title: Diagnosis of Morquio Syndrome in Dried Blood Spots Based on a New MRM-MS Assay.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Centogene AG, Rostock, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Cozma
authors.affiliation: Centogene AG, Rostock, Germany.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Eichler
authors.affiliation: Centogene AG, Rostock, Germany.
authors.forename: Gyula
authors.initials: G
authors.lastname: Wittmann
authors.affiliation: Centogene AG, Rostock, Germany.
authors.forename: Alba
authors.initials: A
authors.lastname: Flores Bonet
authors.affiliation: Centogene AG, Rostock, Germany.
authors.forename: Guido Johannes
authors.initials: GJ
authors.lastname: Kramp
authors.affiliation: Albrecht-Kossel-Institute, University of Rostock, Rostock, Germany.
authors.forename: Anne-Katrin
authors.initials: AK
authors.lastname: Giese
authors.affiliation: Centogene AG, Rostock, Germany; Albrecht-Kossel-Institute, University of Rostock, Rostock, Germany.
authors.forename: Arndt
authors.initials: A
authors.lastname: Rolfs
mesh_terms: D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D009085:Mucopolysaccharidosis IV / Q000097:blood / Q000175:diagnosis*