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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: 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.This technique will significantly simplify the early detection of MPS IVA patients.

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

A. Enzymatic GALNS assay in DBS shows a statistically significant difference (p<0.0001 in two-tailed Man-Whitney test) between samples of healthy controls and the samples of affected MPS IVA patients. B. β- galactosidase in DBS, the proposed control enzyme test, carried out on the control samples and MPS IVA patient samples, show similar activity in both groups.
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pone.0131228.g003: A. Enzymatic GALNS assay in DBS shows a statistically significant difference (p<0.0001 in two-tailed Man-Whitney test) between samples of healthy controls and the samples of affected MPS IVA patients. B. β- galactosidase in DBS, the proposed control enzyme test, carried out on the control samples and MPS IVA patient samples, show similar activity in both groups.

Mentions: The pathological range of GALNS activity was determined on 24 MPS IVA patients and determined to be between 0 and 0.64 μmol/L blood /h (mean ± standard deviation: 0.35 ± 0.21)—see Fig 3.


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)

A. Enzymatic GALNS assay in DBS shows a statistically significant difference (p<0.0001 in two-tailed Man-Whitney test) between samples of healthy controls and the samples of affected MPS IVA patients. B. β- galactosidase in DBS, the proposed control enzyme test, carried out on the control samples and MPS IVA patient samples, show similar activity in both groups.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131228.g003: A. Enzymatic GALNS assay in DBS shows a statistically significant difference (p<0.0001 in two-tailed Man-Whitney test) between samples of healthy controls and the samples of affected MPS IVA patients. B. β- galactosidase in DBS, the proposed control enzyme test, carried out on the control samples and MPS IVA patient samples, show similar activity in both groups.
Mentions: The pathological range of GALNS activity was determined on 24 MPS IVA patients and determined to be between 0 and 0.64 μmol/L blood /h (mean ± standard deviation: 0.35 ± 0.21)—see Fig 3.

Bottom Line: 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.This technique will significantly simplify the early detection of MPS IVA patients.

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