Diagnosis of Morquio Syndrome in Dried Blood Spots Based on a New MRM-MS Assay

Mucopolysaccharidosis IVA (MPS IVA; Morquio A disease) is an autosomal recessive disease caused and characterized by a decreased activity of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), resulting in accumulation of keratan sulfate and chondroitin-6-sulfate in tissues and secondary organ damage...

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Published in:	PloS one Vol. 10; no. 7; p. e0131228
Main Authors:	Cozma, Claudia, Eichler, Sabrina, Wittmann, Gyula, Flores Bonet, Alba, Kramp, Guido Johannes, Giese, Anne-Katrin, Rolfs, Arndt
Format:	Journal Article
Language:	English
Published:	United States Public Library of Science 06-07-2015 Public Library of Science (PLoS)
Subjects:	Assaying Blood Chondroitin sulfate Chromatography Damage detection Diagnosis Disease Enzymes Galactosidase Humans Identification Innovations Keratan sulfate Mass spectrometry Mass Spectrometry - methods Medical diagnosis Medical screening Molecular structure Mucopolysaccharidosis Mucopolysaccharidosis IV - blood Mucopolysaccharidosis IV - diagnosis Mutation N-Acetylgalactosamine Patients Scientific imaging Spectrometry Spots Substrates Sulfate Sulfates Tissues Urine β-Galactosidase
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Summary:	Mucopolysaccharidosis IVA (MPS IVA; Morquio A disease) is an autosomal recessive disease caused and characterized by a decreased activity of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), resulting in accumulation of keratan sulfate and chondroitin-6-sulfate in tissues and secondary organ damage. Recently approved enzyme replacement therapy renders the easy and early identification of MPS IVA of out-most importance. We propose a completely new assay for the stable and reproducible detection of GALNS deficiency in dry blood spots (DBS). For the validation blood samples were taken from 59 healthy individuals and 24 randomly selected genetically confirmed MPS IVA patients. The material extracted from DBS was incubated with a 4-methylumbelliferyl-β-D-galactopyranoside-6-sulfate as a specific substrate. Final enzymatic product, 4-methylumbelliferone, obtained after adding exogenous beta-galactosidase, was quantified by LC/MRM-MS (liquid-chromatography/multiple-reaction-monitoring mass-spectrometry). 4-propyl-5-hydroxy-7-methyl-2h-chromen-2-one was used as internal standard, a compound with a similar molecular structure and fragmentation pattern in negative ion mode as 4-methylumbelliferone. The enzymatic assay yielded a positive and negative predictive value of 1.0 for genetically confirmed MPS IVA patients (GALNS activity of 0.35 ± 0.21 μmol/L/h) and for controls with normal GALNS activity (23.1 ± 5.3 μmol/L /h). With present enzymatic conditions, the reaction yield in dried blood spots is at least 20 fold higher than any previously reported data with other assays. The present LC/MRM-MS based assay for MPS IVA diagnosis provides an easy, highly-standardized, accurate and innovative quantification of the enzymatic product in vitro and distinguishes perfectly between MPS IVA affected patients and normal controls. This technique will significantly simplify the early detection of MPS IVA patients.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Competing Interests: The following authors are employees of Centogene (Rostock, Germany): Claudia Cozma, Sabrina Eichler, Gyula Wittmann, Alba Flores Bonet, Guido Johannes Kramp. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials. Conceived and designed the experiments: AR CC. Performed the experiments: CC AFB. Analyzed the data: CC AR SE. Contributed reagents/materials/analysis tools: CC SE GW AFB GJK AR. Wrote the paper: CC AKG AR.
ISSN:	1932-6203 1932-6203
DOI:	10.1371/journal.pone.0131228