Cardiovascular involvement is commonly reported in various muco- polysaccharidoses. We report a first case of Sanfilippo syndrome type A in a 12-year-old white female who has developed combined progressive mitral valvar regurgitation due to prolapse and aortic regurgitation.

Human α-N-acetylglucosaminidase (Naglu) is a lysosomal enzyme implicated in the rare metabolic storage disorder Mucopolysaccharidosis III type B (MPS IIIB). A deficiency in Naglu results in a buildup of heparan sulfate in lysosomes, which is most detrimental in the central nervous system, causing mental retardation and a shortened lifespan. Enzyme replacement therapy is currently ineffective in treating the neurological symptoms of MPS IIIB due to the inability of Naglu to cross the blood-brain barrier. This laboratory uses a Spodoptera frugiperda insect cell system to express recombinant Naglu conjugated to a synthetic protein transduction domain with the intent to allow Naglu to cross the blood-brain barrier and treat the neurological symptoms. In the present study, we aimed to purify a recombinant Naglu-PTD4 fusion protein in order to assess its capacity to cross cellular membranes. A three-step method involving multi-modal, hydrophobic interaction, and gel filtration chromatography was optimized to achieve pure Naglu-PTD4, in good yield. Cellular uptake by human MPSIIIB fibroblasts of Naglu-PTD4 was not detectable. It is hypothesized that additional amino acids, including a hexahistidine domain, following the PTD4 domain limited the fusion protein’s membrane transduction capacity. Future studies will focus on removing the additional amino acids and adjusting the purification method as necessary. The ultimate goal of this research is to develop a large-scale recombinant Naglu production protocol for enzyme replacement therapy of MPS IIIB. / Graduate

This thesis, which focuses on sleep disturbance in people with neurodevelopmental disabilities, is divided into three sections. Paper one is a systematic review of the extant literature on objective studies of sleep in neurodevelopmental genetic disorders. Twenty papers met inclusion criteria and were subject to quality assessment, of which five were found to be high-quality, thirteen were medium-quality and two were low-quality. Studies were grouped by disorder and although there was some disparity across investigations, generally there was agreement about specific sleep difficulties in each disorder which seem to be part of the behavioural phenotypes. Overall a lack of total sleep, diminished REM sleep, and fragmented, less efficient sleep are prevalent across the disorders. Paper two is an empirical study which employed actigraphy to assess sleep in children with mucopolysaccharidosis type III (MPS III) and typically developing children. Parents completed a sleep diary, a sleep questionnaire and took saliva samples from their child. Actigraphic findings showed that MPS III patients had lengthened sleep onset latencies and greater daytime sleep than controls, but night-time sleep duration was within the normal range. In the MPS III group, some sleep problems correlated with age and progression of the disorder. Analysis of saliva samples revealed that children with MPS III had abnormal melatonin concentrations. Questionnaire responses demonstrated that children with MPS III had more sleep difficulties in all domains compared to controls. Implications for the management of sleep difficulties are discussed. Paper three is a critical appraisal of the research process which includes personal reflections on designing and conducting this research and a discussion of the principal issues which arose. Strengths and limitations of the research, ideas for further research and implications for clinical practice are considered.

Clostridium perfringens is a gram positive spore forming anaerobe and a causative agent of gas gangrene, necrotic enteritis (pig-bel) and food poisoning in humans and other animals. This organism secretes a battery of exotoxins during the course of infection as well as a variety of virulence factors which may help to potentiate the activities of the toxins. Among these virulence factors is the μ-toxin, a family 84 glycoside hydrolase which acts to degrade hyaluronan, a component of human connective tissue. C. perfringens has 53 open reading frames encoding glycoside hydrolases. About half of these glycoside hydrolases are predicted to be secreted. Among these are CpGH84C, a paralogue of the μ-toxin, and CpGH89. CpGH89 shares sequence similarity to the human α-N-acetylglucosaminidase, NAGLU, in which mutations can cause a devastating genetic disease called mucopolysaccharidosis IIIB. One striking feature of the secreted glycoside hydrolase enzymes of C. perfringens is their modularity, with modules predicted to be dedicated to catalysis, carbohydrate-binding, protein-protein interactions and cell wall attachment. The extent of the modularity is remarkable, with some enzymes containing up to eight ancillary modules. In order to help understand the role of carbohydrate-active enzymes produced by bacterial pathogens, this thesis will focus on the structure and function of the modular extracellular glycoside hydrolase enzymes secreted by the disease causing bacterium, C. perfringens. These structure function studies examine two family 32 CBMs (carbohydrate-binding modules), one from the μ-toxin and the other from CpGH84C. As well we examine the complete structure of CpGH84C in order to help further our understanding of the structure of carbohydrate-active enzymes as a whole. Finally, the catalytic module of CpGH89 is characterized and its relationship to the human NAGLU enzyme is discussed.

L’acétylation des résidus de glucosamine terminaux par la N-acétyltransférase lysosomale (HGSNAT) est une étape essentielle de la dégradation catabolique de l’héparan sulfate. Des défauts dans cette réaction causent une maladie de surcharge lysosomale autosomale récessive rare : le désordre de Sanfilippo type C (SFC). À ce jour, 54 mutations ont été rapportées chez des patients SFC, incluant 13 mutations des sites d’épissage, 11 insertions et délétions, 8 mutations non-sens, 18 mutations faux-sens et 4 polymorphismes, avec différentes manifestations phénotypiques. Nous avons identifié 10 d’entre elles et effectué une étude exhaustive portant sur l’éventail des mutations SFC, leur distribution dans la population de patients, ainsi que leur impact potentiel sur la structure de la HGSNAT. Les erreurs d’épissage, les mutations non-sens, les insertions et les délétions devraient toutes entraîner un ARN non fonctionnel qui est rapidement dégradé par des mécanismes de contrôle qualité cellulaire. Les 4 polymorphismes identifiés sont des changements d'acides aminés qui ne modifient pas l'activité enzymatique, la glycosylation ou la localisation et n'ont donc pas de signification au niveau clinique. Au niveau des enzymes, les polymorphismes sont des changements d’acides aminés qui n’affectent pas la fonction, mais dans un contexte d’acides nucléiques ils peuvent être considérés comme des mutations faux-sens. Les dix-huit mutations faux-sens qui ont été exprimées ont produit des protéines inactives, en raison d'erreurs dans leur repliement. Ceci expliquerait donc la progression sévère de la maladie chez les personnes porteuses de ces mutations. Les protéines mutantes mal repliées sont anormalement glycosylées et conservées dans le réticulum endoplasmique. La thérapie par amélioration de l’activité enzymatique par des chaperonnes est une option thérapeutique potentielle, spécifiquement conçue pour exploiter l'activité enzymatique résiduelle de mutants mal repliés, afin d’éliminer les substrats stockés. Nous avons démontré que le traitement de plusieurs lignées de fibroblastes de patients SFC avec le chlorhydrate de glucosamine, un inhibiteur spécifique de la HGSNAT, a partiellement restauré l’activité de l'enzyme mutante, fournissant une preuve de l’utilité future de la thérapie par des chaperonnes dans le traitement de la maladie de SFC. / The acetylation of terminal glucosamine residues by lysosomal N-acetyltransferase (HGSNAT) is an essential part of the catabolic breakdown of heparan sulfate. Defects in this reaction result in the rare autosomal recessive lysosomal storage disorder Sanfilippo syndrome type C (SFC). To date 54 mutations in SFC patients have been reported including 13 splice-site mutations, 11 insertions and deletions, 8 nonsense, 18 missense and 4 polymorphisms, with different phenotypic manifestations. We have identified 10 of them and conducted a comprehensive review discussing the spectrum of Sanfilippo C mutations, their distribution within the patient population as well as how the mutations could potentially affect the structure of HGSNAT. Splicing errors, nonsense mutations, insertions and deletions were all predicted to result in non-functional RNA which is rapidly degraded by cellular quality control mechanisms. The 4 identified polymorphisms resulted in amino acid changes which did not affect the enzyme activity, glycosylation or targeting and were therefore not clinically significant. Polymorphisms, in the context of enzymes are amino acid changes not affecting function, but in the context of nucleic acids can still be considered as missense mutations. Eighteen missense mutations were expressed and shown be inactive due to errors in protein folding providing an explanation for the severe disease progression seen in individuals with these mutations. Misfolded mutants were abnormally glycosylated and retained in the endoplasmic reticulum. Enzyme enhancement/chaperone therapy is a potential treatment option specifically designed to exploit the residual enzyme activity of misfolded mutants in order to clear stored substrates. We demonstrated that treatment of several fibroblast lines of SFC patients with a specific inhibitor of HGSNAT; glucosamine-hydrochloride partially rescued mutant enzyme activity providing a proof of principle for the future use of chaperone therapeutics in the treatment of SFC.

L’acétylation des résidus de glucosamine terminaux par la N-acétyltransférase lysosomale (HGSNAT) est une étape essentielle de la dégradation catabolique de l’héparan sulfate. Des défauts dans cette réaction causent une maladie de surcharge lysosomale autosomale récessive rare : le désordre de Sanfilippo type C (SFC). À ce jour, 54 mutations ont été rapportées chez des patients SFC, incluant 13 mutations des sites d’épissage, 11 insertions et délétions, 8 mutations non-sens, 18 mutations faux-sens et 4 polymorphismes, avec différentes manifestations phénotypiques. Nous avons identifié 10 d’entre elles et effectué une étude exhaustive portant sur l’éventail des mutations SFC, leur distribution dans la population de patients, ainsi que leur impact potentiel sur la structure de la HGSNAT. Les erreurs d’épissage, les mutations non-sens, les insertions et les délétions devraient toutes entraîner un ARN non fonctionnel qui est rapidement dégradé par des mécanismes de contrôle qualité cellulaire. Les 4 polymorphismes identifiés sont des changements d'acides aminés qui ne modifient pas l'activité enzymatique, la glycosylation ou la localisation et n'ont donc pas de signification au niveau clinique. Au niveau des enzymes, les polymorphismes sont des changements d’acides aminés qui n’affectent pas la fonction, mais dans un contexte d’acides nucléiques ils peuvent être considérés comme des mutations faux-sens. Les dix-huit mutations faux-sens qui ont été exprimées ont produit des protéines inactives, en raison d'erreurs dans leur repliement. Ceci expliquerait donc la progression sévère de la maladie chez les personnes porteuses de ces mutations. Les protéines mutantes mal repliées sont anormalement glycosylées et conservées dans le réticulum endoplasmique. La thérapie par amélioration de l’activité enzymatique par des chaperonnes est une option thérapeutique potentielle, spécifiquement conçue pour exploiter l'activité enzymatique résiduelle de mutants mal repliés, afin d’éliminer les substrats stockés. Nous avons démontré que le traitement de plusieurs lignées de fibroblastes de patients SFC avec le chlorhydrate de glucosamine, un inhibiteur spécifique de la HGSNAT, a partiellement restauré l’activité de l'enzyme mutante, fournissant une preuve de l’utilité future de la thérapie par des chaperonnes dans le traitement de la maladie de SFC. / The acetylation of terminal glucosamine residues by lysosomal N-acetyltransferase (HGSNAT) is an essential part of the catabolic breakdown of heparan sulfate. Defects in this reaction result in the rare autosomal recessive lysosomal storage disorder Sanfilippo syndrome type C (SFC). To date 54 mutations in SFC patients have been reported including 13 splice-site mutations, 11 insertions and deletions, 8 nonsense, 18 missense and 4 polymorphisms, with different phenotypic manifestations. We have identified 10 of them and conducted a comprehensive review discussing the spectrum of Sanfilippo C mutations, their distribution within the patient population as well as how the mutations could potentially affect the structure of HGSNAT. Splicing errors, nonsense mutations, insertions and deletions were all predicted to result in non-functional RNA which is rapidly degraded by cellular quality control mechanisms. The 4 identified polymorphisms resulted in amino acid changes which did not affect the enzyme activity, glycosylation or targeting and were therefore not clinically significant. Polymorphisms, in the context of enzymes are amino acid changes not affecting function, but in the context of nucleic acids can still be considered as missense mutations. Eighteen missense mutations were expressed and shown be inactive due to errors in protein folding providing an explanation for the severe disease progression seen in individuals with these mutations. Misfolded mutants were abnormally glycosylated and retained in the endoplasmic reticulum. Enzyme enhancement/chaperone therapy is a potential treatment option specifically designed to exploit the residual enzyme activity of misfolded mutants in order to clear stored substrates. We demonstrated that treatment of several fibroblast lines of SFC patients with a specific inhibitor of HGSNAT; glucosamine-hydrochloride partially rescued mutant enzyme activity providing a proof of principle for the future use of chaperone therapeutics in the treatment of SFC.

Human α-N-Acetylglucosaminidase (Naglu) is a lysosomal acid hydrolase implicated in tthe rare metabolic storage disorder known as mucopolysaccharidosis type IIIB (MPS IIIB; also Sanfilippo syndrome B). Absence of this enzyme results in cytotoxic accumulation of heparan sulphate in the central nervous system, causing mental retardation and a shortened lifespan. Enzyme replacement therapy is not currently effective to treat neurological symptoms due to the inability of exogenous Naglu to access the brain. This laboratory uses a Spodoptera frugiperda (Sf9) insect cell system to express Naglu fused to a synthetic protein transduction domain with the intent to facilitate delivery of Naglu across the blood-brain barrier. The project described herein may be broken down into three main sections. Firstly, the impact of two cryptic splice sites on Naglu expression levels was analyzed in both transiently expressing Sf9 cultures and stably selected cell lines. Secondly, the effectiveness of the native Naglu secretion-signaling peptide in the Sf9 system was examined. Finally, purification of a Naglu fusion protein from suspension culture medium was performed using hydrophobic interaction chromatographic techniques. The ultimate goal of this research is to develop an efficient system for economical, large-scale production of a human recombinant Naglu fusion protein that has the potential to be successfully used for enzyme replacement therapy to treat MPS IIIB. / Graduate