heparitin-sulfate and Mucopolysaccharidosis-I

Mucopolysaccharidosis type I (MPS I) is a rare monogenic disease in which glycosaminoglycans' abnormal metabolism leads to the storage of heparan sulfate and dermatan sulfate in various tissues. It causes its damage and impairment. Patients with the severe form of MPS I usually do not live up to the age of ten. Currently, the therapy is based on multidisciplinary care and enzyme replacement therapy or hematopoietic stem cell transplantation. Applying gene therapy might benefit the MPS I patients because it overcomes the typical limitations of standard treatments. Nanoparticles, including nanoemulsions, are used more and more in medicine to deliver a particular drug to the target cells. It allows for creating a specific, efficient therapy method in MPS I and other lysosomal storage disorders. This article briefly presents the basics of nanoemulsions and discusses the current state of knowledge about their usage in mucopolysaccharidosis type I.

Topics: Enzyme Replacement Therapy; Genetic Therapy; Glycosaminoglycans; Heparitin Sulfate; Humans; Mucopolysaccharidosis I; Mucopolysaccharidosis II

Heparan sulfate (HS) is an essential glycosaminoglycan (GAG) as a component of proteoglycans, which are present on the cell surface and in the extracellular matrix. HS-containing proteoglycans not only function as structural constituents of the basal lamina but also play versatile roles in various physiological processes, including cell signaling and organ development. Thus, inherited mutations of genes associated with the biosynthesis or degradation of HS can cause various diseases, particularly those involving the bones and central nervous system (CNS). Mucopolysaccharidoses (MPSs) are a group of lysosomal storage disorders involving GAG accumulation throughout the body caused by a deficiency of GAG-degrading enzymes. GAGs are stored differently in different types of MPSs. Particularly, HS deposition is observed in patients with MPS types I, II, III, and VII, all which involve progressive neuropathy with multiple CNS system symptoms. While therapies are available for certain symptoms in some types of MPSs, significant unmet medical needs remain, such as neurocognitive impairment. This review presents recent knowledge on the pathophysiological roles of HS focusing on the pathogenesis of MPSs. We also discuss the possible use and significance of HS as a biomarker for disease severity and therapeutic response in MPSs.

Topics: Biomarkers; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Mucopolysaccharidoses; Mucopolysaccharidosis I

Topics: Animals; Cytokines; Glycosaminoglycans; Heparitin Sulfate; Humans; Mucopolysaccharidosis I; Protein Binding

Synthesis of a series of l-iduronic acid (IdoA)- and imino-IdoA-typed C-glycosides for modulating α-l-iduronidase (IDUA) activity is described. In an enzyme inhibition study, IdoA-typed C-glycosides were more potent than imino-IdoA analogs, with the most potent IdoA-typed C-glycoside 27c showing an IC

Topics: Fibroblasts; Glycosides; Heparitin Sulfate; Humans; Iduronidase; Mucopolysaccharidosis I

Mucopolysaccharidoses (MPSs) are complex lysosomal storage disorders that result in the accumulation of glycosaminoglycans (GAGs) in urine, blood, and tissues. Lysosomal enzymes responsible for GAG degradation are defective in MPSs. GAGs including chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), and keratan sulfate (KS) are disease-specific biomarkers for MPSs. This article describes a stable isotope dilution-tandem mass spectrometric method for quantifying CS, DS, and HS in urine samples. The GAGs are methanolyzed to uronic or iduronic acid-N-acetylhexosamine or iduronic acid-N-sulfo-glucosamine dimers and mixed with internal standards derived from deuteriomethanolysis of GAG standards. Specific dimers derived from HS, DS, and CS are separated by ultra-performance liquid chromatography (UPLC) and analyzed by electrospray ionization tandem mass spectrometry (MS/MS) using selected reaction monitoring for each targeted GAG product and its corresponding internal standard. This UPLC-MS/MS GAG assay is useful for identifying patients with MPS types I, II, III, VI, and VII. © 2023 Wiley Periodicals LLC. Basic Protocol: Urinary GAG analysis by ESI-MS/MS Support Protocol 1: Prepare calibration samples Support Protocol 2: Preparation of stable isotope-labeled internal standards Support Protocol 3: Preparation of quality controls for GAG analysis in urine Support Protocol 4: Optimization of the methanolysis time Support Protocol 5: Measurement of the concentration of methanolic HCl.

Topics: Chondroitin Sulfates; Chromatography, Liquid; Dermatan Sulfate; Glycosaminoglycans; Heparitin Sulfate; Humans; Iduronic Acid; Isotopes; Mucopolysaccharidoses; Mucopolysaccharidosis I; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry

Mucopolysaccharidosis type I (MPS I), a lysosomal storage disease caused by a deficiency of α-L-iduronidase, leads to storage of the glycosaminoglycans, dermatan sulfate and heparan sulfate. Available therapies include enzyme replacement and hematopoietic stem cell transplantation. In the last two decades, newborn screening (NBS) has focused on early identification of the disorder, allowing early intervention and avoiding irreversible manifestations. Techniques developed and optimized for MPS I NBS include tandem mass-spectrometry, digital microfluidics, and glycosaminoglycan quantification. Several pilot studies have been conducted and screening programs have been implemented worldwide. NBS for MPS I has been established in Taiwan, the United States, Brazil, Mexico, and several European countries. All these programs measure α-L-iduronidase enzyme activity in dried blood spots, although there are differences in the analytical strategies employed. Screening algorithms based on published studies are discussed. However, some limitations remain: one is the high rate of false-positive results due to frequent pseudodeficiency alleles, which has been partially solved using post-analytical tools and second-tier tests; another involves the management of infants with late-onset forms or variants of uncertain significance. Nonetheless, the risk-benefit ratio is favorable. Furthermore, long-term follow-up of patients detected by neonatal screening will improve our knowledge of the natural history of the disease and inform better management.

Topics: Heparitin Sulfate; Humans; Iduronidase; Infant; Infant, Newborn; Mucopolysaccharidosis I; Neonatal Screening; Tandem Mass Spectrometry

Mucopolysaccharidosis type I is a rare autosomal recessive genetic disease caused by deficient activity of α-L-iduronidase. As a consequence of low or absent activity of this enzyme, glycosaminoglycans accumulate in the lysosomal compartments of multiple cell types throughout the body. Mucopolysaccharidosis type I has been classified into 3 clinical subtypes, ranging from a severe Hurler form to the more attenuated Hurler-Scheie and Scheie phenotypes. Over 200 gene variants causing the various forms of mucopolysaccharidosis type I have been reported. DNA isolated from dried blood spot was used to sequencing of all exons of the IDUA gene from a patient with a clinical phenotype of severe mucopolysaccharidosis type I syndrome. Enzyme activity of α-L-iduronidase was quantified by fluorimetric assay. Additionally, a molecular dynamics simulation approach was used to determine the effect of the Ser633Trp mutation on the structure and dynamics of the α-L-iduronidase. The DNA sequencing analysis and enzymatic activity shows a c.1898C>G mutation associated a patient with a homozygous state and α-L-iduronidase activity of 0.24 μmol/L/h, respectively. The molecular dynamics simulation analysis shows that the p.Ser633Trp mutation on the α-L-iduronidase affect significant the temporal and spatial properties of the different structural loops, the N-glycan attached to Asn372 and amino acid residues around the catalytic site of this enzyme. Low enzymatic activity observed for p.Ser633Trp variant of the α-L-iduronidase seems to lead to severe mucopolysaccharidosis type I phenotype, possibly associated with a perturbation of the structural dynamics in regions of the enzyme close to the active site.

Topics: Abnormalities, Multiple; Catalytic Domain; Crystallography, X-Ray; Dermatan Sulfate; Enzyme Replacement Therapy; Gene Expression; Heparitin Sulfate; Humans; Iduronidase; Infant; Male; Molecular Dynamics Simulation; Mucopolysaccharidosis I; Point Mutation; Principal Component Analysis; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Substrate Specificity

To validate a liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the measurement of glycosaminoglycans (GAGs) in plasma and serum. To establish plasma, cerebrospinal fluid (CSF) and urine reference intervals. To compare GAGs in serum with that in urine and CSF from patients with MPS I.. Dermatan sulfate (DS), heparan sulfate (HS), and chondroitin sulfate (CS) in serum/plasma, urine and CSF were methanolysed into dimers and analyzed using pseudo isotope dilution UPLC-MS/MS assay. Serum, CSF and urine DS and HS were quantified for 11 patients with mucopolysaccharidosis (MPS) type I before and after treatment with Aldurazyme® (laronidase) enzyme replacement therapy (ERT).. The method showed acceptable imprecision and recovery for the quantification of serum/plasma CS, DS, and HS. The serum, urine, and CSF DS and HS concentrations were reduced after 26 weeks of ERT in 4 previously untreated patients. Serum DS and HS concentrations normalized in some patients, and were mildly elevated in others after ERT. In contrast, urine and CSF DS and HS values remained elevated above the reference ranges. Compared with serum GAGs, urine and CSF DS and HS were more sensitive biomarkers for monitoring the ERT treatment of patients with MPS I.

Topics: Chromatography, Liquid; Dermatan Sulfate; Enzyme Replacement Therapy; Glycosaminoglycans; Heparitin Sulfate; Humans; Mucopolysaccharidosis I; Tandem Mass Spectrometry

To evaluate the performance of a 2-tiered newborn screening method for mucopolysaccharidosis type I (MPS I) in North Carolina.. The screening algorithm included a flow injection analysis-tandem mass spectrometry assay as a first-tier screening method to measure α-L-iduronidase (IDUA) enzyme activity and Sanger sequencing of the IDUA gene on dried blood spots as a second-tier assay. The screening algorithm was revised to incorporate the Collaborative Laboratory Integrated Reports, an analytical interpretive tool, to reduce the false-positive rate. A medical history, physical examination, IDUA activity, and urinary glycosaminoglycan (GAG) analysis were obtained on all screen-positive infants.. A total of 62 734 specimens were screened with 54 screen-positive samples using a cut-off of 15% of daily mean IDUA activity. The implementation of Collaborative Laboratory Integrated Reports reduced the number of specimens that screened positive to 19 infants. Of the infants identified as screen-positive, 1 had elevated urinary GAGs and a homozygous pathogenic variant associated with the severe form of MPS I. All other screen-positive infants had normal urinary GAG analysis; 13 newborns had pseudodeficiency alleles, 3 newborns had variants of unknown significance, and 2 had heterozygous pathogenic variants.. An infant with severe MPS I was identified and referred for a hematopoietic stem cell transplant. Newborn IDUA enzyme deficiency is common in North Carolina, but most are due to pseudodeficiency alleles in infants with normal urinary GAG analysis and no evidence of disease. The pilot study confirmed the need for second-tier testing to reduce the follow-up burden.

Topics: Algorithms; Dermatan Sulfate; Genetic Testing; Genetic Variation; Glycosaminoglycans; Heparitin Sulfate; Humans; Iduronidase; Infant, Newborn; Mucopolysaccharidosis I; Neonatal Screening; North Carolina; Referral and Consultation; Sequence Analysis; Tandem Mass Spectrometry

Certain recessively inherited diseases result from an enzyme deficiency within lysosomes. In mucopolysaccharidoses (MPS), a defect in glycosaminoglycan (GAG) degradation leads to GAG accumulation followed by progressive organ and multiple system dysfunctions. Current methods of GAG analysis used to diagnose and monitor the diseases lack sensitivity and throughput. Here we report a LC-MS method with accurate metabolite mass analysis for identifying and quantifying biomarkers for MPS type I without the need for extensive sample preparation. The method revealed 225 LC-MS features that were >1000-fold enriched in urine, plasma and tissue extracts from untreated MPS I mice compared to MPS I mice treated with iduronidase to correct the disorder. Levels of several trisaccharides were elevated >10000-fold. To validate the clinical relevance of our method, we confirmed the presence of these biomarkers in urine, plasma and cerebrospinal fluid from MPS I patients and assessed changes in their levels after treatment.

Topics: Animals; Biomarkers; Chromatography, Liquid; Disease Models, Animal; Female; Glycosaminoglycans; Heparitin Sulfate; Humans; Iduronidase; Male; Mice; Mucopolysaccharidosis I; Trisaccharides

Residual disease, primarily involving musculoskeletal tissue, is a common problem in patients with neuronopathic mucopolysaccharidosis type I (MPS I, Hurler or severe Hurler-Scheie phenotype) after a successful hematopoietic cell transplantation (HCT). The concentration of the GAG derived biomarkers heparan sulfate (HS) and dermatan sulfate (DS), may reflect residual disease and is used for monitoring biochemical response to therapies. This study investigates the response of HS and DS in blood and urine to HCT in MPS I patients.. In 143 blood- and urine samples of 17 neuronophatic MPS I patients, collected prior and post successful HCT, the concentration of the disaccharides derived after full enzymatic digestion of HS and DS were analyzed by multiplex liquid chromatography tandem-mass spectrometry (LC-MS/MS).. Median follow up after HCT was 2.4years (range 0-11years). HCT led to a rapid decrease of both HS and DS. However, only 38% of the patients reached normal HS levels in blood and even less patients (6%) reached normal DS levels. In none of the patients normalization of HS or DS was observed in urine.. Biomarker response after HCT is incomplete, which may reflect residual disease activity. Novel therapeutic strategies should aim for full metabolic correction to minimize clinical manifestations.

Topics: Biomarkers; Cell Transplantation; Child; Child, Preschool; Chromatography, Liquid; Dermatan Sulfate; Female; Hematopoietic Stem Cell Transplantation; Heparitin Sulfate; Humans; Infant; Infant, Newborn; Male; Mucopolysaccharidosis I; Tandem Mass Spectrometry

Application of metabolic phenotyping could expand the pathophysiological knowledge of mucopolysaccharidoses (MPS) and may reveal the comprehensive metabolic impairments in MPS. However, few studies applied this approach to MPS.. We applied targeted and untargeted metabolic profiling in urine samples obtained from a French cohort comprising 19 MPS I and 15 MPS I treated patients along with 66 controls. For that purpose, we used ultra-high-performance liquid chromatography combined with ion mobility and high-resolution mass spectrometry following a protocol designed for large-scale metabolomics studies regarding robustness and reproducibility. Furthermore, 24 amino acids have been quantified using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Keratan sulfate, Heparan sulfate and Dermatan sulfate concentrations have also been measured using an LC-MS/MS method. Univariate and multivariate data analyses have been used to select discriminant metabolites. The mummichog algorithm has been used for pathway analysis.. The studied groups yielded distinct biochemical phenotypes using multivariate data analysis. Univariate statistics also revealed metabolites that differentiated the groups. Specifically, metabolites related to the amino acid metabolism. Pathway analysis revealed that several major amino acid pathways were dysregulated in MPS. Comparison of targeted and untargeted metabolomics data with in silico results yielded arginine, proline and glutathione metabolisms being the most affected.. This study is one of the first metabolic phenotyping studies of MPS I. The findings might help to generate new hypotheses about MPS pathophysiology and to develop further targeted studies of a smaller number of potentially key metabolites.

Topics: Adolescent; Adult; Aged; Algorithms; Amino Acids; Arginine; Case-Control Studies; Child; Child, Preschool; Chromatography, High Pressure Liquid; Dermatan Sulfate; Female; Glutathione; Heparitin Sulfate; Humans; Infant; Keratan Sulfate; Male; Mass Spectrometry; Metabolome; Metabolomics; Middle Aged; Mucopolysaccharidosis I; Multivariate Analysis; Phenotype; Proline

Mucopolysaccharidosis (MPS) type-IH is a lysosomal storage disease that results from mutations in the IDUA gene causing the accumulation of glycosaminoglycans (GAGs). Historically, children with the severe phenotype, MPS-IH (Hurler syndrome) develop progressive neurodegeneration with death in the first decade due to cardio-pulmonary complications. New data suggest that inflammation may play a role in MPS pathophysiology. To date there is almost no information on the pathophysiologic changes within the cerebral spinal fluid (CSF) of these patients. We evaluated the CSF of 25 consecutive patients with MPS-IH. While CSF glucose and total protein were within the normal range, we found a significantly mean elevated CSF opening pressure at 24 cm H

Topics: Adaptor Proteins, Signal Transducing; Adolescent; Biomarkers; Cerebrospinal Fluid Pressure; Chemokine CCL2; Chemokine CXCL12; Child; Child, Preschool; Female; Gene Expression; Glucose; Heparitin Sulfate; Humans; Iduronidase; Infant; Interleukin-8; Male; Mucopolysaccharidosis I; Mutation; Nerve Tissue Proteins

Antibody formation can interfere with effects of enzyme replacement therapy (ERT) in lysosomal storage diseases. Biomarkers are used as surrogate marker for disease burden in MPS I, but large systematic studies evaluating the response of biomarkers to ERT are lacking. We, for the first time, investigated the response of a large panel of biomarkers to long term ERT in MPS I patients and correlate these responses with antibody formation and antibody mediated cellular uptake inhibition.. A total of 428 blood and urine samples were collected during long-term ERT in 24 MPS I patients and an extensive set of biomarkers was analyzed, including heparan sulfate (HS) and dermatan sulfate (DS) derived disaccharides; total urinary GAGs (DMBu); urinary DS:CS ratio and serum heparin co-factor II thrombin levels (HCII-T). IgG antibody titers and the effect of antibodies on cellular uptake of the enzyme were determined for 23 patients.. Median follow-up was 2.3 years. In blood, HS reached normal levels more frequently than DS (50% vs 12.5%, p=0.001), though normalization could take several years. DMBu normalized more rapidly than disaccharide levels in urine (p=0.02). Nineteen patients (83%) developed high antibody titers. Significant antibody-mediated inhibition of enzyme uptake was observed in 8 patients (35%), and this correlated strongly with a poorer biomarker response for HS and DS in blood and urine as well as for DMBu, DS:CS-ratio and HCII-T (all p<0.006).. This study shows that, despite a response of all studied biomarkers to initiation of ERT, some biomarkers were less responsive than others, suggesting residual disease activity. In addition, the correlation of cellular uptake inhibitory antibodies with a decreased biomarker response demonstrates a functional role of these antibodies which may have important clinical consequences.

Topics: Adolescent; Adult; Biomarkers; Child; Child, Preschool; Dermatan Sulfate; Disaccharides; Enzyme Replacement Therapy; Female; Follow-Up Studies; Heparin Cofactor II; Heparitin Sulfate; Humans; Iduronidase; Immunoglobulin G; Infant; Infant, Newborn; Male; Mucopolysaccharidosis I; Recombinant Proteins; Thrombin; Young Adult

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disorder due to deficiency of alpha iduronidase (IDUA). Progressive storage of dermatan and heparan sulfate throughout the body lead to a multiorgan presentation including short stature, dysostosis multiplex, corneal clouding, hearing loss, coarse facies, hepatosplenomegaly, and intellectual disability. Diagnosis of MPS I is based on IDUA enzyme analysis in leukocytes or dried blood spots (DBS) followed by molecular confirmation of the IDUA gene mutations in individuals with low enzyme activity. The advent of mass spectrometry methods for enzyme analysis in DBS has enabled high-throughput screening for MPS I in symptomatic individuals and newborn infants. The following unit provides the detailed analytical protocol for measurement of IDUA activity in DBS using tandem mass spectrometry.

Topics: Dermatan Sulfate; Dried Blood Spot Testing; Enzyme Replacement Therapy; Gene Expression; Heparitin Sulfate; Humans; Iduronidase; Infant; Infant, Newborn; Leukocytes, Mononuclear; Mucopolysaccharidosis I; Mutation; Neonatal Screening; Tandem Mass Spectrometry

Mucopolysaccharidoses (MPS) are characterized by mental retardation constantly present in the severe forms of Hurler (MPS I), Hunter (MPS II) and Sanfilippo (MPS III) diseases. On the contrary, mental retardation is absent in Morquio (MPS IV) and Maroteaux-Lamy (MPS VI) diseases and absent or only minimal in the attenuated forms of MPS I, II and III. Considering that MPS patients affected by mental disease accumulate heparan sulfate (HS) due to specific enzymatic defects, we hypothesized a possible correlation between urinary HS-derived glucosamine (GlcN) accumulated in tissues and excreted in biological fluids and mental retardation. 83 healthy subjects were found to excrete HS in the form of fragments due to the activity of catabolic enzymes that are absent or impaired in MPS patients. On the contrary, urinary HS in 44 patients was observed to be composed of high molecular weight polymer and fragments of various lengths depending on MPS types. On this basis we correlated mental retardation with GlcN belonging to high and low molecular weight HS. We demonstrate a positive relationship between the accumulation of high molecular weight HS and mental retardation in MPS severe compared to attenuated forms. This is also supported by the consideration that accumulation of other GAGs different from HS, as in MPS IV and MPS VI, and low molecular weight HS fragments do not impact on central nervous system disease.

Topics: Adolescent; Adult; Child; Child, Preschool; Female; Glucosamine; Heparitin Sulfate; Humans; Infant; Intellectual Disability; Male; Molecular Weight; Mucopolysaccharidoses; Mucopolysaccharidosis I; Mucopolysaccharidosis III; Reference Values; Young Adult

Mucopolysaccharidosis I Hurler (MPSI-H) is a pediatric lysosomal storage disease caused by genetic deficiencies in IDUA, coding for α-l-iduronidase. Idua(-/-) mice share similar clinical pathology with patients, including the accumulation of the undegraded glycosaminoglycans (GAGs) heparan sulfate (HS), and dermatan sulfate (DS), progressive neurodegeneration, and dysostosis multiplex. Hematopoietic stem cell transplantation (HSCT) is the most effective treatment for Hurler patients, but reduced intensity conditioning is a risk factor in transplantation, suggesting an underlying defect in hematopoietic cell engraftment. HS is a co-receptor in the CXCL12/CXCR4 axis of hematopoietic stem and progenitor cell (HSPC) migration to the bone marrow (BM), but the effect of HS alterations on HSPC migration, or the functional role of HS in MPSI-H are unknown. We demonstrate defective WT HSPC engraftment and migration in Idua(-/-) recipient BM, particularly under reduced intensity conditioning. Both intra- but especially extracellular Idua(-/-) BM HS was significantly increased and abnormally sulfated. Soluble heparinase-sensitive GAGs from Idua(-/-) BM and specifically 2-O-sulfated HS, elevated in Idua(-/-) BM, both inhibited CXCL12-mediated WT HSPC transwell migration, while DS had no effect. Thus we have shown that excess overly sulfated extracellular HS binds, and sequesters CXCL12, limiting hematopoietic migration and providing a potential mechanism for the limited scope of HSCT in Hurler disease.

Topics: Animals; Bone Marrow; Cell Movement; Chemokine CXCL12; Graft Survival; Hematopoiesis; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells; Heparitin Sulfate; Humans; Mice, Inbred C57BL; Mice, Knockout; Mucopolysaccharidosis I; Stem Cell Niche

N-glycosylation is a major posttranslational modification that endows proteins with various functions. It is established that N-glycans are essential for the correct folding and stability of some enzymes; however, the actual effects of N-glycans on their activities are poorly understood. Here, we show that human α-l-iduronidase (hIDUA), of which a dysfunction causes accumulation of dermatan/heparan sulfate leading to mucopolysaccharidosis type I, uses its own N-glycan as a substrate binding and catalytic module. Structural analysis revealed that the mannose residue of the N-glycan attached to N372 constituted a part of the substrate-binding pocket and interacted directly with a substrate. A deglycosylation study showed that enzyme activity was highly correlated with the N-glycan attached to N372. The kinetics of native and deglycosylated hIDUA suggested that the N-glycan is also involved in catalytic processes. Our study demonstrates a previously unrecognized function of N-glycans.

Topics: Amino Acid Sequence; Binding Sites; Biocatalysis; Circular Dichroism; Crystallography, X-Ray; Dermatan Sulfate; Electrophoresis, Polyacrylamide Gel; Heparitin Sulfate; Humans; Iduronidase; Kinetics; Mannose; Models, Molecular; Molecular Sequence Data; Mucopolysaccharidosis I; Mutation; Polysaccharides; Protein Binding; Protein Structure, Tertiary; Sequence Homology, Amino Acid; Substrate Specificity

Mucopolysaccharidosis type I (MPS I) results in a defective breakdown of the glycosaminoglycans (GAGs) heparan sulfate and dermatan sulfate, which leads to a progressive disease. Enzyme replacement therapy (ERT) results in clearance of these GAGs from a range of tissues and can significantly ameliorate several symptoms. The biochemical efficacy of ERT is generally assessed by the determination of the total urinary excretion of GAGs. However, this has limitations. We studied the concentrations of heparan sulfate and dermatan sulfate derived disaccharides (HS and DS, respectively) in the plasma and urine of seven patients and compared these levels with total urinary GAGs (uGAGs) levels.. Plasma and urine samples were collected at different time points relative to the weekly ERT for three non-consecutive weeks in seven MPS I patients who had been treated with ERT for at least 2.5 years. Heparan and dermatan sulfate in plasma and urine were enzymatically digested into disaccharides, and HS and DS levels were determined by HPLC-MS/MS analysis. uGAGs were measured by the DMB test.. The levels of HS and DS were markedly decreased compared with the levels before the initiation of ERT. However, the concentrations of DS in plasma and of both HS and DS in urine remained significantly elevated in all studied patients, while in six patients the level of total uGAGs had normalized. The concentrations of plasma and urinary HS during the weekly ERT followed a U-shaped curve. However, the effect size is small. The concentrations of plasma and urinary DS and uGAGs appeared to be in a steady state.. HS and DS are sensitive biomarkers for monitoring the biochemical treatment efficacy of ERT and remain elevated despite long-term treatment. This finding may be related to the labeled dose or antibody status of the patient. The timing of the sample collection is not relevant, at least at the current dose of 100 IU/kg/weekly.

Topics: Adolescent; Adult; Aged; Biomarkers; Child; Child, Preschool; Dermatan Sulfate; Disaccharides; Enzyme Replacement Therapy; Female; Glycosaminoglycans; Heparitin Sulfate; Humans; Infant; Infant, Newborn; Male; Middle Aged; Mucopolysaccharidosis I; Young Adult

Mucopolysaccharide diseases (MPS) are caused by deficiency of glycosaminoglycan (GAG) degrading enzymes, leading to GAG accumulation. Neurodegenerative MPS diseases exhibit cognitive decline, behavioural problems and shortened lifespan. We have characterised neuropathological changes in mouse models of MPSI, IIIA and IIIB to provide a better understanding of these events.Wild-type (WT), MPSI, IIIA and IIIB mouse brains were analysed at 4 and 9 months of age. Quantitative immunohistochemistry showed significantly increased lysosomal compartment, GM2 ganglioside storage, neuroinflammation, decreased and mislocalised synaptic vesicle associated membrane protein, (VAMP2), and decreased post-synaptic protein, Homer-1, in layers II/III-VI of the primary motor, somatosensory and parietal cortex. Total heparan sulphate (HS), was significantly elevated, and abnormally N-, 6-O and 2-O sulphated compared to WT, potentially altering HS-dependent cellular functions. Neuroinflammation was confirmed by significantly increased MCP-1, MIP-1α, IL-1α, using cytometric bead arrays. An overall genotype effect was seen in all parameters tested except for synaptophysin staining, neuronal cell number and cortical thickness which were not significantly different from WT. MPSIIIA and IIIB showed significantly more pronounced pathology than MPSI in lysosomal storage, astrocytosis, microgliosis and the percentage of 2-O sulphation of HS. We also observed significant time progression of all genotypes from 4-9 months in lysosomal storage, astrocytosis, microgliosis and synaptic disorganisation but not GM2 gangliosidosis. Individual genotype*time differences were disparate, with significant progression from 4 to 9 months only seen for MPSIIIB with lysosomal storage, MPSI with astrocytocis and MPSIIIA with microgliosis as well as neuronal loss. Transmission electron microscopy of MPS brains revealed dystrophic axons, axonal storage, and extensive lipid and lysosomal storage. These data lend novel insight to MPS neuropathology, suggesting that MPSIIIA and IIIB have more pronounced neuropathology than MPSI, yet all are still progressive, at least in some aspects of neuropathology, from 4-9 months.

Topics: Animals; Carrier Proteins; Cytokines; Disease Models, Animal; Disease Progression; Female; G(M2) Ganglioside; Glycosaminoglycans; Heparitin Sulfate; Homer Scaffolding Proteins; Immunohistochemistry; Lysosomes; Male; Mice; Mucopolysaccharidosis I; Mucopolysaccharidosis III; Neurons; Parietal Lobe; Somatosensory Cortex; Vesicle-Associated Membrane Protein 2

Mucopolysaccharidoses (MPSs) are a group of lysosomal storage disorders (LSDs) caused by a defect in the degradation of glycosaminoglycans (GAGs). The accumulation of GAGs in MPS patients results in extensive, severe and progressive disease. Disease modifying therapy is available for three of the MPSs and is being developed for the other types. Early initiation of treatment, before the onset of irreversible tissue damage, clearly provides a favorable disease outcome. However, early diagnosis is difficult due to the rarity of these disorders in combination with the wide variety of clinical symptoms. Newborn screening (NBS) is probably the optimal approach, and several screening techniques for different MPSs have been studied. Here we describe a relatively simple and sensitive method to measure levels of dermatan and heparan sulfate derived disaccharides in dried blood spots (DBS) with HPLC-MS/MS, and show that this reliably separates MPS I, II and MPS III newborns from controls and heterozygotes.. Newborn DBS of 11 MPS I, 1 MPS II, and 6 MPS III patients, with phenotypes ranging from severe to relatively attenuated, were collected and levels of dermatan and heparan sulfate derived disaccharides in these DBS were compared with levels in DBS of newborn MPS I and MPS III heterozygotes and controls.. The levels of dermatan and heparan sulfate derived disaccharides were clearly elevated in all newborn DBS of MPS I, II and III patients when compared to controls. In contrast, DBS of MPS I and III heterozygotes showed similar disaccharide levels when compared to control DBS.. Our study demonstrates that measurement of heparan and dermatan sulfate derived disaccharides in DBS may be suitable for NBS for MPS I, II and MPS III. We hypothesize that this same approach will also detect MPS VI, and VII patients, as heparan sulfate and/or dermatan sulfate is also the primary storage products in these disorders.

Topics: Biomarkers; Child; Child, Preschool; Dermatan Sulfate; Disaccharides; Heparitin Sulfate; Humans; Infant; Infant, Newborn; Mucopolysaccharidoses; Mucopolysaccharidosis I; Mucopolysaccharidosis II; Mucopolysaccharidosis III; Neonatal Screening; Reproducibility of Results; Tandem Mass Spectrometry

Mucopolysaccharidoses (MPSs) are complex storage disorders caused by specific lysosomal enzyme deficiencies, resulting in the accumulation of glycosaminoglycans (GAGs) in urine, plasma, as well as in various tissues. We devised and validated a straightforward, but accurate and precise tandem mass spectrometry methodology coupled to high performance liquid chromatography (LC-MS/MS) for the quantification of GAGs in urine. The method is applicable to the investigation of patients with MPS I, II, and VI, by quantifying dermatan sulfate (DS) and heparan sulfate (HS) in urine. We analyzed urine samples from 28 MPS patients, aged 1 to 42 years, and 55 control subjects (41 days to 18 years old). Levels of DS and HS in urine from healthy controls of all ages were below the limit of quantification. The levels of DS and HS in urine from 6 treated patients with MPS I were lower than in 6 untreated patients in DS (0.7-45 vs 9.3-177 mg/mmol creat) and HS (0-123 mg/mmol creatinine vs 38-418 mg/mmol creatinine); similar results were obtained for 9 patients with MPS II and 7 patients with MPS VI. Analyses were performed on as little as 250 μL of urine. Methanolysis took 75 min per sample; the total analysis run time for each LC-MS/MS injection was 8 min. Results indicate that the method is applicable to a wide variety of situations in which high accuracy and precision are required, including the evaluation of the effectiveness of existing and emerging treatments.

Topics: Adolescent; Adult; Biomarkers; Case-Control Studies; Child; Child, Preschool; Chromatography, Liquid; Creatinine; Dermatan Sulfate; Enzyme Replacement Therapy; Female; Glycosaminoglycans; Heparitin Sulfate; Humans; Infant; Male; Mucopolysaccharidosis I; Mucopolysaccharidosis II; Mucopolysaccharidosis VI; Reference Values; Tandem Mass Spectrometry; Young Adult

New therapies for the treatment of mucopolysaccharidoses that target the brain, including intrathecal enzyme replacement, are being explored. Quantitative analysis of the glycosaminoglycans (GAGs) that accumulate in these disorders is required to assess the disease burden and monitor the effect of therapy in affected patients. Because current methods lack the required limit of quantification and specificity to analyze GAGs in small volumes of cerebrospinal fluid (CSF), we developed a method based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS).. Samples of CSF (25 μL) were evaporated to dryness and subjected to methanolysis. The GAGs were degraded to uronic acid-N-acetylhexosamine dimers and mixed with internal standards derived from deuteriomethanolysis of GAG standards. Specific dimers derived from heparan, dermatan and chondroitin sulfates (HS, DS and CS) were separated by UPLC and analyzed by electrospray ionization MS/MS using selected reaction monitoring for each targeted GAG product and its corresponding internal standard.. CSF from control pediatric subjects (n = 22) contained <0.38 mg/L HS, 0.26 mg/L DS, and 2.8 mg/L CS, whereas CSF from patients with Hurler syndrome (n = 7) contained concentrations of DS and HS that were at least 6-fold greater than the upper control limits. These concentrations were reduced by 17.5% to 82.5% after allogeneic transplantation and treatment with intrathecal and intravenous enzyme replacement therapy.. The method described here has potential value in monitoring patients with mucopolysaccharidoses receiving treatment targeted to the brain.

Topics: Biomarkers; Calibration; Child; Chondroitin Sulfates; Chromatography, High Pressure Liquid; Dermatan Sulfate; Deuterium; Dimerization; Enzyme Replacement Therapy; Hematopoietic Stem Cell Transplantation; Heparitin Sulfate; Hexosamines; Humans; Indicator Dilution Techniques; Injections, Intravenous; Injections, Spinal; Mucopolysaccharidosis I; Reference Standards; Reference Values; Tandem Mass Spectrometry; Uronic Acids

Mucopolysaccharide (MPS) diseases are characterized by accumulation of glycosaminoglycans (GAGs) due to deficiencies in lysosomal enzymes responsible for GAG breakdown. Using a murine model of MPSI Hurler (MPSIH), we have quantified the heparan sulfate (HS) accumulation resulting from α-l-iduronidase (Idua) deficiency. HS levels were significantly increased in liver and brain tissue from 12-week-old Idua(-/-) mice by 87- and 20-fold, respectively. In addition, HS chains were shown to contain significantly increased N-, 2-O-, and 6-O-sulfation. Disaccharide compositional analyses also uncovered an HS disaccharide uniquely enriched in MPSIH, representing the terminal iduronic acid residue capping the non-reducing end of the HS chain, where no further degradation can occur in the absence of Idua. Critically, we identified that excess HS, some of which is colocalized to the Golgi secretory pathway, acts as a positive regulator of HS-sulfation, increasing the N-sulfotransferase activity of HS-modifying N-deacetylase/N-sulfotransferase enzymes. This mechanism may have severe implications during disease progression but, now identified, could help direct improved therapeutic strategies.

Topics: Animals; Disease Models, Animal; Golgi Apparatus; Heparitin Sulfate; Humans; Iduronic Acid; Iduronidase; Mice; Mice, Knockout; Mucopolysaccharidosis I; Sulfotransferases

Mucopolysaccharide (MPS) diseases are lysosomal storage disorders caused by deficiencies of enzymes catabolising glycosaminoglycans (GAGs). Abnormal GAG accumulation leads to symptoms including severe progressive neurological decline, skeletal deformities, organomegally, respiratory compromise and premature death. Treatment is available for some MPS diseases; enzyme replacement therapy for MPS I, II and VI, and haematopoietic stem cell transplantation for MPS I, VI and VII. These treatments are reliant on early diagnosis of the disease and accurate monitoring of treatment outcomes. Blood enzyme levels and total urinary GAGs are commonly used biomarkers in diagnosis of MPS but are not good measures of treatment outcome. Serum heparin cofactor II-thrombin complex (HCII-T), which is a GAG regulated serpin-protease complex, has recently been identified as a promising biomarker for MPS diseases. Here we present an assessment of the HCII-T biomarker in mouse models of MPS I, IIIA and IIIB, which suggests that HCII-T is a reliable marker for MPS I when measured in serum or dried blood spots stored for over a year at 4 degrees C, but that murine MPS IIIA and IIIB cannot be reliably detected using this biomarker. We also show that HCII-T formation in vivo is dependent on the presence of excess intravenous dermatan sulphate (DS), whilst intravenous heparan sulphate (HS), does not promote complex formation effectively. This suggests that HCII-T will prove effective as a biomarker for MPS I, II, VI and VII diseases, storing dermatan sulphate but may not be as appropriate for MPS III, storing heparan sulphate. With careful sample preparation, HCII-T ELISA could prove to be a useful biomarker for both newborn screening and measurement of treatment outcomes in selected MPS diseases.

Topics: Animals; Biomarkers; Blood Specimen Collection; Dermatan Sulfate; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Heparin Cofactor II; Heparitin Sulfate; Humans; Mice; Mice, Inbred C57BL; Mucopolysaccharidosis I; Mucopolysaccharidosis III; Thrombin

Mucopolysaccharidosis I (MPS I) is an autosomal recessive disorder caused by deficiency of alpha-L-iduronidase leading to accumulation of its catabolic substrates, dermatan sulfate (DS) and heparan sulfate (HS), in lysosomes. This results in progressive multiorgan dysfunction and death in early childhood. The recent success of enzyme replacement therapy (ERT) for MPS I highlights the need for biomarkers that reflect response to such therapy. To determine which biochemical markers are better, we determined serum and urine DS and HS levels by liquid chromatography tandem mass spectrometry in ERT-treated MPS I patients. The group included one Hurler, 11 Hurler/Scheie, and two Scheie patients. Seven patients were treated from week 1, whereas the other seven were treated from week 26. Serum and urine DS (DeltaDi-4S/6S) and HS (DeltaDiHS-0S, DeltaDiHS-NS) were measured at baseline, week 26, and week 72. Serum DeltaDi-4S/6S, DeltaDiHS-0S, and DeltaDiHS-NS levels decreased by 72%, 56%, and 56%, respectively, from baseline at week 72. Urinary glycosaminoglycan level decreased by 61.2%, whereas urine DeltaDi-4S/6S, DeltaDiHS-0S, and DeltaDiHS-NS decreased by 66.8%, 71.8%, and 71%, respectively. Regardless of age and clinical severity, all patients showed marked decrease of DS and HS in blood and urine samples. We also evaluated serum DS and HS from dried blood-spot samples of three MPS I newborn patients, showing marked elevation of DS and HS levels compared with those in control newborns. In conclusion, blood and urine levels of DS and HS provide an intrinsic monitoring and screening tool for MPS I patients.

Topics: Adolescent; Adult; Biomarkers; Child; Child, Preschool; Chromatography, Liquid; Dermatan Sulfate; Enzyme-Linked Immunosorbent Assay; Female; Heparitin Sulfate; Humans; Infant, Newborn; Male; Mass Screening; Mucopolysaccharidosis I; Neonatal Screening; Tandem Mass Spectrometry; Young Adult

Bone morphogenetic proteins (BMPs) and their endogenous antagonists are important for brain and bone development and tumor initiation and progression. Heparan sulfate (HS) proteoglycans (HSPG) modulate the activities of BMPs and their antagonists. How glycosaminoglycans (GAGs) influence BMP activity in various malignancies and in inherited abnormalities of GAG metabolism, and the structural features of GAGs essential for modulation of BMP signaling, remain incompletely defined. We examined whether chemically modified soluble heparins, the endogenous HS in malignant cells and the HS accumulated in Hurler syndrome cells influence BMP-4 signaling and activity. We show that both exogenous (soluble) and endogenous GAGs modulate BMP-4 signaling and activity, and that this effect is dependent on specific sulfate residues of GAGs. Our studies suggest that endogenous sulfated GAGs promote the proliferation and impair differentiation of malignant human cells, providing the rationale for investigating whether pharmacological agents that inhibit GAG synthesis or function might reverse this effect. Our demonstration of impairment of BMP-4 signaling by GAGs in multipotent stem cells in human Hurler syndrome identifies a mechanism that might contribute to the progressive neurological and skeletal abnormalities in Hurler syndrome and related mucopolysaccharidoses.

Topics: Bone Morphogenetic Protein 4; Bone Morphogenetic Protein Receptors; Bone Morphogenetic Proteins; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cells, Cultured; Glycoproteins; Heparin; Heparitin Sulfate; Humans; Inhibitor of Differentiation Protein 1; Intercellular Signaling Peptides and Proteins; Mucopolysaccharidosis I; Multipotent Stem Cells; Osteosarcoma; Phosphorylation; Promoter Regions, Genetic; Recombinant Proteins; Signal Transduction; Smad1 Protein; Time Factors

Mucopolysaccharidosis I is a lysosomal storage disorder caused by a deficiency of the lysosomal hydrolase alpha-l-iduronidase, which is required for the degradation of heparan sulphate and dermatan sulphate. Given the wide spectrum of disease severity in mucopolysaccharidosis I patients, one of the challenges for managing the disorder is to accurately predict clinical phenotype. Enzyme replacement therapy by intravenous infusion is unlikely to make a significant impact on central nervous system pathology and patients displaying this clinical manifestation may respond better to bone marrow transplantation. In order to predict whether mucopolysaccharidosis I patients are going to develop central nervous system pathology, we investigated a number of biochemical parameters in cultured skin fibroblasts from patients of different genotype/phenotype. Residual levels of alpha-l-iduronidase activity and protein were determined using sensitive immune-quantification assays and fibroblast cell extracts from patients with central nervous system pathology generally had lower levels of alpha-l-iduronidase than patients with no evidence of central nervous system disease. A total of 15 oligosaccharides, derived from heparan sulphate and dermatan sulphate, was measured in fibroblast extracts using electrospray-ionisation tandem mass spectrometry and all were shown to discriminate mucopolysaccharidosis I from controls. Of these, two trisaccharides were able to group patients based on the presence/absence of central nervous system disease. Moreover, a ratio of alpha-l-iduronidase activity to these trisaccharides provided clear discrimination between mucopolysaccharidosis I patients with and without central nervous system pathology. We suggest that this type of analysis may be very useful for predicting disease severity in mucopolysaccharidosis I patients.

Topics: Adolescent; Adult; Cells, Cultured; Central Nervous System; Child; Child, Preschool; Dermatan Sulfate; Fibroblasts; Forecasting; Genotype; Heparitin Sulfate; Humans; Iduronidase; Infant; Mass Spectrometry; Mucopolysaccharidosis I; Oligosaccharides; Phenotype; Skin

In mucopolysaccharidosis-I (MPS-I), alpha-L-iduronidase deficiency leads to progressive heparan sulfate (HS) and dermatan sulfate (DS) glycosaminoglycan (GAG) accumulation. The functional consequences of these accumulated molecules are unknown. HS critically influences tissue morphogenesis by binding to and modulating the activity of several cytokines (eg, fibroblast growth factors [FGFs]) involved in developmental patterning. We recently isolated a multipotent progenitor cell from postnatal human bone marrow, which differentiates into cells of all 3 embryonic lineages. The availability of multipotent progenitor cells from healthy volunteers and patients with MPS-I (Hurler syndrome) provides a unique opportunity to directly examine the functional effects of abnormal HS on cytokine-mediated stem-cell proliferation and survival. We demonstrate here that abnormally sulfated HS in Hurler multipotent progenitor cells perturb critical FGF-2-FGFR1-HS interactions, resulting in defective FGF-2-induced proliferation and survival of Hurler multipotent progenitor cells. Both the mitogenic and survival-promoting activities of FGF-2 were restored by substitution of Hurler HS by normal HS. This perturbation of critical HS-cytokine receptor interactions may represent a mechanism by which accumulated HS contributes to the developmental pathophysiology of Hurler syndrome. Similar mechanisms may operate in the pathogenesis of other diseases where structurally abnormal GAGs accumulate.

Topics: Case-Control Studies; Cell Proliferation; Cell Survival; Cells, Cultured; Chromatography, High Pressure Liquid; Fibroblast Growth Factor 2; Heparitin Sulfate; Humans; Mucopolysaccharidosis I; Multipotent Stem Cells; Receptor Protein-Tyrosine Kinases; Receptor, Fibroblast Growth Factor, Type 1; Receptors, Fibroblast Growth Factor

Mucopolysaccharidosis I (MPS I) is a lysosomal disorder characterized by a deficiency of the enzyme alpha-L: -iduronidase (IDUA), which is responsible for the degradation of glycosaminoglycans (GAGs). This deficiency leads to the accumulation of dermatan and heparan sulphate in lysosomes. Presently available treatments include bone marrow transplantation and enzyme replacement therapies, both of which are limited in their effects. In this work, knockout (KO) MPS I mice were treated with a nonviral vector containing the human IDUA cDNA. KO mice were transfected by hydrodynamic injection of pRIDUA in the caudal vein (i.v., n = 3) or by intraperitoneal injection of pRIDUA/Superfect complexes (i.p., n = 3). GAG concentration and IDUA activity were analysed in the kidneys, spleen, lungs, brain and liver. The expression of IDUA in the organs of i.v.- and i.p.-treated mice was also analysed by real-time reverse-transcription (RT) PCR and compared by relative quantification. The concentration of GAGs in the organs differed between KO and wild-type mice. In the spleen and liver, GAG levels were lower in i.v.- and i.p.-treated KO mice than in control nontreated animals. Real-time RT-PCR showed that the transgene is expressed in all the analysed organs of i.p.- and i.v.-treated KO mice. Enzyme activity was similarly observed in all the organs analysed. Our data suggest that this kind of transfection may be a useful tool for studies of nonviral protocols for gene therapy of MPS.

Topics: Animals; Bone Marrow Transplantation; Dermatan Sulfate; Disease Models, Animal; DNA, Complementary; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Glycosaminoglycans; Heparitin Sulfate; Humans; Lysosomes; Mice; Mice, Inbred C57BL; Mice, Knockout; Mucopolysaccharidosis I; Plasmids; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tissue Distribution; Transduction, Genetic; Transfection

Bone marrow transplantation is the therapy of choice in patients affected by MPS I (Hurler syndrome), but a high incidence of rejection limits the success of this treatment. The deficiency of alpha-L-iduronidase (EC 1.2.3.76), one of the enzymes responsible for the degradation of glycosaminoglycans, results in accumulation of heparan and dermatan sulphate in these patients. Heparan sulphate and dermatan sulphate are known to be important components of the bone marrow microenvironment and critical for haematopoietic cell development. In this study we compared the ability of marrow stromal cells from MPS I patients and healthy donors to support normal haematopoiesis in Dexter-type long term culture. We found an inverse stroma/supernatant ratio in the number of clonogenic progenitors, particularly the colony-forming unit granulocyte-machrophage in MPS I cultures when compared to normal controls. No alteration in the adhesion of haematopoietic cells to the stroma of MPS I patients was found, suggesting that the altered distribution in the number of clonogenic progenitors is probably the result of an accelerated process of differentiation and maturation. The use of alpha-L-iduronidase gene-corrected marrow stromal cells re-established normal haematopoiesis in culture, suggesting that correction of the bone marrow microenvironment with competent enzyme prior to transplantation might help establishment of donor haematopoiesis.

Topics: Adolescent; Antigens, CD34; Bone Marrow; Bone Marrow Cells; Cell Adhesion; Cell Proliferation; Cells, Cultured; Child; Child, Preschool; Collagen; Dermatan Sulfate; Hematopoietic Stem Cells; Heparitin Sulfate; Humans; Iduronidase; Infant; Mucopolysaccharidosis I; Stem Cells; Stromal Cells; Time Factors

There is little information about MPS I-related complications during laronidase therapy. We describe the first autopsy report of a young male MPS I patient who died of infection-induced cardiopulmonary failure following 2 years of weekly treatment with laronidase.

Topics: Adult; Autopsy; Fatal Outcome; Glycosaminoglycans; Heart Failure; Heparitin Sulfate; Humans; Iduronidase; Infections; Male; Mucopolysaccharidosis I; Pharmaceutical Preparations; Recombinant Proteins

The catabolism of glycosaminoglycans begins with endohydrolysis of polysaccharides to oligosaccharides followed by the sequential action of an array of exoenzymes to reduce these oligosaccharides to monosaccharides and inorganic sulfate. In a lysosomal storage disorder known as mucopolysaccharidosis I, caused by a deficiency of the exohydrolase alpha-l-iduronidase, fragments of two different glycosaminoglycans, dermatan sulfate and heparan sulfate, have been shown to accumulate. Oligosaccharides isolated from the urine of a mucopolysaccharidosis I patient using anion exchange and gel filtration chromatography were identified as di-, tri-, tetra-, penta-, and hexasaccharides using electrospray ionization-tandem mass spectrometry and shown to have nonreducing terminal alpha-l-iduronate residues, susceptible to digestion with alpha-l-iduronidase. The presence of odd and even oligosaccharides suggests both endo-beta-glucuronidase and endo-N-acetylhexosaminidase activities toward both glycosaminoglycans. Cultured skin fibroblasts from mucopolysaccharidosis I patients accumulate the same dermatan sulfate-and heparan sulfate-derived di- and trisaccharides as identified in urine, and supplementation of culture medium with recombinant alpha-l-iduronidase reduced their level to that of unaffected control fibroblasts. A dermatan-derived tetrasaccharide not elevated in mucopolysaccharidosis I fibroblasts transiently increased in these fibroblasts in the presence of recombinant alpha-l-iduronidase, indicating it is an intermediate product of catabolism. These oligosaccharides were elevated in urine samples from mucopolysaccharidosis I patients, and we suggest that these glycosaminoglycan-derived oligosaccharides may be useful biochemical markers for the identification and the clinical management of mucopolysaccharidosis I patients.

Topics: Animals; Cells, Cultured; Cricetinae; Dermatan Sulfate; Fibroblasts; Glucuronidase; Glycosaminoglycans; Heparitin Sulfate; Humans; Iduronic Acid; Iduronidase; Mucopolysaccharidosis I; Skin; Spectrometry, Mass, Electrospray Ionization

Mucopolysaccharidosis (MPS) type I (alpha-iduronidase deficiency) is characterized by storage and massive urinary excretion of dermatan sulfate and heparan sulfate; it may be distinguished into three different subtypes based on age at onset and severity of the clinical symptoms. We report on progressive white matter involvement documented by serial MR imaging in a patient with the MPS type I, severe skeletal involvement and preserved mental capabilities (intermediate phenotype or Hurler/Scheie syndrome).The natural history of white matter abnormalities in patients with MPS is still unclear; based on the present study, it appears that degenerative changes of the white matter mimicking a leukodystrophy may mark the course of MPS type I. We also suggest that the degree of MR changes in patients with MPS does not always reflect their neurological impairment.

Topics: Adolescent; Atrophy; Bone and Bones; Bone Diseases, Metabolic; Brain; Cerebral Ventricles; Cognition Disorders; Dermatan Sulfate; Diagnosis, Differential; Female; Heparitin Sulfate; Humans; Iduronidase; Leukodystrophy, Globoid Cell; Magnetic Resonance Imaging; Mucopolysaccharidosis I; Nerve Fibers, Myelinated; Phenotype

Hurler disease resulting from a deficiency in alpha-L-iduronidase, which causes an accumulation of dermatan sulfate and heparan sulfate glycosaminoglycans, is characterized by connective tissue and skeletal deformations, cardiomyopathy, cardiac valve defects, and progressive coronary artery stenosis. In this report, we present evidence that accumulation of dermatan sulfate but not heparan sulfate moieties is linked to impaired elastic fiber assembly that, in turn, contributes substantially to the development of the clinical phenotype in Hurler disease. Our data suggest that dermatan sulfate-bearing moieties bind to and cause functional inactivation of the 67-kd elastin-binding protein, a molecular chaperone for tropoelastin, which normally facilitates its secretion and assembly into elastic fibers. We demonstrate that, in contrast to normal skin fibroblasts and cells from Sanfilippo disease, which accumulate heparan sulfate, Hurler fibroblasts show reduced expression of elastin-binding protein and do not assemble elastic fibers, despite an adequate synthesis of tropoelastin and sufficient production of a microfibrillar scaffold of elastic fibers. Because cultured Hurler fibroblasts proliferate more quickly than their normal counterparts and the addition of exogenous insoluble elastin reduces their proliferation, we suggest that cell contacts with insoluble elastin play an important role in controlling their proliferation.

Topics: Adolescent; Cell Count; Cell Division; Cells, Cultured; Child; Child, Preschool; Coronary Vessels; Dermatan Sulfate; Elastic Tissue; Elastin; Female; Fetus; Fibroblasts; Fibronectins; Fluorescent Antibody Technique, Indirect; Heparitin Sulfate; Humans; Infant; Male; Mitral Valve; Mucopolysaccharidosis I; Mucopolysaccharidosis III; Receptors, Cell Surface; Skin; Tropoelastin

To evaluate whether in vivo accumulations of heparan sulfate caused by inborn errors in the metabolism of glycosaminoglycans lead to the formation of neurofibrillary tangles and/or senile plaques, as seen in Alzheimer disease (AD), we studied postmortem brains from 9 patients, ages 1 to 42 years, with mucopolysaccharidosis (MPS). The brains of patients with Hurler's syndrome (MPS I: n = 5) and Sanfilippo's syndrome (MPS III; n = 4) as well as from caprine MPS IIID and murine MPS VII models were evaluated by thioflavine-S staining and by immunohistochemistry using antibodies directed against heparan sulfate proteoglycans, hyperphosphorylated tau, amyloid-beta peptide precursor proteins (APP), and amyloid-beta peptides (A beta [1-40], and A beta [1-42]). A two-site sandwich enzyme-linked immunosorbent assay (ELISA) was also utilized to compare levels of total soluble and insoluble A beta (1-40) and A beta (1-42) obtained from temporal cortex of MPS patients. Although no neurofibrillary tangles, senile plaques, or tau-positive lesions were detected in any of the MPS brains studied here, antibodies directed against A beta (1-40) intensely and diffusely stained the cytoplasm of cells throughout the brains of the MPS patients and the caprine MPS model. The ELISA assay also demonstrated a significant 3-fold increase in the level of soluble A beta (1-40) in the MPS brains compared with normal control brains. Thus, at least some of the metabolic defects that lead to accumulations of glycosaminoglycans in MPS also are associated with an increase in immunoreactive A beta (1-40) within the cytoplasmic compartment where they could contribute to the dysfunction and death of affected cells in these disorders, but not induce the formation of plaques and tangles. Models of MPS may enable mechanistic studies of the role A beta and glycosaminoglycans play in the amyloidosis that is a neuropathological feature of AD.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Amyloid beta-Peptides; Animals; Antibodies, Monoclonal; Brain; Child; Child, Preschool; Enzyme-Linked Immunosorbent Assay; Female; Glycosaminoglycans; Goats; Heparitin Sulfate; Humans; Infant; Male; Mice; Middle Aged; Mucopolysaccharidoses; Mucopolysaccharidosis I; Mucopolysaccharidosis III; Mucopolysaccharidosis VII; Peptide Fragments

Identification with specific heparan sulfate-lyases, heparitinase I and heparinase of the constitutional unsaturated disaccharide (delta Di-SHS) derived from heparan sulfate (HS) isomers and heparin was achieved using high-performance liquid chromatography (HPLC) with a sulfonated styrene-divinylbenzene copolymer. Eight delta Di-SHS products derived from HS isomers were identified. Enzymatic digestion with heparitinase I and heparinase converts heterogeneous sulfated HS isomers and heparin into different delta Di-SHS. The practical application of these enzymes was examined using specific enzymes and HPLC. In a patient with Hurler syndrome, eight individual delta i-SHS were identified in urinary HS isomers.

Topics: Chromatography, High Pressure Liquid; Disaccharides; Electrophoresis, Polyacrylamide Gel; Heparitin Sulfate; Humans; Hydrolysis; Isomerism; Mucopolysaccharidosis I; Polysaccharide-Lyases; Reproducibility of Results; Spectrophotometry, Ultraviolet

A 37-year-old man with coarse facies, stiff joints, corneal clouding, and normal intelligence sought medical attention. The diagnosis of a systemic mucopolysaccharidosis (MPS) type I-S (Scheie's syndrome) was confirmed by the presence of lysosomal alpha-L-iduronidase deficiency and excessive urinary dermatan and heparan sulfate excretion. The corneal button after perforating keratoplasty of the right eye demonstrated mucopolysaccharides consisting of numerous vacuoles containing fibrillogranular and partly membranebound material in epithelial cells, histiocytes, keratocytes, and extracellular matrix. Endothelial cells were distinctly free of storage material. The epithelial basement membrane showed frequent breaks, whereas Bowman's layer was only slightly attenuated. Irregular collagen fibrils and fibrous long-spacing collagen were noted near degenerating distended keratocytes. The Descemet's membrane was normal. The literature of six reported histopathological examinations of the cornea in Scheie's syndrome is reviewed. Detection of fibrous long-spacing collagen seems to be a typical abnormality of the cornea in MPS I-S.

Topics: Adult; Cornea; Dermatan Sulfate; Fibroblasts; Heparitin Sulfate; Humans; Iduronidase; Keratoplasty, Penetrating; Leukocytes; Male; Mucopolysaccharidosis I

Vitamin A decreased the urinary excretion of total mucopolysaccharides in a patient with Hurler-Scheie compound (type IH-S mucopolysaccharidosis). Vitamin A was administered orally in daily doses of 1,000 to 2,000 IU/kg body weight for 10 years. Adverse clinical responses such as irritability, bone pain, dizziness, vomiting and diarrhea appeared in the patient and were controlled by reduction of the dose administered. No clinical improvement was observed, although it is possible that the clinical course of the disease may have been retarded.

Topics: Adolescent; Chondroitin Sulfates; Dermatan Sulfate; Dose-Response Relationship, Drug; Female; Glycosaminoglycans; Heparitin Sulfate; Humans; Mucopolysaccharidosis I; Syndrome; Vitamin A

Crude glycosaminoglycan (GAG) fraction was directly precipitated with cetylpyridinium chloride without prior dialysis of urine of orthopedic patients. The crude GAG fraction was then fractionated with trichloroacetic acid (TCA). The TCA-insoluble peptide-bound GAG fraction thus obtained was treated with alkali to eliminate the peptide moiety for enzymatic analysis. The GAG compositions of this fraction and the TCA-soluble fraction were determined by digestion with mucopolysaccharidases (chondroitinase AC, chondroitinase B, chondroitinase C, heparitinase and Streptomyces hyaluronidase). When the amount of the crude GAG fraction was small, no significant amount of the TCA-insoluble peptide-bound GAG fraction was obtained. The GAG composition of this case was also determined by the same procedures after direct alkali-treatment of the crude GAG fraction. The data indicated that the proportion of the TCA-insoluble peptide-bound GAG fraction was very small. The alkali-treated TCA-insoluble peptide-bound GAG fraction contained a larger proportion of heparan sulfate than the TCA-soluble GAG fraction. It was clearly demonstrated that the patients with Werner's syndrome and mucopolysaccharidosis I-S (Scheie) excreted large amounts of hyaluronic acid and dermatan sulfate respectively, into urines. It was indicated in most cases that major urinary GAG were chondroitin 4-sulfate, chondroitin 6-sulfate plus chondroitin and heparan sulfate, while minor ones were dermatan sulfate and hyaluronic acid. In addition, the data suggested a wide range of the degree of desulfation or urinary GAG, and the presence of significant amounts of keratan sulfate plus acidic glycopeptides in the urinary GAG fractions. The present data provided more precise information on urinary GAG from orthopedic patients than those reported previously.

Topics: Adolescent; Adult; Aged; Bone Diseases; Chemical Fractionation; Chondroitin Sulfates; Dermatan Sulfate; Female; Glucuronidase; Glycosaminoglycans; Heparitin Sulfate; Humans; Hyaluronic Acid; Lyases; Male; Middle Aged; Mucopolysaccharidosis I; Werner Syndrome

Glycosaminoglycan isolated from urine of a patient with the Hurler-Scheie compound syndrome consisted of dermatan sulfate (60%), heparan sulfate (34%) and chondroitin sulfate (6%). About 60% of both dermatan and chondroitin sulfates had molecular weight 8,000-10,000, while 95% of the heparan sulfate had molecular weight less than 6,000. The total sulfate content of the glycosaminoglycans increased with decrease in molecular weight. N-sulfate content in the heparan sulfate, however, had no relation to molecular weight, and was 0.33 mole per mole of glucosamine on the average. About 70% of the heparan sulfate with the lowest molecular weight (1,500) were composed of three repeating disaccharide units of heparan sulfate and two acetyl, one N-sulfate and three O-sulfate groups linked to the units. The dermatan sulfate contained 1.0-1.2 moles of sulfate per mole of galactosamine. Of the excess sulfate 45-65% were bound to iduronate residues and the rest to C-6 of N-acetylgalactosamine 4-sulfate residues. Most of the dermatan sulfate (83.2-100%) had nonsulfated iduronic acid at the non-reducing end. This finding is consistent with the defect of iduronidase in this disease.

Topics: Dermatan Sulfate; Glycosaminoglycans; Heparitin Sulfate; Humans; Molecular Weight; Mucopolysaccharidosis I

A fluorogenic substrate for alpha-L-iduronidase, 4-methylumbelliferyl alpha-L-iduronide, has been newly synthesized and the enzyme activity has been measured in urine samples obtained from normal persons and patients suffering from mucopolysaccharidosis. Urine samples derived from a patient with Scheie syndrome showed greatly reduced activity compared with a normal adult at a similar age. This patient exhibited a high level of urinary excretion of dermatan sulfate and heparan sulfate, which could be interpreted in terms of her low alpha-L-iduronidase activity. The use of the fluorogenic substrate has some advantages over existing methods because of the high sensitivity and the relative ease of handling, and it should be useful not only for diagnosis but also for following the purification process of the enzyme.

Topics: Adult; Child, Preschool; Dermatan Sulfate; Female; Fluorometry; Glycoside Hydrolases; Heparitin Sulfate; Humans; Hymecromone; Iduronic Acid; Iduronidase; Male; Middle Aged; Mucopolysaccharidoses; Mucopolysaccharidosis I

Topics: Animals; Aorta; Cattle; Cells, Cultured; Fibroblasts; Glucosamine; Glycosaminoglycans; Glycoside Hydrolases; Heparitin Sulfate; Humans; Mucopolysaccharidosis I; Mucopolysaccharidosis III; Oligosaccharides; Skin; Uronic Acids

Topics: Adolescent; Adult; Brain; Child; Child, Preschool; Female; Glycosaminoglycans; Heparitin Sulfate; Humans; Iduronidase; Infant; Intellectual Disability; Liver; Male; Molecular Weight; Mucopolysaccharidoses; Mucopolysaccharidosis I; Mucopolysaccharidosis III; Organ Specificity; Spleen; Sulfatases

The mucopolysaccharidoses are a group of genetic diseases characterized by storage of incompletely degraded glycosaminoglycans. Such storage causes marked distortion of many tissues with consequent severe somatic changes and mental retardation. Storage of glycosaminoglycans results from markedly diminished activity of specific hydrolases requisite for the normal degradation of glycosaminoglycans. The specific enzymic defects have been identified in nine different diseases. In some cases evidence has been obtained indicating the existence of additional allelic diseases based on the same enzyme. The knowledge obtained from these studies has made prenatal diagnosis possible and has led to the possibility that therapy may be undertaken utilizing enzyme replacement.

Topics: Acetylglucosaminidase; Alleles; Arylsulfatases; Chondroitin Sulfates; Dermatan Sulfate; Glucuronidase; Glycosaminoglycans; Heparitin Sulfate; History, 20th Century; Humans; Iduronidase; Mucopolysaccharidoses; Mucopolysaccharidosis I; Sulfatases

Multidisciplinary studies were conducted on the brain and other tissues of patients who died with the antemortem diagnosis of mucopolysaccharidosis (MPS) of one of the following types; type V, Scheie disease (MPS-V); type I, Hurler disease (MPS-I): and type II, Hunter disease (MPS-II). The principal new finding in the brain of the patient with MPS-V is the presence of lesions in the periadventitial mesenchymal tissue of the white matter, similar to those of MPS-I, while the nerve cells in MPS-V are histologically normal, in contradistinction to MPS-I, in which the neuronal abnormality is severe. Electron microscopical studies of the brain in MPS-I demonstrated numerous complex membranous inclusions in the neurons, whereas the neurons in MPS-V contained only a small number of lipofuscin-like inclusions and typical lipofuscin granules. There was a threefold increase of glycosaminoglycans (GAG) in the brain of MPS-I, but only a slight increase in the MPS-V; GAG in the liver and spleen of all patients was noticeably increased. alpha-L-iduronidase activity was not detectable in the brain and liver of patients with MPS-I and MPS-V, thus suggesting a similar enzymatic defect.

Topics: Adult; Brain; Cerebral Cortex; Child; Dermatan Sulfate; Diagnosis, Differential; Female; Glycosaminoglycans; Heparitin Sulfate; Humans; Iduronidase; Liver; Male; Mucopolysaccharidoses; Mucopolysaccharidosis I; Mucopolysaccharidosis II

Glycosaminoglycans were isolated from the urines of 46 patients with mucopolysaccharidosis; 11 with Type I (Hurler), 8 with Type II (Hunter), 16 with Type III (Sanfilippo A and B), 9 with Type V (Scheie), one with Type VI (Marateaux-Lamy), and one unclassified. All 46 patients excreted in their urine excessive amounts of dermatan sulfate, heparan sulfate or both. In addition, patients of certain types excreted excessive amounts of chondroitin sulfates A and/or C. There is a trend in each type of the disease towards the same carbazole/orcinol ratio, glucosamine/galactosamine ratio and glycosaminoglycan composition. Molecular weight distribution of the urinary glycosaminoglycans by gel filtration from Sephadex G-200 is characteristic for each different type of mucopolysaccharidosis and is distinguished from normal controls and patients without mucopolysaccharidosis. Preparation of elution diagrams from Sephadex G-200 allows an estimation of the composition of the glycosamino-glycans. Practically all heparan sulfate and a sizable part of dermatan sulfate from the urinary glycosaminoglycans of all these patients have been highly degraded. In all the patients in which the specific enzyme defect was demonstrated, the assignment of the type of mucopolysaccharidosis, on the basis of the elution diagrams, was correct. Patients with mucopolysaccharidosis Type V displayed two conspicuously different types of elution patterns, suggesting heterogeneity. Indeed, only a portion of these patients showed alpha-L- iduronidase deficiency. Carriers had normal urinary glycosaminoglycan output and composition and exhibited normal elution diagrams.

Topics: Adolescent; Adult; Bone and Bones; Carbohydrate Metabolism, Inborn Errors; Child; Child, Preschool; Chondroitin; Dermatan Sulfate; Female; Glycosaminoglycans; Heparitin Sulfate; Humans; Hypertrichosis; Infant; Intellectual Disability; Joint Diseases; Male; Mucopolysaccharidoses; Mucopolysaccharidosis I; Mucopolysaccharidosis II; Retinitis Pigmentosa

Heparan sulfates were isolated from the urine of normal individuals and patients with genetic mucopolysaccharidoses after exhaustive digestion with chondroitinase ABC. Electrophoresis of these preparations on cellulose acetate membrane revealed one spot corresponding in mobility to reference heparan sulphate in barium acetate buffer, while electrophoresis in 0.1 M HCl resulted in two distinct spots for each case; one corresponded in migration rate to reference heparan sulfate, and the other was faster in mobility than reference heparan sulfate but slightly retarded when compared with reference heparin. On thin-layer gel filtration on Sephadex G-200 (superfine) heparan sulfate from normal urine was polydispersed in character and its molecular size was larger than those of other preparations. Heparan sulfates from Hunter's and Sanfilippo's urine were monodispersed and small in molecular size. The molecular size of heparan sulfate from Sanfilippo's urine was the smallest of all. Heparin sulfate from Hurler's urine appeared to be composed of two populations; one corresponded in molecular size to heparan sulfate from normal urine, and the other corresponded to that of Hunter's urine.

Topics: Adolescent; Child; Child, Preschool; Chromatography, Gel; Chromatography, Thin Layer; Electrophoresis, Cellulose Acetate; Galactosamine; Glucosamine; Glycosaminoglycans; Heparitin Sulfate; Humans; Male; Molecular Weight; Mucopolysaccharidoses; Mucopolysaccharidosis I; Mucopolysaccharidosis II; Mucopolysaccharidosis III

Topics: Carbohydrate Metabolism, Inborn Errors; Diagnosis, Differential; Female; Glycosaminoglycans; Heparitin Sulfate; Humans; Infant; Infant, Newborn; Intellectual Disability; Male; Mucopolysaccharidosis I; Mucopolysaccharidosis IV