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      A Multiplex Assay for the Diagnosis of Mucopolysaccharidoses and Mucolipidoses


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          Diagnosis of the mucopolysaccharidoses (MPSs) generally relies on an initial analysis of total glycosaminoglycan (GAG) excretion in urine. Often the dimethylmethylene blue dye-binding (DMB) assay is used, although false-negative results have been reported. We report a multiplexed diagnostic test with a high sensitivity for all MPSs and with the potential to identify patients with I-cell disease (ML II) and mucolipidosis III (ML III).


          Urine samples of 100 treatment naive MPS patients were collected and analyzed by the conventional DMB assay and a multiplex assay based on enzymatic digestion of heparan sulfate (HS), dermatan sulfate (DS) and keratan sulfate (KS) followed by quantification by LC-MS/MS. Specificity was calculated by analyzing urine samples from a cohort of 39 patients suspected for an inborn error of metabolism, including MPSs.


          The MPS cohort consisted of 18 MPS I, 16 MPS II, 34 MPS III, 10 MPS IVA, 3 MPS IVB, 17 MPS VI and 2 MPS VII patients. All 100 patients were identified by the LC-MS/MS assay with typical patterns of elevation of HS, DS and KS, respectively (sensitivity 100%). DMB analysis of the urine was found to be in the normal range in 10 of the 100 patients (sensitivity 90%). Three out of the 39 patients were identified as false-positive, resulting in a specificity of the LS-MS/MS assay of 92%. For the DMB this was 97%. All three patients with MLII/MLIII had elevated GAGs in the LC-MS/MS assay while the DMB test was normal in 2 of them.


          The multiplex LC-MS/MS assay provides a robust and very sensitive assay for the diagnosis of the complete spectrum of MPSs and has the potential to identify MPS related disorders such as MLII/MLIII. Its performance is superior to that of the conventional DMB assay.

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          The frequency of lysosomal storage diseases in The Netherlands.

          We have calculated the relative frequency and the birth prevalence of lysosomal storage diseases (LSDs) in The Netherlands based on all 963 enzymatically confirmed cases diagnosed during the period 1970-1996. The combined birth prevalence for all LSDs is 14 per 100,000 live births. Glycogenosis type II is the most frequent LSD with a birth prevalence of 2.0 per 100,000 live births, representing 17% of all diagnosed cases. Within the group of lipidoses, metachromatic leukodystrophy (MLD) is the most frequent LSD. MLD was diagnosed in 24% of lipidoses and the calculated birth prevalence was 1.42 per 100,000 for all types combined. Krabbe disease, diagnosed in 17% of cases, also belongs to the more frequent lipid storage diseases in The Netherlands with a birth prevalence of 1.35 per 100,000. The birth prevalence of Gaucher disease, commonly regarded as the most frequent lipid storage disease is 1.16 per 100,000 for all types combined. The combined birth prevalence for all lipid storage diseases is 6.2 per 100,000 live births. Within the group of mucopolysaccharidoses (MPSs), MPS I has the highest calculated birth prevalence of 1.19 per 100,000 (25% of all cases of MPS diagnosed), which is slightly more frequent than MPS IIIA with an estimated birth prevalence of 1.16 per 100,000. As a group, MPS III comprises 47% of all MPS cases diagnosed and the combined birth prevalence is 1.89 per 100,000 live births. The birth prevalence of MPS II is 0.67 per 100,000 (1.30 per 100,000 male live births). All other MPSs are rare. The combined birth prevalence for all MPSs is 4.5 per 100,000 live births. Mucolipidoses and oligosaccharidoses are very rare with birth prevalences between 0.04 and 0.20 for individual diseases. Only 49 cases were diagnosed between 1970 and 1996. Their combined birth prevalence is 1.0 per 100,000 live births.
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            Increased levels of glycosaminoglycans during septic shock: relation to mortality and the antibacterial actions of plasma.

            Glycosaminoglycans (GAGs) are structurally heterogeneous negatively charged polysaccharides. Endothelial GAGs, also known as glycocalyx, are involved in capillary permeability. In rat venules stimulated with proinflammatory substances ex vivo, the GAG-containing proteoglycan, syndecan-1, is shed from the endothelium. We wanted to investigate if we could trace the same response during septic shock as reflected in the circulating GAG levels. Arterial plasma samples were collected from 18 consecutive septic shock patients admitted to our intensive care unit. Plasma GAGs were measured with an Alcian blue slot binding assay, and syndecan-1 levels were measured with enzyme-linked immunosorbent assay. Effects of GAGs on the antibacterial activity of plasma were assessed by a radial diffusion assay. The median plasma GAG level was significantly higher in the septic shock patients than in matched controls (median [interquartile range], 2.7 microg/mL [1.9 - 4.8 microg/mL] vs. 1.8 microg/mL [1.7 - 2.0 microg/mL]). Furthermore, the GAG levels were significantly higher in nonsurvivors (4.6 microg/mL [3.1 - 8.8 microg/mL], n = 8) than survivors (1.8 microg/mL [1.6 - 2.6 microg/mL], n = 10). The syndecan-1 levels were also increased in the patients compared with controls (246 ng/mL [180 - 496 ng/mL] vs. 26 ng/mL [23 - 31 ng/mL]) and correlated to the cardiovascular Sequential Organ Failure Assessment (SOFA) score. The GAGs inhibited the endogenous antibacterial activity of plasma as well as isolated antimicrobial peptides. The concentrations required were in the same range as the GAG levels measured in the patients. These results show that the GAG levels are increased in septic shock patients, possibly reflecting peripheral endothelial cell damage. We also found that GAGs in relevant concentrations neutralize antimicrobial peptides in plasma.
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              Dimethylmethylene blue-based spectrophotometry of glycosaminoglycans in untreated urine: a rapid screening procedure for mucopolysaccharidoses.

              Glycosaminoglycans (GAGs) are measured in urine to screen for mucopolysaccharidoses. Other assay procedures are only qualitative (spot tests), can give false-negative results (spot tests, turbidity tests), or are relatively laborious (uronic acid-carbazole test). The present spectrophotometric procedure, based on the color reaction with dimethylmethylene blue (DMB), can be performed directly on untimed urine samples without prior precipitation. Reference values were age dependent. We tested urines of 27 patients with various mucopolysaccharidoses and compared results by three other procedures (cetylpyridinium chloride turbidity tests at pH 4.8 and at pH 7.0, and the uronic acid-carbazole test). In the DMB assay, GAGs were increased in 26 of the 27 patients. The exception was a Morquio A patient, whose activity of the defective enzyme was higher than in classical Morquio patients. Uronic acid, measured in precipitated GAG by the carbazole test, was increased in 23 of the 25 patients so tested. In the turbidity test at pH 7.0, values were increased in 24 of the 27 patients. In contrast, with the citrate-buffered (pH 4.8) turbidity measurement, GAG content was increased in only 19 of the 27 patients. This rapid and easy DMB method is a reliable screening procedure for mucopolysaccharidoses and compares well with procedures used hitherto.

                Author and article information

                Role: Editor
                PLoS One
                PLoS ONE
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                25 September 2015
                : 10
                : 9
                : e0138622
                [1 ]Department of Pediatric Metabolic Diseases, Emma Children’s Hospital and Amsterdam Lysosome Center ‘Sphinx’, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
                [2 ]Laboratory for Genetic Metabolic Diseases, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
                [3 ]Translational Metabolic Laboratory, Departments of Neurology & Laboratory Medicine, Radboud University Medical Centre, Nijmegen, The Netherlands
                [4 ]Department of Pediatrics, Center for Lysosomal and Metabolic Diseases, Erasmus Medical Center, Rotterdam, The Netherlands
                [5 ]Department of Clinical Genetics, Center for Lysosomal and Metabolic Diseases, Erasmus Medical Center, Rotterdam, The Netherlands
                University of Patras, GREECE
                Author notes

                Competing Interests: Biomarin pharmaceutical inc financially supported development of the assay. Furthermore, this does not alter adherence to PLOS ONE policies on sharing data and materials. The authors have declared that no competing interests exist.

                Conceived and designed the experiments: EJL FAW NVV. Performed the experiments: TW HVL WK NVV. Analyzed the data: EJL. Contributed reagents/materials/analysis tools: DJL EO ATVDP GJR RAW FAW. Wrote the paper: EJL FAW NVV.

                Copyright @ 2015

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

                : 13 May 2015
                : 31 August 2015
                Page count
                Figures: 5, Tables: 3, Pages: 13
                Biomarin pharmaceutical inc financially supported development of the assay. Validation of the diagnostic accuracy was not funded by this sponsor. The authors received no other funding for this work. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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