Mucopolysaccharidoses result from genetic mutations in lysosomal enzymes required for degradation of glycosaminoglycans. The deficiency in any of eight lysosomal enzymes needed to degrade heparan sulfate leads to an accumulation of both non-degraded and partially degraded polysaccharides within the lysosomes of many tissues. Interestingly, six of these deficient enzymes can be treated by a relatively new approach – substrate reduction therapy (SRT), which aims to reduce the synthesis of the substrate for the deficient enzyme being targeted. I developed a cell-based high throughput screen assay for the identification of compounds that decrease the expression of the first modifying enzyme in HS biosynthesis, N-deacetylase/N-sulfotransferase 1, by inhibiting the transcription of its mRNA. From the high throughput screen, I identified several compounds, with a previous history of use in humans, which significantly decreased the endogenous NDST1 expression and therefore, could be considered as potential SRT agents for up to six Mucopolysaccharidoses.

Mucopolysaccharidoses result from genetic mutations in lysosomal enzymes required for degradation of glycosaminoglycans. The deficiency in any of eight lysosomal enzymes needed to degrade heparan sulfate leads to an accumulation of both non-degraded and partially degraded polysaccharides within the lysosomes of many tissues. Interestingly, six of these deficient enzymes can be treated by a relatively new approach – substrate reduction therapy (SRT), which aims to reduce the synthesis of the substrate for the deficient enzyme being targeted. I developed a cell-based high throughput screen assay for the identification of compounds that decrease the expression of the first modifying enzyme in HS biosynthesis, N-deacetylase/N-sulfotransferase 1, by inhibiting the transcription of its mRNA. From the high throughput screen, I identified several compounds, with a previous history of use in humans, which significantly decreased the endogenous NDST1 expression and therefore, could be considered as potential SRT agents for up to six Mucopolysaccharidoses.

Thèse de doctorat : Médecine. Virologie : Nantes : 2006. / Bibliogr.

submitted by Chair Siu Fan. / Thesis (M.Sc.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 75-83). / Chapter 1 --- INTRODUCTION --- p.1 / Chapter 2 --- LITERATIRE REVIEW --- p.3 / Chapter 2.1 --- Causes and clinical syndromes in MPS --- p.3 / Chapter 2.1.1 --- MPS --- p.4 / Chapter 2.1.2 --- Classification of MPS --- p.4 / Chapter 2.1.2.1 --- MPS I (Hurler's syndrome) --- p.5 / Chapter 2.1.2.2 --- MPS IS (Scheie syndrome) --- p.5 / Chapter 2 .1.2.3 --- MPS II (Hunter's disease) --- p.6 / Chapter 2.1.2.4 --- MPS Type III (The Sanfilippo diseases) --- p.6 / Chapter 2.1.2.5 --- MPS Type IV (Morquio's disease) --- p.6 / Chapter 2.1.2.6 --- MPS Type VI (Maroteaux 一 Lamy syndrome) --- p.7 / Chapter 2.1.2.7 --- MPS Type VII (Sly syndrome) --- p.8 / Chapter 2.1.3 --- Treatment and prospects for MPS --- p.8 / Chapter 2.1.3.1 --- To manage the handicaps and disabilities --- p.8 / Chapter 2.1.3.2 --- Enzyme replacement --- p.9 / Chapter 2.1.3.3 --- Bone marrow transplantation --- p.10 / Chapter 2.2 --- Basic aspects of GAG --- p.10 / Chapter 2.2.1 --- Distributions of GAG --- p.12 / Chapter 2.2.2 --- Functions and Roles of GAG --- p.12 / Chapter 2.2.3 --- Stepwise degradation of GAGs --- p.12 / Chapter 2.2.4 --- Source of urinary GAG --- p.13 / Chapter 2.2.5 --- Common features of GAGS --- p.14 / Chapter 2.2.6 --- Factors affecting the excretion pattern ot GAG --- p.16 / Chapter 2.3 --- Methods for MPS Diagnosis --- p.16 / Chapter 2.3.1 --- Qualitative urine methods for screening and typing --- p.16 / Chapter 2.3.1.1 --- Spot tests --- p.16 / Chapter 2.3.1.2 --- Precipitation methods --- p.16 / Chapter 2.3.1.3 --- One-dimensional electrophoresis --- p.17 / Chapter 2.3.1.4 --- Two-dimensional electrophoresis --- p.17 / Chapter 2.3.1.5 --- Thin layer chromatography --- p.17 / Chapter 2.3.2 --- Quantitative methods for urinary GAG --- p.17 / Chapter 2.3.2.1 --- Measurement of hexuronic acid --- p.18 / Chapter 2.3.2.2 --- HPLC or Column chromatography --- p.18 / Chapter 2.3.2.3 --- Dye-binding methods --- p.19 / Chapter 2.3.3 --- Cytological studies --- p.19 / Chapter 2.3.4 --- Tissue culture --- p.20 / Chapter 2.3.5 --- Tissue biopsy --- p.20 / Chapter 2.3.6 --- Prenatal diagnosis of the MPS --- p.21 / Chapter 2.4 --- Bacterial GAG hydrolytic enzymes --- p.23 / Chapter 2.5 --- Summary of Literature Review --- p.25 / Chapter 3. --- AIMS OF STUDY --- p.26 / Chapter 4. --- MATERIALS AND METHODS --- p.26 / Chapter 4.1 --- Sample collection --- p.26 / Chapter 4.2 --- Materials &-Equipment --- p.26 / Chapter 4.3 --- Preparation of Reagents and Standards --- p.27 / Chapter 4.3.1 --- Stock DMB reagent solutions --- p.27 / Chapter 4.3.2 --- Working DMB solution --- p.27 / Chapter 4.3.3 --- GAG standards --- p.27 / Chapter 4.3.4 --- Reagents for electrophoresis --- p.27 / Chapter 4.3.4.1 --- 0.1M barium acetate solution --- p.27 / Chapter 4.3.4.2 --- 15% ethanolic barium acetate --- p.28 / Chapter 4.3.4.3 --- 50% ethanolic barium acetate --- p.28 / Chapter 4.3.4.4 --- Alcian blue working solution --- p.28 / Chapter 4.3.4.5 --- 0.1M Tris Buffer --- p.28 / Chapter 4.3.4.6 --- CTB Tris solution --- p.28 / Chapter 4.3.4.7 --- 2.0M lithium chloride --- p.28 / Chapter 4.3.5 --- Reagents for enzymatic degradation --- p.28 / Chapter 4.3.5.1 --- Reconstitution of CSE enzyme --- p.28 / Chapter 4.3.5.2 --- Reconstitution of DSE enzyme --- p.29 / Chapter 4.3.5.3 --- Reconstitution ofHSE I enzyme --- p.29 / Chapter 4.4 --- Methods --- p.31 / Chapter 4.4.1 --- Cobas Bio DMB method --- p.31 / Chapter 4.4.2 --- Cobas Fara DMB method --- p.31 / Chapter 4.4.3 --- Evaluation of methods --- p.31 / Chapter 4.4.3.1 --- To study the matrix effect --- p.31 / Chapter 4.4.3.2 --- Calibration --- p.31 / Chapter 4.4.3.3 --- Precision performance --- p.34 / Chapter 4.4.3.4 --- Linearity check --- p.34 / Chapter 4.4.3.5 --- Detection Limit --- p.34 / Chapter 4.4.3.6 --- Recovery study --- p.35 / Chapter 4.4.3.7 --- Correlation with Cobas Bio to develop the reference range --- p.35 / Chapter 4.4.4 --- Electrophoresis method --- p.36 / Chapter 4.4.4.1 --- Sample preparation --- p.36 / Chapter 4.4.4.2 --- Electrophoresis procedure --- p.36 / Chapter 4.4.5 --- Enzymatic degradation method --- p.37 / Chapter 4.4.5.1 --- Digestion of GAG in aqueous and urine matrix --- p.37 / Chapter 4.4.5.2 --- To optimize the amount of enzyme used to degrade GAG --- p.38 / Chapter 4.4.5.3 --- To study the specificity of GAG degrading enzyme --- p.39 / Chapter 4.4.5.4 --- To study the interaction of GAG --- p.40 / Chapter 4.4.5.5 --- To study the stability of enzyme CSE and DSE --- p.40 / Chapter 4.4.5.6 --- Study MPS patient sample --- p.41 / Chapter 5 --- Results --- p.42 / Chapter 5.1 --- Performance characteristics of the DMB method --- p.42 / Chapter 5.1.1 --- Matrix effect --- p.42 / Chapter 5.1.2 --- Calibration --- p.42 / Chapter 5.1.3 --- Precision performance --- p.42 / Chapter 5.1.4 --- Linearity Range --- p.42 / Chapter 5.1.5 --- Detection limit --- p.42 / Chapter 5.1.6 --- Recovery --- p.47 / Chapter 5.1.7 --- Correlation of Cobas Fara with Cobas Bio --- p.47 / Chapter 5.2 --- Results of GAG enzymatic degradation --- p.50 / Chapter 5.2.2 --- To optimise the amount of enzyme for GAG degradation --- p.57 / Chapter 5.2.3 --- The specificity of GAG degrading enzymes --- p.57 / Chapter 5.2.4 --- The interaction of GAG --- p.57 / Chapter 5.2.5 --- The stability of enzymes --- p.57 / Chapter 5.2.6 --- MPS patient study --- p.57 / Chapter 5.2.6.1 --- Type I/II/VI/VII --- p.57 / Chapter 5.2.6.2 --- MPS Type III patient 1 --- p.64 / Chapter 5.2.6.3 --- MPS Type IIIC patient 2 --- p.64 / Chapter 6. --- DISCUSSION --- p.67 / Chapter 6.1 --- Automated DMB method on Cobas Fara --- p.67 / Chapter 6 2 --- GAG specific degradation enzymes --- p.70 / Chapter 7. --- CONCLUSION & SUGGESTION FOR FUTURE STUDIES --- p.73 / Chapter 8. --- REFERENCES --- p.75

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.

La médecine de précision (MP) est un nouveau paradigme qui révolutionne la pratique médicale actuelle et remodèle complètement la médecine de demain. La MP aspire à placer le patient au centre du parcours de soins en y intégrant les données médicales et biologiques individuelles tout en tenant compte de la grande diversité interindividuelle. La prédiction des états pathologiques chez les patients nécessite une compréhension dynamique et systémique. Les erreurs innées du métabolisme (EIM) sont des troubles génétiques résultant de défauts dans une voie biochimique donnée en raison de la déficience d'une enzyme, de son cofacteur ou d’un transporteur. Les EIM ne sont plus considérées comme des maladies monogéniques mais tendent à être plus complexes et multifactorielles. Le profil métabolomique permet le dépistage d’une pathologie, la recherche de biomarqueurs et l’exploration des voies métaboliques mises en jeu. Dans ce travail de thèse, nous avons utilisé l’approche métabolomique qui est particulièrement pertinente pour les EIM compte tenu de leur physiopathologie de base qui est étroitement liée au métabolisme. Ce travail a permis la mise en place d’une méthodologie métabolomique non ciblée basée sur une stratégie analytique multidimensionnelle comportant la spectrométrie de masse à haute résolution couplée à la chromatographie liquide ultra-haute performance et la mobilité ionique. La mise en place de la méthodologie de prétraitement, d’analyse et d’exploitation des données générées avec des outils de design expérimental et d’analyses multivariées ont été aussi établies. Enfin, cette approche a été appliquée pour l’exploration des EIM avec les mucopolysaccharidoses comme preuve de concept. Les résultats obtenus suggèrent un remodelage majeur du métabolisme des acides aminés dans la mucopolysaccharidose de type I. En résumé, la métabolomique pourrait être un outil complémentaire pertinent en appui à l’approche génomique dans l’exploration des EIM. / The new field of precision medicine is revolutionizing current medical practice and reshaping future medicine. Precision medicine intends to put the patient as the central driver of healthcare by broadening biological knowledge and acknowledging the great diversity of individuals. The prediction of physiological and pathological states in patients requires a dynamic and systemic understanding of these interactions. Inborn errors of metabolism (IEM) are genetic disorders resulting from defects in a given biochemical pathway due to the deficiency of an enzyme, its cofactor or a transporter. IEM are no longer considered to be monogenic diseases, which adds another layer of complexity to their characterization and diagnosis. To meet this need for faster screening, the metabolic profile can be a promising candidate given its ability in disease screening, biomarker discovery and metabolic pathway investigation. In this thesis, we used a metabolomic approach which is particularly relevant for IEM given their basic pathophysiology that is tightly related to metabolism. This thesis allowed the implementation of an untargeted metabolomic methodology based on a multidimensional analytical strategy including high-resolution mass spectrometry coupled with ultra-high-performance liquid chromatography and ion mobility. This work also set a methodology for preprocessing, analysis and interpretation of the generated data using experimental design and multivariate data analysis. Finally, the strategy is applied to the exploration of IEM with mucopolysaccharidoses as a proof of concept. The results suggest a major remodeling of the amino acid metabolisms in mucopolysaccharidosis type I. In summary, metabolomic is a relevant complementary tool to support the genomic approach in the functional investigations and diagnosis of IEM.

INTRODUÇÃO: As mucopolissacaridoses (MPSs), consideradas doenças de depósito lisossomal, são classificadas, de acordo com a deficiência enzimática, em sete tipos de doenças metabólicas hereditárias de caráter crônico, progressivo e sistêmico. OBJETIVOS: Caracterizar os achados clínicos e avaliar a evolução dos pacientes com diferentes tipos de MPS. MÉTODOS: Trata-se de um estudo retro e prospectivo de 30 pacientes com diferentes tipos de MPSs atendidos na Unidade de Genética do Instituto da Criança do Hospital das Clínicas da Universidade de São Paulo. Para a realização do estudo foi elaborado um protocolo de pesquisa, que compreendeu de uma avaliação clínica e laboratorial. RESULTADOS: Foram estudados 30 pacientes, assim distribuídos: seis MPS I, dois MPS II, sete MPS III, sete MPS IV, seis MPS VI e dois MPS VII. Dezesseis pacientes eram femininos e 14 masculinos. A idade variou entre 2 anos e 22 anos e 4 meses, média de 12 anos e 1 mês. As complicações clínicas detectadas foram: cardiopatia (90%), opacidade de córneas (70%), infecções de repetição (63%), síndrome da apnéia noturna do sono (SAOS) (37%), diarréia (33%), convulsão (17%) e hipertensão artéria sistêmica (HAS) (10%). Cinqüenta por cento dos pacientes foram internados em enfermaria e 13% em UTI, sendo que a s infecções respiratórias foram os principais motivos para as internações hospitalares. O uso do CPAP foi necessário em quatro pacientes, a média de idade para iniciar o uso foi 9 anos e 3 meses. Vinte pacientes foram operados e submetidos a 37 procedimentos anestésicos e 46 procedimentos cirúrgicos (média de 2,3 cirurgia/paciente), sendo que quatro pacientes apresentaram intercorrências: dificuldade de intubação (3), HAS (1) e sangramento (1). A obstrução das vias aéreas superiores e a complicação cardíaca resultaram no óbito de dois pacientes: um MPS I, aos 8 anos e 9 meses, e um com MPS II, aos 12 anos. Quanto aos exames laboratoriais, o teste de toluidina apresentou resultados falsonegativos em 44%, a dosagem de glicosaminoglicanos (GAGs) urinários foi aumentada em todos os pacientes, a cromatografia de GAGs foi normal em quatro pacientes com MPS IV e dosagem enzimática definiu o diagnóstico e o tipo de MPS. Em relação aos outros exames, as alterações radiológicas, disostose múltipla, foram detectadas em 100% dos pacientes, 90% apresentaram achados ecocardigráficos, 27% SAOS de grau acentuado na polissonografia e 57% ventriculomegalia na tomografia computadorizada de crânio. CONCLUSÃO: Os pacientes necessitam de um acompanhamento periódico, por meio de uma equipe multidisciplinar, com o objetivo de se estabelecer um manejo adequado das freqüentes complicações clinicas / INTRODUCTION: mucopolysaccharidoses (MPSs) are a group of lysosomal storage disorders classified, according to specific enzyme deficiency, into seven types of hereditary metabolic diseases of chronic, progressive, and systemic character. OBJECTIVES: to characterize clinical findings and to evaluate the evolution of patients with different types of MPSs seen at the Genetics Division of the Children\'s Hospital of the University of Sao Paulo Clinical Hospital. For the present study, we devised a research protocol including clinical evaluation and laboratory tests. RESULTS: we studied 30 patients, including six MPS I, two MPS II, seven MPS III, seven MPS IV, six MPS VI, and two MPS VII. Sixteen patients were female, and 14 male. Age ranged between 2 years and 22 years and 4 months, mean 12 years and 1 month. Clinical complications detected included: cardiopathy (90%), corneal opacity (70%), recurrent infections (63%), sleep apnea syndrome (SAS) (37%), diarrhea (33%), seizures (17%), and systemic arterial hypertension (SAH) (10%). Fifty percent of patients were admitted to regular wards, and 13% to intensive care units, respiratory infections being the most frequent reason for hospital admission. CPAP was required for four patients; mean age for onset of CPAP use was 9 years and 3 months. Twenty patients underwent surgery, leading to a total 37 anesthetic procedures and 46 surgical procedures (mean 2.3 surgeries/patient); four patients had complications during surgery; these included intubation difficulties (3), SAH (1), and bleeding (1). Obstruction of the upper airways and cardiac complications led to the death of two patients, one MPS I (age 8 years and 9 months) and one MPS II (age 12 years). Regarding laboratory tests, toluidine blue staining showed false-negative results in 44% of patients, urine glycosaminoglycan (GAG) levels were increased in all patients, GAG chromatography was normal in four patients with MPS IV, and enzyme quantification defined the diagnosis and type of MPS. Radiological alterations (multiple dysostosis) were detected in all patients; 90% of patients showed echocardiographic alterations; polysomnography showed marked SAS in 27% of patients; and cranial CAT scans showed ventriculomegaly in 57% of patients. CONCLUSION: MPS patients require regular follow-up by a multidisciplinary team in order to achieve adequate management of frequent clinical complications

Introdução: As mucopolissacaridoses (MPS) são um conjunto de sete doenças genéticas incluídas dentro das Doenças Lisossômicas que por sua vez fazem parte dos Erros Inatos do Metabolismo (EIM). São doenças multissistêmicas que afetam todo o organismo, com variações conforme o tipo de MPS, sendo que algumas delas possuem tratamento específico. Quase todas comprometem, em graus variados, o sistema osteoarticular, e praticamente todos os pacientes apresentam excreção alterada de glicosaminoglicanos (GAGs) na urina. As MPS são doenças raras que podem ser subdiagnosticadas em função do pouco conhecimento dos profissionais de saúde sobre elas, do pouco acesso aos métodos de triagem e diagnóstico e da sua ampla heterogeneidade clínica, podendo ocorrer formas atenuadas nas quais pode ser difícil de levantar a suspeita clínica de MPS. Material e métodos: o presente estudo foi realizado no período de março de 2012 à janeiro de 2014, tendo incluído 55 pacientes atendidos em serviços de Reumatologia e/ou Ortopedia de Porto Alegre, RS, Brasil e que, apresentavam como principal queixa, manifestações articulares sem etiologia definida. Esses pacientes foram inicialmente investigados através da avaliação quantitativa e qualitativa dos GAGs urinários. Resultados e Discussão: entre os 55 casos investigados, em 1 paciente de 15 anos de idade foi observada na análise dos GAGs urinários excreção aumentada e alteração do padrão qualitativo, sendo posteriormente confirmado o diagnóstico de uma forma atenuada de MPS II, a qual não havia sido suspeitada anteriormente. Conclusão: embora a proporção de pacientes identificados com MPS na amostra estudada tenha sido pequena (1/55), este estudo mostra que ocorre subdiagnóstico dessas doenças e que a triagem sistemática pode contribuir para a identificação de pacientes, os quais podem se beneficiar das medidas de tratamento disponíveis. / Introduction: Mucopolysaccharidoses (MPS) are a set of 7genetic diseases including Lysosomal Diseases , which in turn are part of Inborn Errors of Metabolism (IEM). The MPS are multisystemic conditions that affect the entire body, with variations depending on the type, some of which have specific treatment. Almost all affect, in variable degrees, the osteo-articular system, and virtually all patients have abnormal excretion of glycosaminoglycans (GAGs) in urine. The MPS are rare diseases that are being underdiagnosed due to the little knowledge of health professionals about them, the poor access to screening and diagnostic methods and their extensive clinical heterogeneity. Also, attenuated forms may occur in which it may be difficult to raise the clinical suspicion of MPS. Material and Methods: The present study was conducted from March 2012 to January 2014 and included 55 patients from Rheumatology and/or Orthopedics services of Porto Alegre, RS, Brazil and which had, as main complaint, articular manifestations without defined etiology. These patients were screened by quantitative and qualitative assessment of urinary GAGs. Results and Discussion: Among the 55 cases investigated, in 1 patient 15 years of age was observed in the analysis of urinary GAG excretion and increased change in qualitative standard and subsequently confirmed the diagnosis of an attenuated form of MPS II, which had not previously been suspected. Conclusion: Although the proportion of patients with MPS identified in the study sample was small (1/55), this study shows that occurs underdiagnosis of these diseases and that systematic screening can help to identify patients who may benefit from measures treatment available.

INTRODUÇÃO: As mucopolissacaridoses (MPSs), consideradas doenças de depósito lisossomal, são classificadas, de acordo com a deficiência enzimática, em sete tipos de doenças metabólicas hereditárias de caráter crônico, progressivo e sistêmico. OBJETIVOS: Caracterizar os achados clínicos e avaliar a evolução dos pacientes com diferentes tipos de MPS. MÉTODOS: Trata-se de um estudo retro e prospectivo de 30 pacientes com diferentes tipos de MPSs atendidos na Unidade de Genética do Instituto da Criança do Hospital das Clínicas da Universidade de São Paulo. Para a realização do estudo foi elaborado um protocolo de pesquisa, que compreendeu de uma avaliação clínica e laboratorial. RESULTADOS: Foram estudados 30 pacientes, assim distribuídos: seis MPS I, dois MPS II, sete MPS III, sete MPS IV, seis MPS VI e dois MPS VII. Dezesseis pacientes eram femininos e 14 masculinos. A idade variou entre 2 anos e 22 anos e 4 meses, média de 12 anos e 1 mês. As complicações clínicas detectadas foram: cardiopatia (90%), opacidade de córneas (70%), infecções de repetição (63%), síndrome da apnéia noturna do sono (SAOS) (37%), diarréia (33%), convulsão (17%) e hipertensão artéria sistêmica (HAS) (10%). Cinqüenta por cento dos pacientes foram internados em enfermaria e 13% em UTI, sendo que a s infecções respiratórias foram os principais motivos para as internações hospitalares. O uso do CPAP foi necessário em quatro pacientes, a média de idade para iniciar o uso foi 9 anos e 3 meses. Vinte pacientes foram operados e submetidos a 37 procedimentos anestésicos e 46 procedimentos cirúrgicos (média de 2,3 cirurgia/paciente), sendo que quatro pacientes apresentaram intercorrências: dificuldade de intubação (3), HAS (1) e sangramento (1). A obstrução das vias aéreas superiores e a complicação cardíaca resultaram no óbito de dois pacientes: um MPS I, aos 8 anos e 9 meses, e um com MPS II, aos 12 anos. Quanto aos exames laboratoriais, o teste de toluidina apresentou resultados falsonegativos em 44%, a dosagem de glicosaminoglicanos (GAGs) urinários foi aumentada em todos os pacientes, a cromatografia de GAGs foi normal em quatro pacientes com MPS IV e dosagem enzimática definiu o diagnóstico e o tipo de MPS. Em relação aos outros exames, as alterações radiológicas, disostose múltipla, foram detectadas em 100% dos pacientes, 90% apresentaram achados ecocardigráficos, 27% SAOS de grau acentuado na polissonografia e 57% ventriculomegalia na tomografia computadorizada de crânio. CONCLUSÃO: Os pacientes necessitam de um acompanhamento periódico, por meio de uma equipe multidisciplinar, com o objetivo de se estabelecer um manejo adequado das freqüentes complicações clinicas / INTRODUCTION: mucopolysaccharidoses (MPSs) are a group of lysosomal storage disorders classified, according to specific enzyme deficiency, into seven types of hereditary metabolic diseases of chronic, progressive, and systemic character. OBJECTIVES: to characterize clinical findings and to evaluate the evolution of patients with different types of MPSs seen at the Genetics Division of the Children\'s Hospital of the University of Sao Paulo Clinical Hospital. For the present study, we devised a research protocol including clinical evaluation and laboratory tests. RESULTS: we studied 30 patients, including six MPS I, two MPS II, seven MPS III, seven MPS IV, six MPS VI, and two MPS VII. Sixteen patients were female, and 14 male. Age ranged between 2 years and 22 years and 4 months, mean 12 years and 1 month. Clinical complications detected included: cardiopathy (90%), corneal opacity (70%), recurrent infections (63%), sleep apnea syndrome (SAS) (37%), diarrhea (33%), seizures (17%), and systemic arterial hypertension (SAH) (10%). Fifty percent of patients were admitted to regular wards, and 13% to intensive care units, respiratory infections being the most frequent reason for hospital admission. CPAP was required for four patients; mean age for onset of CPAP use was 9 years and 3 months. Twenty patients underwent surgery, leading to a total 37 anesthetic procedures and 46 surgical procedures (mean 2.3 surgeries/patient); four patients had complications during surgery; these included intubation difficulties (3), SAH (1), and bleeding (1). Obstruction of the upper airways and cardiac complications led to the death of two patients, one MPS I (age 8 years and 9 months) and one MPS II (age 12 years). Regarding laboratory tests, toluidine blue staining showed false-negative results in 44% of patients, urine glycosaminoglycan (GAG) levels were increased in all patients, GAG chromatography was normal in four patients with MPS IV, and enzyme quantification defined the diagnosis and type of MPS. Radiological alterations (multiple dysostosis) were detected in all patients; 90% of patients showed echocardiographic alterations; polysomnography showed marked SAS in 27% of patients; and cranial CAT scans showed ventriculomegaly in 57% of patients. CONCLUSION: MPS patients require regular follow-up by a multidisciplinary team in order to achieve adequate management of frequent clinical complications

Dermatan sulphate (DS) is a sulphated glycosaminoglycan (GAG) that is widely distributed as proteoglycan throughout the extracellular matrix and at cell surfaces where it plays an important role in many key biological processes. The intra-cellular catabolism of DS commences with endohydrolysis of the polysaccharide chains to oligosaccharides, which are then sequentially degraded from the non-reducing terminus by lysosomal exoenzymes to monosaccharides and inorganic sulphate for transport out of the lysosome and re-utilisation by the cell. Both endo-β-N-acetylhexosaminidase (Hyal-1 hyaluronidase) and endo-β-glucuronidase activities towards DS have been proposed. The present study was undertaken to: 1) determine the substrate specificities and sub-cellular locations of these endoglycosidase activities; and 2) compare endoglycosidase activities and substrate specificities in the mucopolysaccharidoses, where a defect in one of the lysosomal exoenzymes required to degrade DS results in the lysosomal accumulation of partially degraded DS oligosaccharide fragments. To this end, a series of oligosaccharide substrates designed to represent aspects of the physiological substrate was prepared, and an assay was developed to measure endoglycosidase activities and determine their substrate specificities by quantifying specific oligosaccharide products. Assay substrates rich in glucuronic acid (GlcA) or iduronic acid (IdoA) were prepared by limited chondroitinase ABC digestion of chondroitin sulphate A and DS, respectively. The resulting tetra-to hexadecasaccharides were separated by size-exclusion chromatography and characterised by electrospray ionisation-tandem mass spectrometry (ESI-MS/MS). These substrates, which were not susceptible to degradation by lysosomal exoenzymes, were then incubated with Chinese hamster ovary (CHO)-K1 cell homogenate (source of endoglycosidase activity), and the oligosaccharide products generated from the non-reducing end of the substrate were measured by ESI-MS/MS. Endo-β-N-acetylhexosaminidase and endohexuronidase activities were detected towards the oligosaccharide substrates, with both activities preferentially degrading the GlcA-rich substrates and only minor activity observed towards IdoA-rich substrate. The endo-β-N-acetylhexosaminidase activity had a minimum-sized substrate requirement of a hexasaccharide and was observed to sequentially remove tetrasaccharides from the non-reducing end of oligosaccharides, whereas the endohexuronidase activity had a minimum substrate of an octasaccharide, acted randomly and was comparatively low. The activities displayed the same acidic pH optimum and responded in the same manner to changes in buffer composition and substrate concentration, and to the presence of divalent cations, NaCl, detergent and protease inhibitors. Both activities were modestly affected by the hyaluronidase inhibitor, apigenin. Percoll density gradient sub-cellular fractionation confirmed that the activities were primarily in the lysosomes and late endosomes. The endo-β-N-acetylhexosaminidase and endohexuronidase activities detected here in CHO-K1 cells are consistent with the Hyal-1 and endo-β-glucuronidase enzymes described previously. These data suggest that Hyal-1 and endo-β-glucuronidase are predominantly lysosomal enzymes that act in concert to degrade the low-sulphate, GlcA-rich domains of DS, but are less active towards the highly sulphated regions containing IdoA. To test the hypothesis that endoglycosidase activities are altered in the mucopolysaccharidoses, an attempt was made to compare Hyal-1- and endo-β-glucuronidase-like activities and their substrate specificities in mucopolysaccharidosis (MPS)-affected and unaffected control skin fibroblasts. However, no activity was detected towards octa- to hexadecasaccharide substrates in control fibroblast homogenates, and in homogenates of MPS fibroblasts deficient in the lysosomal exoenzymes α-L-iduronidase and N-acetylgalactosamine-4-sulphatase, despite the fact that: 1) what appear to be the products of Hyal-1 and endo-β-glucuronidase activities towards endogenous DS could be detected in the lysosomes of the MPS cells by sub-cellular fractionation; and 2) the ESI-MS/MS assay was demonstrated sensitive enough to detect endoglycosidase activities in homogenates of a number of different mouse tissues (including whole skin). We hypothesise that this absence of detectable endoglycosidase activity in skin fibroblasts results from enzyme non-recognition of the exogenous assay substrates tested, and hence that these cells contain heretofore undescribed Hyal-1 and endo-β-glucuronidase isoforms with unique substrate specificities. In conclusion, the development of an ESI-MS/MS assay to measure the products of endoglycosidase activities has enabled the characterisation of these activities towards DS. This strategy may be useful for the future study of endoglycosidase activities towards a variety of other GAGs such as heparan sulphate, where particular oligosaccharide structures have been shown to possess unique biological activities. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1374435 / Thesis (Ph.D.) - University of Adelaide, School of Paediatrics and Reproductive Health, 2009