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

<div><p>Background</p><p>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.</p><p>Methodology</p><p>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.</p><p>Findings</p><p>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.</p><p>Interpretation</p><p>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.</p></div

Influence of storage conditions on GALNS activity in DBS.

<p>Stability tests show rthat filtercards with DBS stored in zip bags maintain their enzymatic activity when stored at– 20°C and 4°C up to 35 days, while samples stored at a temperature of 27°C and above lose over 40% of their activity in the first 5 days. Ideal sample transport should be at a maximum of 22°C and storage at 4°C or below.</p

TripleQuad MRM-MS detection of 4-MU.

<p>Hydroxyl-chromen-2H-one compounds can be detected in Q1 scan as a (M-H)^- ion and, under specific collision energy, the hetero-cycle is broken with a neutral loss of the fragment containing–COO•. We propose that the remaining fragment undergoes a molecular rearrangement to obtain a more stable structure. For enzymatic product detection and quantification, two transitions are monitored: 175/119 (4-MU) and 217/160 (internal standard). <b>A.</b> Collision induced dissociation fragmentation spectrum (MS²) of the analyte (4-MU), obtained using an ABSciex 5500, and MRM-MS transition spectrum monitored during the MPS IV assays (175/119); B. Collision induced dissociation fragmentation spectrum (MS2) of the internal standard (4-propyl-5-hydroxy-7-methyl-2H-chromen-2-one), obtained using an ABSciex 5500, and MRM-MS transition spectrum monitored during the MPS IV assays (217/160).</p

Diagnosis of Morquio Syndrome in Dried Blood Spots Based on a New MRM-MS Assay - Fig 3

<p><b>A. Enzymatic GALNS assay in DBS</b> 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>B. β- galactosidase in DBS</b>, the proposed control enzyme test, carried out on the control samples and MPS IVA patient samples, show similar activity in both groups.</p

GALNS mutations of patients diagnosed and selected as reference for the MPS IVA enzymatic assay development.

<p>^aNot described in HGMD, present in CentoMD [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131228#pone.0131228.ref020" target="_blank">20</a>]</p><p>^bPatients carry two heterozygous mutations</p><p>^cPatient carries three heterozygous mutations/variant</p><p>GALNS mutations of patients diagnosed and selected as reference for the MPS IVA enzymatic assay development.</p

Comparison of cardiac troponin I levels.

<p>Presented are patients with Fabry disease (FD) and left ventricular hypertrophy (LVH) (n = 17) versus FD patients without LVH (n = 18) and non-FD patients with LVH of other cause (n = 17). The cut-off level for cTnI in diagnosing myocardial infarction (≥0.04 ng/ml) is indicated in the figure.</p

Patients with Fabry disease: comparison of patients with cTnI elevation ≥0.04 ng/ml versus without.

<p>Patients with Fabry disease: comparison of patients with cTnI elevation ≥0.04 ng/ml versus without.</p

Characteristics in 17 age- and sex-matched individuals with left ventricular hypertrophy due to hypertension.

<p>Characteristics in 17 age- and sex-matched individuals with left ventricular hypertrophy due to hypertension.</p