Estimated birth prevalence of mucopolysaccharidoses in Brazil.

Several studies have been published on the frequency of the mucopolysaccharidoses (MPS) in different countries. The objective of the present study was to estimate the birth prevalence (BP) of MPS in Brazil. MPS diagnosis registered at MPS-Brazil Network and in Instituto Vidas Raras were reviewed. BP was estimated by (a) the number of registered patients born between 1994 and 2015 was divided by the number of live births (LBs), and (b) a sample of 1,000 healthy individuals was tested for the most frequent variant in IDUA gene in MPS I (p.Trp402Ter) to estimate the frequency of heterozygosity and homozygosity. (a) The BP based on total number of LBs was (cases per 100,000 LBs): MPS overall: 1.25; MPS I: 0.24; MPS II: 0.37; MPS III: 0.21; MPS IV: 0.14; MPS VI: 0.28; MPS VII: 0.02. (b) The overall frequency of p.Trp402Ter was 0.002. Considering the frequency of heterozygotes for the p.Trp402Ter IDUA variant in the RS state, the frequency of this variant among MPS I patients and the relative frequency of the different MPSs, we estimated the birth prevalence of MPS in total and of each MPS type, as follows: MPS overall: 4.62; MPS I: 0.95; MPS II: 1.32; MPS III: 0.56; MPS IV: 0.57; MPS VI: 1.02; MPS VII: 0.05. This study provided original data about BP and relative frequency of the MPS types, in Brazil, based on the frequency of the commonest IDUA pathogenic variant and in the records of two large patient databases.

Validation of Liquid Chromatography-Tandem Mass Spectrometry-Based 5-Plex Assay for Mucopolysaccharidoses.

Mucopolysaccharidoses (MPSs) are rare lysosomal storage diseases caused by the accumulation of undegraded glycosaminoglycans in cells and tissues. The effectiveness of early intervention for MPS has been reported. Multiple-assay formats using tandem mass spectrometry have been developed. Here, we developed a method for simultaneous preparation and better measurement of the activities of five enzymes involved in MPSs, i.e., MPS I, MPS II, MPS IIIB, MPS IVA, and MPS VI, which were validated using 672 dried blood spot samples obtained from healthy newborns and 23 patients with MPS. The mean values of the enzyme activities and standard deviations in controls were as follows: α-iduronidase (IDUA), 4.19 ± 1.53 µM/h; iduronate-2-sulfatase (I2S), 8.39 ± 2.82 µM/h; N-acetyl-α-glucosaminidase (NAGLU), 1.96 ± 0.57 µM/h; N-acetylgalactosamine-6-sulfatase (GALNS), 0.50 ± 0.20 µM/h; and N-acetylgalactosamine-4-sulfatase (ARSB), 2.64 ± 1.01 µM/h. All patients displayed absent or low enzyme activity. In MPS I, IIIB, and VI, each patient group was clearly separated from controls, whereas there was some overlap between the control and patient groups in MPS II and IVA, suggesting the occurrence of pseudo-deficiencies. Thus, we established a multiplex assay for newborn screening using liquid chromatography tandem mass spectrometry, allowing simultaneous pretreatment and measurement of five enzymes relevant to MPSs.

Glycosaminoglycans analysis in blood and urine of patients with mucopolysaccharidosis.

To explore the correlation between glycosaminoglycan (GAG) levels and mucopolysaccharidosis (MPS) type, we have evaluated the GAG levels in blood of MPS II, III, IVA, and IVB and urine of MPS IVA, IVB, and VI by tandem mass spectrometry. Dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS; mono-sulfated KS, di-sulfated KS), and the ratio of di-sulfated KS in total KS were measured. Patients with untreated MPS II had higher levels of DS and HS in blood while untreated MPS III had higher levels of HS in blood than age-matched controls. Untreated MPS IVA had higher levels of KS in blood and urine than age-matched controls. The ratio of blood di-sulfated KS/total KS in untreated MPS IVA was constant and higher than that in controls for children up to 10 years of age. The ratio of urine di-sulfated KS/total KS in untreated MPS IVA was also higher than that in age-matched controls, but the ratio in untreated MPS IVB was lower than controls. ERT reduced blood DS and HS in MPS II, and urine KS in MPS IVA patients, although GAGs levels remained higher than the observed in age-matched controls. ERT did not change blood KS levels in MPS IVA. MPS VI under ERT still had an elevation of urine DS level compared to age-matched controls. There was a positive correlation between blood and urine KS in untreated MPS IVA patients but not in MPS IVA patients treated with ERT. Blood and urine KS levels were secondarily elevated in MPS II and VI, respectively. Overall, measurement of GAG levels in blood and urine is useful for diagnosis of MPS, while urine KS is not a useful biomarker for monitoring therapeutic efficacy in MPS IVA.

[Maroteaux-Lamy syndrome: a case report]. / Syndrome de Maroteaux-Lamy : à propos d'un cas.

The Maroteaux-Lamy disease, or mucopolysaccharidosis type VI is an inherited metabolic disorder severe and rare. It is caused by a deficiency of the enzyme arylsulfatase B. It is characterized by a heterogeneous clinical, radiological and genetic. We report the case of a Maroteaux-Lamy syndrome of in a child aged 7 years whose diagnosis was suspected clinically by the combination of a dysmorphic syndrome, a failure to thrive not harmonious, hepatomegaly and normal intelligence. Radiological exams have objectified dysostosis multiplex. Biochemical analysis of urine showed the abnormal presence of dermatan sulfate. The determination of leukocyte enzyme activity confirmed the diagnosis by showing arylsulfatase B deficiency. Hence the diagnosis of syndrome Maroteaux-Lamy in its mild form (type B) was selected.

Tandem mass spectrometry for the direct assay of lysosomal enzymes in dried blood spots: application to screening newborns for mucopolysaccharidosis VI (Maroteaux-Lamy syndrome).

We report a new assay of N-acetylgalactosamine-4-sulfatase (aryl sulfatase B) activity in dried blood spots (DBS) for the early detection of mucopolysaccharidosis VI (Maroteaux-Lamy syndrome) in newborn screening. The assay uses a synthetic substrate consisting of N-acetylgalactosamine-4-sulfate moiety glycosidically linked to a hydrophobic residue and furnished with a tert-butyloxycarbamido group as a marker for specific mass spectrometric fragmentation. Incubation with aryl sulfatase B present in DBS converts the substrate to a desulfated product which is detected by electrospray tandem mass spectrometry and quantified using a homologous internal standard. Assay and workup procedures were optimized to be compatible with the work flow in newborn screening laboratories. Analysis of DBS from human newborns showed clear distinction of aryl sulfatase B activity from 89 healthy individuals where it ranged between 1.4 and 16.9 µmol/(h L of blood), with an average activity of 7.4 µmol/(h L of blood), and an MPS-VI patient that had an activity of 0.12 µmol/(h L of blood). Results are also reported for the aryl sulfatase B assay in DBS from groups of normal felines and felines affected with MPS-VI.

Enzyme replacement therapy for mucopolysaccharidosis VI: evaluation of long-term pulmonary function in patients treated with recombinant human N-acetylgalactosamine 4-sulfatase.

Pulmonary function is impaired in untreated mucopolysaccharidosis type VI (MPS VI). Pulmonary function was studied in patients during long-term enzyme replacement therapy (ERT) with recombinant human arylsulfatase B (rhASB; rhN-acetylgalactosamine 4-sulfatase). Pulmonary function tests prior to and for up to 240 weeks of weekly infusions of rhASB at 1 mg/kg were completed in 56 patients during Phase 1/2, Phase 2, Phase 3 and Phase 3 Extension trials of rhASB and the Survey Study. Forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1) and, in a subset of patients, maximum voluntary ventilation (MVV), were analyzed as absolute volume in liters. FEV1 and FVC showed little change from baseline during the first 24 weeks of ERT, but after 96 weeks, these parameters increased over baseline by 11% and 17%, respectively. This positive trend compared with baseline continued beyond 96 weeks of treatment. Improvements from baseline in pulmonary function occurred along with gains in height in the younger group (5.5% change) and in the older patient group (2.4% change) at 96 weeks. Changes in MVV occurred earlier within 24 weeks of treatment to approximately 15% over baseline. Model results based on data from all trials showed significant improvements in the rate of change in pulmonary function during 96 weeks on ERT, whereas little or no improvement was observed for the same time period prior to ERT. Thus, analysis of mean percent change data and longitudinal modeling both indicate that long-term ERT resulted in improvement in pulmonary function in MPS VI patients.

Improving arylsulfatase activity determination in dried blood spots: Screening and diagnostic approaches for Maroteaux-Lamy syndrome (MPS VI).

BACKGROUND: Mucopolysaccharidosis type VI can be screened by measuring the lysosomal arylsulfatase B (ARSB) residual enzyme activity in dried blood spots (DBS) using synthetic substrates. However, we have found experimental obstacles when determining ARSB activity with the fluorescent method due to the significant quenching effect rendered by DBS components. METHODS: We adapted the methods originally described by Chamoles et al. [1] and Civallero et al. [2] and put forward 2 distinct approaches for ARSB activity quantification from DBS samples by measuring the 4-methylumbelliferone (ß-MU) fluorescence generated from the ARSB 4-methylumbelliferone sulfate (ß-MUS) substrate. RESULTS: We demonstrate the high throughput feasibility of a novel approach for measuring ARSB activities by incorporating tailor-made calibration curves according to each patient's DBS sample quenching properties. The second method is used to calculate ARSB activities by measuring the fluorescence and absorbance parameters in each reaction sample with a single DBS-free calibration curve. CONCLUSIONS: The quantitative correlation between the DBS sample absorbance and its quenching effect can be used to calculate predictive ARSB activities and would serve as an affordable first tier screening test. The method described herein demonstrates the critical importance of adapting the ß-MU calibration curves to each patient's unique DBS sample matrix and its positive impact on the accuracy and reliability of ARSB activity measurements.

Development, validation, and clinical implementation of an assay to measure total antibody response to naglazyme (galsulfase).

Naglazyme (galsulfase, rhASB) was developed as enzyme replacement therapy for mucopolysaccharidosis type VI. Naglazyme generated an IgG antibody response in most patients. To better characterize Naglazyme immunogenicity, a solution phase bridged immunoassay was developed to measure total antibody response regardless of isotype. Overnight incubation of serum dilutions with rhASB labeled with biotin and ruthenium-based tags allowed antibody-antigen complexes to form prior to capture on a streptavidin plate. Neat serum was tolerated in the assay, with a 1:10 screening dilution implemented for testing. At this dilution, the assay was sensitive to 75 ng/ml anti-rhASB. Titers were reported as the highest dilution factor with signal above a 95% confidence interval from naïve individual sera. Precise measurement of titers, within two consecutive dilution factors, was observed across analysts and days. Clinical samples showed similar positive/negative results between the IgG ELISA and the total antibody ECLA, although with an imperfect correlation. Improvements in assay performance and implementation strategy altered some positive clinical samples to negative and vice versa. Comparison of the titer readout for clinical samples with the screening signal illustrates a range of relationships for signal versus sample dilution factor, confirming that signal from a screening dilution cannot directly predict the reported titer.

Morphology of leukocytes from cats affected with alpha-mannosidosis and mucopolysaccharidosis VI (MPS VI).

The morphology and ultrastructure of circulating white blood cells from six Persian and from five Russian Blue/Siamese cats deficient in lysosomal activity of alpha-mannosidase and arylsulfatase B, respectively, were studied and compared to cells from corresponding normal and carrier cats. In cats with mannosidosis, light microscopic examination revealed vacuoles in lymphocytes and monocytes, whereas electron microscopic studies demonstrated additional vacuoles in neutrophils, eosinophils, and basophils. In cats with mucopolysaccharidosis VI (MPS VI), vacuoles containing metachromatic granules were observed in lymphocytes, neutrophils, eosinophils, and monocytes. Ultrastructural studies of these cells identified the accumulation of fibrillar material, which often was associated with lamellated membrane structures.

Empty sella syndrome in a boy with mucopolysaccharidosis type VI (Maroteaux-Lamy).

An 11-year-old boy with mucopolysaccharidosis type VI is presented. Because of optic atrophy, a pneumencephalogram was performed which revealed a relatively large hydrocephalus internus and a very wide, empty sella that filled with air. A Spitz-Holter valve was inserted into a lateral ventricle. STH, TSH, and cortisol response to stress, and thyroid function were normal. LH and prolactin were measurable but close to the lower normal limit. Prolactin did not rise during surgical stress. Those analyses, together with the clinical aspect, rule out severe pituitary malfunction.