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

Cozma C, Eichler S, Wittmann G, Flores Bonet A, Kramp GJ, Giese AK, Rolfs A - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Centogene AG, Rostock, Germany.

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.

Methodology: 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.

Findings: 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.

Interpretation: 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.

No MeSH data available.


Related in: MedlinePlus

Total Ion Chromatogram profiles of 4-MU at constant concentration of internal standard obtained with TripleQuad MRM-MSforA—a blank sample or filter paper incubated in the same manner as the blood samples (that contain 4-MU present at the beginning of the enzymatic reaction as a byproduct of the synthesis); B—pathological blood sample (with similar TIC profile as the blank sample, used as a quality control in each assay); C—a normal activity blood sample (healthy control with an average GALNS activity used as a quality control in each assay); D—a high activity blood sample (a sample with atypically high activity of lysosomal enzymes).
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pone.0131228.g002: Total Ion Chromatogram profiles of 4-MU at constant concentration of internal standard obtained with TripleQuad MRM-MSforA—a blank sample or filter paper incubated in the same manner as the blood samples (that contain 4-MU present at the beginning of the enzymatic reaction as a byproduct of the synthesis); B—pathological blood sample (with similar TIC profile as the blank sample, used as a quality control in each assay); C—a normal activity blood sample (healthy control with an average GALNS activity used as a quality control in each assay); D—a high activity blood sample (a sample with atypically high activity of lysosomal enzymes).

Mentions: For all the MPS 4a tests, the standard curves preparation also incorporated steps that mimic the those used in the preparation of investigated blood samples (dilution in stop buffer, mixing with the internal standard, liquid—liquid extraction) thus measurement differences caused by traces of salts or low extraction yield were eliminated. Examples of TIC (total ion chromatogram) obtained by LC/MRM-MS are shown in Fig 2 for a blank sample (A new blank sample is measured in every measurement as background to be subtracted from the values obtained for the samples investigated. This step is necessary due to the free 4-MU present in the substrate before the start of the reaction), a pathological sample (similar in profile with the blank sample), a normal control sample with average GALNS activity, and a sample with high lysosomal enzyme activity (2 x higher than the average).


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

Cozma C, Eichler S, Wittmann G, Flores Bonet A, Kramp GJ, Giese AK, Rolfs A - PLoS ONE (2015)

Total Ion Chromatogram profiles of 4-MU at constant concentration of internal standard obtained with TripleQuad MRM-MSforA—a blank sample or filter paper incubated in the same manner as the blood samples (that contain 4-MU present at the beginning of the enzymatic reaction as a byproduct of the synthesis); B—pathological blood sample (with similar TIC profile as the blank sample, used as a quality control in each assay); C—a normal activity blood sample (healthy control with an average GALNS activity used as a quality control in each assay); D—a high activity blood sample (a sample with atypically high activity of lysosomal enzymes).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492791&req=5

pone.0131228.g002: Total Ion Chromatogram profiles of 4-MU at constant concentration of internal standard obtained with TripleQuad MRM-MSforA—a blank sample or filter paper incubated in the same manner as the blood samples (that contain 4-MU present at the beginning of the enzymatic reaction as a byproduct of the synthesis); B—pathological blood sample (with similar TIC profile as the blank sample, used as a quality control in each assay); C—a normal activity blood sample (healthy control with an average GALNS activity used as a quality control in each assay); D—a high activity blood sample (a sample with atypically high activity of lysosomal enzymes).
Mentions: For all the MPS 4a tests, the standard curves preparation also incorporated steps that mimic the those used in the preparation of investigated blood samples (dilution in stop buffer, mixing with the internal standard, liquid—liquid extraction) thus measurement differences caused by traces of salts or low extraction yield were eliminated. Examples of TIC (total ion chromatogram) obtained by LC/MRM-MS are shown in Fig 2 for a blank sample (A new blank sample is measured in every measurement as background to be subtracted from the values obtained for the samples investigated. This step is necessary due to the free 4-MU present in the substrate before the start of the reaction), a pathological sample (similar in profile with the blank sample), a normal control sample with average GALNS activity, and a sample with high lysosomal enzyme activity (2 x higher than the average).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Centogene AG, Rostock, Germany.

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.

Methodology: 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.

Findings: 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.

Interpretation: 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.

No MeSH data available.


Related in: MedlinePlus