Retrospective chart review of urinary glycosaminoglycan excretion and long-term clinical outcomes of enzyme replacement therapy in patients with mucopolysaccharidoses.

BACKGROUND: Mucopolysaccharidoses (MPS) are a group of rare, inherited metabolic diseases that result from a deficiency in one of several lysosomal enzymes essential for stepwise glycosaminoglycan (GAG) degradation, leading to GAG accumulation and widespread cellular pathology and clinical disease. Although disease presentation is heterogeneous, the clinical hallmarks are largely comparable across several MPS subtypes. Extensive data have shown that the level of urinary GAG (uGAG) excretion above normal is strongly correlated with disease severity and clinical outcomes in MPS diseases. Thus, change in uGAG excretion may have significant value as a potential primary endpoint in clinical trials of MPS diseases that are too rare to study using traditional clinical endpoints. METHODS: A retrospective medical chart review was undertaken of patients with MPS I, II, and VI who had been treated long term with enzyme replacement therapy (ERT). The relationship between uGAG reduction and clinical outcomes relevant to the major clinical manifestations of these MPS diseases was evaluated. A multi-domain responder index (MDRI) score was calculated, measuring the following 4 domains: 6-min walk test, pulmonary function, growth rate, and Clinician Global Impression of Change. For each domain, a minimal important difference (MID) was defined based on published information of these outcome measures in MPS and other diseases. RESULTS: Of the 50 patients evaluated, 18 (36%) had MPS I, 23 (46%) had MPS II, and 9 (18%) had MPS VI. Forty-two were clinical practice patients and 8 had participated in clinical trials. Across all MPS subtypes, the mean (± SD) uGAG level at baseline was 66.0 ± 51.5 mg/mmol creatinine (n = 48) and there was a mean reduction of 54.6% following ERT. Analysis of the MDRI score based on the MID defined for each domain showed a greater magnitude of improvement in patients with increased uGAG reduction when compared with those patients with lower uGAG reduction for all assessed uGAG thresholds, and a trend toward a higher likelihood of positive mean MDRI score in patients with a uGAG reduction ≥40%. CONCLUSIONS: In this retrospective study, uGAG reduction was associated with long-term clinical outcomes as assessed by a number of approaches, supporting the use of uGAG reduction as a biomarker primary endpoint.

Normalization of glycosaminoglycan-derived disaccharides detected by tandem mass spectrometry assay for the diagnosis of mucopolysaccharidosis.

Mucopolysaccharidosis (MPS) is caused by the deficiency of a specific hydrolytic enzyme that catalyzes the step-wise degradation of glycosaminoglycans (GAGs). In this study, we propose an empirical method to calculate levels of GAG-derived disaccharides based on the quantity (peak areas) of chondroitin sulfate (CS) with the aim of making a diagnosis of MPS more accurate and reducing the occurrence of false positive and false negative results. In this study, levels of urinary GAG-derived disaccharides were measured in 67 patients with different types of MPS and 165 controls without MPS using a tandem mass spectrometry assay. Two different methods of reporting GAG-derived disaccharides were assessed; normalization to urinary CS (in µg/mL), and normalization to µg/mg creatinine. CS-normalization yielded more consistent values than creatinine-normalization. In particular, levels of urinary dermatan sulfate (DS), heparan sulfate (HS), and keratan sulfate (KS) significantly varied because of changes in urine creatinine levels, which were proportional to age but inversely proportional to DS, HS, and KS measurements. Using CS-normalization revealed the actual status of DS, HS, and KS without the influence of factors such as age, urine creatinine, and other physiological conditions. It could discriminate between the patients with MPS and controls without MPS, and also to evaluate changes in GAG levels pre- and post-enzyme replacement therapy.

Fast, sensitive method for trisaccharide biomarker detection in mucopolysaccharidosis type 1.

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.

Urinary metabolic phenotyping of mucopolysaccharidosis type I combining untargeted and targeted strategies with data modeling.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Clinical features of Mexican patients with Mucopolysaccharidosis type I.

Mucopolysaccharidosis type I (MPS-I) is an autosomal recessive lysosomal storage disorder caused by a deficiency or absence of α--iduronidase, which is involved in the catabolism of glycosaminoglycans (GAGs). This deficiency leads to the accumulation of GAGs in several organs. Given the wide spectrum of the disease, MPS-I has historically been classified into 3 clinical subtypes - severe (Hurler syndrome), intermediate (Hurler-Scheie syndrome), and mild (Scheie syndrome) - none of which is determined by residual enzyme activity. Eleven Mexican patients with MPS-I from northwestern México were evaluated. Diagnoses were confirmed through quantification of GAGs in urine and enzyme assay for α--iduronidase. Regardless of phenotype, all patients had various degrees of infiltrated facies, short stature, dysostosis multiplex, joint contractures, and corneal opacity typical of the disease. A better understanding of the spectrum of this disease can assist in diagnosis, treatment, and improvement in the quality of life for these patients.

Incomplete biomarker response in mucopolysaccharidosis type I after successful hematopoietic cell transplantation.

BACKGROUND: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.

Deleterious effects of interruption followed by reintroduction of enzyme replacement therapy on a lysosomal storage disorder.

Temporary interruption of enzyme replacement therapy (ERT) in patients with different lysosomal storage disorders may happen for different reasons (adverse reactions, issues with reimbursement, logistic difficulties, and so forth), and the impact of the interruption is still uncertain. In the present work, we studied the effects of the interruption of intravenous ERT (Laronidase, Genzyme) followed by its reintroduction in mice with the prototypical lysosomal storage disorder mucopolysaccharidosis type I, comparing to mice receiving continuous treatment, untreated mucopolysaccharidosis type I mice, and normal mice. In the animals which treatment was temporarily interrupted, we observed clear benefits of treatment in several organs (liver, lung, heart, kidney, and testis) after reintroduction, but a worsening in the thickness of the aortic wall was detected. Furthermore, these mice had just partial improvements in behavioral tests, suggesting some deterioration in the brain function. Despite worsening is some disease aspects, urinary glycosaminoglycans levels did not increase during interruption, which indicates that this biomarker commonly used to monitor treatment in patients should not be used alone to assess treatment efficacy. The deterioration observed was not caused by the development of serum antienzyme antibodies. All together our results suggest that temporary ERT interruption leads to deterioration of function in some organs and should be avoided whenever possible.

Obstructive sleep apnea syndrome after hematopoietic stem cell transplantation in children with mucopolysaccharidosis type I.

BACKGROUND: Obstructive sleep apnea syndrome (OSAS) is very common in mucopolysaccharidosis I (MPS I). Hematopoietic stem cell transplantation (HSCT) is the preferred treatment for patients with severe MPS I diagnosed early in life. The protective effect of HSCT on the development of long term OSAS is not known. METHODS: Overnight polysomnography (PSG) and biomarker data were analyzed during the annual follow-up in consecutive MPS I patients treated with HSCT. RESULTS: The data of 13 patients (6 boys) were analyzed. Median age at HSCT was 17 (range 14-19) months, median age at PSG was 9.0 (4.5-14.5) years, and median time elapsed since HSCT was 7.6 (2.4-13.2) years. A significant correlation was observed between time elapsed since HSCT and the apnea-hypopnea index (AHI, r(2)=0.493, p=+0.003) and the oxygen desaturation index (r(2)=0.424, p=+0.02). Patients older than 10 years of age had a higher mean AHI (25.8/h vs 1.4/h, p=0.0008), a lower mean pulse oximetry (94.7% vs 97.2%, p=0.01) and a higher mean hypopnea index (18.8 vs 0.71/h, p=0.016) as compared to those younger than 10 years of age. No correlation was observed between the AHI and the metabolic clearance, assessed by urine glycosaminoglycan (GAG) excretion and residual enzyme activity, although there was a positive trend for the urinary GAG/higher normal value for age ratio (p=0.09). CONCLUSION: HSCT does not offer long term protection against OSAS in MPS I with OSAS being documented in all patients after a time elapse since HSCT exceeding 10 years. The potential benefit of additional enzyme replacement therapy needs to be assessed.

Sleep disordered breathing in mucopolysaccharidosis I: a multivariate analysis of patient, therapeutic and metabolic correlators modifying long term clinical outcome.

BACKGROUND: The lysosomal storage disorder, mucopolysaccharidosis I (MPS I), commonly manifests with upper airway obstruction and sleep disordered breathing (SDB). The success of current therapies, including haematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT) may be influenced by a number of factors and monitored using biomarkers of metabolic correction. We describe the pattern of SDB seen in the largest MPS I cohort described to date and determine therapies and biomarkers influencing the severity of long-term airway disease. METHODS: Therapeutic, clinical and biomarker data, including longitudinal outcome parameters from 150 sleep oximetry studies were collected in 61 MPS I (44 Hurler, 17 attenuated) patients between 6 months pre to 16 years post-treatment (median follow-up 22 months). The presence and functional nature of an immune response to ERT was determined using ELISA and a cellular uptake inhibition assay. Multivariate analysis was performed to determine significant correlators of airway disease. RESULTS: The incidence of SDB in our cohort is 68%, while 16% require therapeutic intervention for airway obstruction. A greater rate of progression (73%) and requirement for intervention is seen amongst ERT patients in contrast to HSCT treated individuals (24%). Multivariate analysis identifies poorer metabolic clearance, as measured by a rise in the biomarker urinary dermatan sulphate: chondroitin sulphate (DS:CS) ratio, as a significant correlator of increased presence and severity of SDB in MPS I patients (p = 0.0017, 0.008). Amongst transplanted Hurler patients, delivered enzyme (leukocyte iduronidase) at one year is significantly raised in those without SDB (p = 0.004). Cellular uptake inhibitory antibodies in ERT treated patients correlate with reduced substrate clearance and occurrence of severe SDB (p = 0.001). CONCLUSION: We have identified biochemical and therapeutic factors modifying airway disease across the phenotypic spectrum in MPS I. Interventions maximising substrate reduction correlate with improved long-term SDB, while inhibitory antibodies impact on biochemical and clinical outcomes. Monitoring and tolerisation strategies should be re-evaluated to improve detection and minimise the inhibitory antibody response to ERT in MPS I and other lysosomal storage diseases. Future studies should consider the use of sleep disordered breathing as an objective parameter of clinical and metabolic improvement.

Effects of enzyme replacement therapy started late in a murine model of mucopolysaccharidosis type I.

Mucopolysaccharidosis type I (MPS I) is a progressive disorder caused by deficiency of α-L-iduronidase (IDUA), which leads to storage of heparan and dermatan sulphate. It is suggested that early enzyme replacement therapy (ERT) leads to better outcomes, although many patients are diagnosed late and don't receive immediate treatment. This study aims to evaluate the effects of late onset ERT in a MPS I murine model. MPS I mice received treatment from 6 to 8 months of age (ERT 6-8mo) with 1.2mg laronidase/kg every 2 weeks and were compared to 8 months-old wild-type (Normal) and untreated animals (MPS I). ERT was effective in reducing urinary and visceral GAG to normal levels. Heart GAG levels and left ventricular (LV) shortening fraction were normalized but cardiac function was not completely improved. While no significant improvements were found on aortic wall width, treatment was able to significantly reduce heart valves thickening. High variability was found in behavior tests, with treated animals presenting intermediate results between normal and affected mice, without correlation with cerebral cortex GAG levels. Cathepsin D activity in cerebral cortex also did not correlate with behavior heterogeneity. All treated animals developed anti-laronidase antibodies but no correlation was found with any parameters analyzed. However, intermediary results from locomotion parameters analyzed are in accordance with intermediary levels of heart function, cathepsin D, activated glia and reduction of TNF-α expression in the cerebral cortex. In conclusion, even if started late, ERT can have beneficial effects on many aspects of the disease and should be considered whenever possible.

Biomarker responses correlate with antibody status in mucopolysaccharidosis type I patients on long-term enzyme replacement therapy.

BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

Mutation c.1190-1delG/N in intron 8 and c.1708G>C/N in exon 12 not reported in the IDUA gene developed a clinical phenotype of Scheie syndrome / Mutación c.1190-1delG/N en el intrón 8 y c.1708G>C/N en el exón 12 no reportada en el gen de IDUA desarrolló un fenotipo clínico de síndrome de Scheie

Mucopolysaccharidoses are a group of lysosomal storage disorders caused by deficiency of enzymes catalyzing the degradation of glycosaminoglycans. Mucopoly-saccharidosis I can present a wide range of phenotypic characteristics with three major recognized clinical entities: Hurler and Scheie syndromes represent phenotypes at the severe and mild ends of the clinical spectrum, respectively, and the Hurler-Scheie syndrome is intermediate in phenotypic expression. These are caused by the deficiency or absence of α-L-iduronidase, essential to the metabolism of both dermatan and heparan sulfate, and it is encoded by the IDUA gene. We report the case of a 34-year-old male patient with enzymatic deficiency of α-L-iduronidase, accumulation of its substrate and a previously unreported mutation in the IDUA gene that developed a phenotype of Scheie syndrome.

Las mucopolisacaridosis son un grupo de trastornos de almacenamiento lisosomal causada por la deficiencia de enzimas que catalizan la degradación de glicosaminoglicanos. La mucopolisacaridosis tipo I puede presentar un amplio rango de características fenotípicas englobadas en tres entidades clínicas reconocidas: los síndromes de Hurler y Scheie representan los fenotipos graves y leves del espectro clínico, respectivamente y el síndrome de Hurler-Scheie intermedio en la expresión fenotípica. Estos son causados por la deficiencia o ausencia de la α-L-iduronidasa esencial para el metabolismo del dermatán y el heparán sulfato y es codificada por el gen IDUA. Se presenta el caso de paciente masculino de 34 años de edad con deficiencia enzimática de α-L-iduronidasa, acumulación de su sustrato y una mutación en el gen IDUA, no reportada previamente, que desarrolló un fenotipo del síndrome de Scheie.

Mutation c.1190-1delG/N in intron 8 and c.1708G>C/N in exon 12 not reported in the IDUA gene developed a clinical phenotype of Scheie syndrome.

Mucopolysaccharidoses are a group of lysosomal storage disorders caused by deficiency of enzymes catalyzing the degradation of glycosaminoglycans. Mucopoly-saccharidosis I can present a wide range of phenotypic characteristics with three major recognized clinical entities: Hurler and Scheie syndromes represent phenotypes at the severe and mild ends of the clinical spectrum, respectively, and the Hurler-Scheie syndrome is intermediate in phenotypic expression. These are caused by the deficiency or absence of alpha-L-iduronidase, essential to the metabolism of both dermatan and heparan sulfate, and it is encoded by the lDUA gene. We report the case of a 34-year-old male patient with enzymatic deficiency of alpha-L-iduronidase, accumulation of its substrate and a previously unreported mutation in the IDUA gene that developed a phenotype of Scheie syndrome.

Plasma and urinary levels of dermatan sulfate and heparan sulfate derived disaccharides after long-term enzyme replacement therapy (ERT) in MPS I: correlation with the timing of ERT and with total urinary excretion of glycosaminoglycans.

INTRODUCTION: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Evidence of a progressive motor dysfunction in Mucopolysaccharidosis type I mice.

Mucopolysaccharidosis (MPS) type I (Hurler syndrome) is a lysosomal storage disorder characterized by deficiency of alpha-L-iduronidase (IDUA), intracellular storage of glycosaminoglycans (GAGs) and progressive neurological pathology. The MPS I mouse model provides an opportunity to study the pathophysiology of this disorder and to determine the efficacy of novel therapies. Previous work has demonstrated a series of abnormalities in MPS I mice behavior, but so far some important brain functions have not been addressed. Therefore, in the present study we aimed to determine if MPS I mice have motor abnormalities, and at what age they become detectable. MPS I and normal male mice from 2 to 8 months of age were tested in open-field for locomotor activity, hindlimb gait analysis and hang wire performance. We were able to detect a progressive reduction in the crossings and rearings in the open field test and in the hang wire test in MPS I mice from 4 months, as well as a reduction in the gait length at 8 months. Histological examination of 8-month old mice cortex and cerebellum revealed storage of GAGs in Purkinje cells and neuroinflammation, evidenced by GFAP immunostaining. However TUNEL staining was negative, suggesting that death does not occur. Our findings suggest that MPS I mice have a progressive motor dysfunction, which is not caused by loss of neuron cells but might be related to a neuroinflammatory process.

Longitudinal observations of serum heparin cofactor II-thrombin complex in treated Mucopolysaccharidosis I and II patients.

Monitoring of therapeutic response in mucopolysaccharidosis (MPS) patients is problematic as most biomarkers are specific for either disease complications or specific organ system involvement. Recent studies have indicated that serum heparin-cofactor II-thrombin complex (HCII-T) may serve as an important biomarker in the group of MPSs where dermatan sulphate is stored. This complex forms when blood coagulates in the presence of glycosaminoglycans (GAGs) where the ultimate amount of HCII-T that forms reflects the concentration of circulating GAGs. We have studied serum HCII-T levels in 9 MPS I and 11 MPS II treated patients and have compared values to studies of urinary GAGs. In severe MPS I patients treated with either transplantation or enzyme replacement therapy (ERT), serum HCII-T levels never reach the range of normal despite normalization of uGAGs in some patients. Some attenuated MPS I patients have normalization of HCII-T but require a protracted exposure time relative to the drop in urinary GAGs. Treated MPS II patients show a clear correlation of serum HCII-T levels with the presence of antibodies to Idursulfase, with antibody positive patients showing an early drop in HCII-T levels with eventual increases in levels often to levels above those seen at baseline. This is contrasted by a robust and persistent drop in uGAGs. Antibody negative MPS II patients show a drop in HCII-T levels on treatment but levels never normalize despite normalization of uGAGs. This study highlights the utility and biologic relevance of serum HCII-T levels in monitoring therapy in these disorders.

Efficient analysis of urinary glycosaminoglycans by LC-MS/MS in mucopolysaccharidoses type I, II and VI.

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.

Effect of 6 years of enzyme replacement therapy on plasma and urine glycosaminoglycans in attenuated MPS I patients.

Enzyme-replacement therapy (ERT) is a new option for the clinical management of MPS I. However, no detailed data are available on the structural characterization of glycosaminoglycans (GAGs) in the urine and plasma of patients before ERT and during treatment regimens. Before ERT and over a two-week period of enzyme infusion, GAGs in urine and plasma were analyzed in two patients with the Hurler-Scheie form of MPS I subjected to ERT for 6 years. In both patients before ERT, high amounts of a GAG were found in the urine, composed in particular of a high molecular mass polymer (approximately 13,000-13,500) consisting of approximately 75-78% iduronic acid and rich in 4-sulfated disaccharides (DeltaDi4s) and attributable to DS. Furthermore, a high amount of this GAG was directly detected in the blood. Plasma GAGs in MPS I patients subjected to ERT were found to be comparable to those of normal subjects with the absence of heparan sulfate and of DS. On the contrary, a polysaccharide possessing a high molecular mass, approximately 11,500-12,000, lower than the polymer extracted before ERT but slightly higher than the controls (approximately 11,000), was found in the urine of both patients. This macromolecule was characterized as a mixture of DS/chondroitin sulfate based on the high percentage of 4-sulfated disaccharide (4s/6s ratio of approximately 3.1) and iduronic acid ( approximately 60%). These results are indicative of the incapacity of ERT at the standard dose to definitively eliminate DS from the urine. Finally, a variable effect of ERT depending on each administration was also observed.

Ultrastructural analysis of dermal fibroblasts in mucopolysaccharidosis type I: Effects of enzyme replacement therapy and hematopoietic cell transplantation.

In mucopolysaccharidosis type I (MPS I; alpha-L-iduronidase deficiency), glycosaminoglycans (GAGs) accumulate in different cell types, causing characteristic vacuolization. Hematopoietic cell transplantation (HCT) and enzyme replacement therapy (ERT) both aim to restore tissue morphology by delivering alpha-L-iduronidase to the deficient cells. The authors investigated the efficacy of both therapies on dermal fibroblast morphology in 12 patients by electron microscopy of repeated skin biopsies before and during 2 years of ERT as well as before and 6 months after HCT. Cell vacuolization was rated according to a semi-quantitative scoring system. At baseline all patients showed an increased vacuolization score as compared to controls. In addition the vacuolization score was significantly higher in patients with the severe phenotype of the disease (n = 7) compared to patients with attenuated phenotypes (n = 5) (p = .009). After initiation of ERT a significant decrease in cell vacuolization was observed (p = .012). However, the response rate varied among patients, as the vacuolization score remained high during the first year of ERT in 3 patients with the severe phenotype. In all patients who received a successful HCT (n = 3) only minimal disturbances in cell morphology were observed afterward. In conclusion, both ERT and HCT are capable of restoring, at least partially, dermal fibroblast morphology in MPS I.