human NAGLU, determined to a resolution of 2.3 Å. The crystal structure reveals a novel homotrimeric configuration, maintained primarily by hydrophobic and electrostatic interactions via domain II of three contiguous domains from the N- to C-terminus. The active site cleft is located between domains II and III. Catalytic glutamate residues, E316 and E446, are located at the top of the (α/ß)8 barrel structure in domain II. We utilized the three-dimensional structure of NAGLU to map several MPS III-B mutations, and hypothesize their functional consequences. Revealing atomic level structural information about this critical lysosomal enzyme paves the way for the design of novel therapeutics to target the underlying causes of MPS III-B.

Lysosomal Proteins as a Therapeutic Target in Neurodegeneration.

Several proteins that are mutated in lysosomal storage diseases are linked to neurodegenerative disease. This review focuses on some of these lysosomal enzymes and transporters, as well as current therapies that have emerged from the lysosomal storage disease field. Given the deeper genetic understanding of lysosomal defects in neurodegeneration, we explore why some of these orphan disease drug candidates are also attractive targets in subpopulations of individuals with neurodegenerative disease.

Biochemical, machine learning and molecular approaches for the differential diagnosis of Mucopolysaccharidoses.

This study was aimed to construct classification and regression tree (CART) model of glycosaminoglycans (GAGs) for the differential diagnosis of Mucopolysaccharidoses (MPS). Two-dimensional electrophoresis and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used for the qualitative and quantitative analysis of GAGs. Specific enzyme assays and targeted gene sequencing were performed to confirm the diagnosis. Machine learning tools were used to develop CART model based on GAG profile. Qualitative and quantitative CART models showed 96.3% and 98.3% accuracy, respectively, in the differential diagnosis of MPS. The thresholds of different GAGs diagnostic of specific MPS types were established. In 60 MPS positive cases, 46 different mutations were identified in six specific genes. Among 31 different mutations identified in IDUA, nine were nonsense mutations and two were gross deletions while the remaining were missense mutations. In IDS gene, four missense, two frameshift, and one deletion were identified. In NAGLU gene, c.1693C > T and c.1914_1914insT were the most common mutations. Two ARSB, one case each of SGSH and GALNS mutations were observed. LC-MS/MS-based GAG pattern showed higher accuracy in the differential diagnosis of MPS. The mutation spectrum of MPS, specifically in IDUA and IDS genes, is highly heterogeneous among the cases studied.

Knockdown of ß-N-acetylglucosaminidase gene disrupts molting process in Heortia vitessoides Moore.

ß-N-acetylglucosaminidase (NAG) is a key enzyme in insect chitin metabolism and plays an important role in many physiological activities of insects. The HvNAG1 gene was identified from the Heortia vitessoides Moore (Lepidoptera: Crambidae) cDNA library and its expression patterns were determined using quantitative real-time polymerase chain reaction. The results indicated that HvNAG1 mRNA levels were high in the midgut and before molting, and 20E could induce its expression. Subsequently, the HvNAG1 gene was knocked down via RNA interference to identify its functions. We found that 3 µg of dsNAG1 resulted in optimal interference at 48 and 72 hr after injection, causing a decrease in NAG1 protein content, which resulted in abnormal or lethal phenotypes, and a sharp decrease in the survival rate. These results indicate that HvNAG1 plays a key role in the molting process of H. vitessoides. However, the silencing of HvNAG1 had no significant effect on the chitin metabolism-related genes tested in this study. Our present study provides a reference for further research on the utility of key genes involved in the chitin metabolic pathway in the insect molting process.

High-level expression of ß-N-Acetylglucosaminidase BsNagZ in Pichia pastoris to obtain GlcNAc.

ß-N-Acetylglucosaminidases (NAGase) can remove N-acetylglucosamine (GlcNAc) from the non-reducing end of chitin or chitosan. GlcNAc has many important physiological functions in organism, which can be used for the treatment of rheumatoid arthritis clinically and be used as food antioxidant, infant food additive and diabetic sweetener. Thus, it is very important to develop genetic-engineering strains with high-yield NAGase to hydrolyze chitin into GlcNAc. Here, the NAGase gene of Bacillus subtilis 168 (BsnagZ) was synthesized according to the codon bias of Pichia pastoris and expressed in P. pastoris. The expression level of BsNagZ in P. pastoris increased over the induced time and the highest activity reached 0.76 U/mL at the 7th day. The recombinant BsNagZ was purified for characterization. The optimal temperature and pH are 60 °C and 6.0, respectively. It can both keep over 80% activities after pre-incubation at 55 °C for one hour and at 4 °C for 12 h from pH 4.5 to 10.0. To further improve the expression level of BsNagZ, a recombinant strain with four copy BsnagZs was screened using a high concentration of zeocin. The highest BsNagZ activity reached 3.2 U/mL at the 12th day, which was fourfold higher than that of single-copy strain. Combined with commercial chitinase CtnSg, GlcNAc can be produced by recombinant BsNagZ when used colloidal chitin as the substrate. Our study highlights that the NAGase was first successfully expressed in P. pastoris and GlcNAc can be produced via NAGase hydrolyzing the colloidal chitin.

Integrative analysis of oncogenic fusion genes and their functional impact in colorectal cancer.

BACKGROUND: Fusion genes are good candidates of molecular targets for cancer therapy. However, there is insufficient research on the clinical implications and functional characteristics of fusion genes in colorectal cancer (CRC). METHODS: In this study, we analysed RNA sequencing data of CRC patients (147 tumour and 47 matched normal tissues) to identify oncogenic fusion genes and evaluated their role in CRC. RESULTS: We validated 24 fusion genes, including novel fusions, by three algorithms and Sanger sequencing. Fusions from most patients were mutually exclusive CRC oncogenes and included tumour suppressor gene mutations. Eleven fusion genes from 13 patients (8.8%) were determined as oncogenic fusion genes by analysing their gene expression and function. To investigate their oncogenic impact, we performed proliferation and migration assays of CRC cell lines expressing fusion genes of GTF3A-CDK8, NAGLU- IKZF3, RNF121- FOLR2, and STRN-ALK. Overexpression of these fusion genes increased cell proliferation except GTF3A-CDK8. In addition, overexpression of NAGLU-IKZF3 enhanced migration of CRC cells. We demonstrated that NAGLU-IKZF3, RNF121-FOLR2, and STRN-ALK had tumourigenic effects in CRC. CONCLUSION: In summary, we identified and characterised oncogenic fusion genes and their function in CRC, and implicated NAGLU-IKZF3 and RNF121-FOLR2 as novel molecular targets for personalised medicine development.

Mucopolysaccharidosis IIIB and mild skeletal anomalies: coexistence of NAGLU and CYP26B1 missense variations in the same patient in a Chinese family.

BACKGROUND: Sanfilippo type B syndrome (mucopolysac-charidosis type IIIB; MPS IIIB) is an autosomal recessive lysosomal storage disorder. It is caused by a critically reduced α-2-acetamido-2-deoxy-D-glucoside acetamidodeoxy glucohydrolase (α-N-acetylglucosaminidase or NAGLU) activity. Recently, an autosomal recessive disorder of skeletal dysplasia associated with CYP26B1 was reported in three families, in which the patients were all homozygous variations. However, the co-occurrence of two rare diseases in a person is very rare. Here, we reported one patient with two novel pathogenic missense variations in NAGLU and CYP26B1. CASE PRESENTATION: We found an infant with biallelic variation both in NAGLU-compound heterozygous c.1843C > T (p. R615C) and c.1224C > A (p. H408Q) as well as in CYP26B1-compound heterozygous c.529G > A (p. E177K) and c.525C > A (p. H175Q). All variations were novel but predicted pathogenicity according to American College of Medical Genetics and Genomics (ACMG) guidelines. The main phenotypes of the infant were quite different from those previously reported, and some were combinations of the two rare diseases, including epilepsy, early onset epileptic encephalopathy, hypermyotonia, skull deformity, dilatation of the lateral ventricles and premature closure of fontanel. His NAGLU enzyme activity was significantly decreased. CONCLUSIONS: NAGLU and CYP26B1 mutations were related to MPS IIIB and skeletal dysplasia, respectively. Here, we first reported the pathogenic mutations of two genes concurrent in one patient, which not only expands the phenotype and genotype spectra of NAGLU and CYP26B1, but more importantly indicates the possibility of simultaneous occurrence of two rare diseases in one patient. This interesting finding should be attributed to the use of whole exome sequencing (WES), which indicates that we should be aware of the importance of WES in diagnosing rare diseases.