Usefulness of urinary glycosaminoglycans assay for a mucopolysaccharidosis-specific screening.

BACKGROUND: Mucopolysaccharidoses (MPS), a group of inherited metabolic disorders characterized by the accumulation of glycosaminoglycans, can be diagnosed early through newborn screening programs. Establishing newborn screening in Morocco is a challenging task for multiple economic and social reasons. Screening in a Moroccan population using 1,9-dimethylmethylene blue urinary glycosaminoglycan (GAG) assays may allow for an earlier diagnosis of MPS. We studied the feasibility of implementing screening in Moroccan children as an alternative to national newborn screening. We determined the reference ranges for GAGs in the Moroccan population, their stability during transport, the effectiveness of this test as a screening procedure for MPS in patients, and its use as a screening test for MPS in the Imssouane region, where the rate of consanguineous marriage is 38%. METHODS: Using dimethylmethylene blue assays, urine samples of 47 MPS patients were analyzed, together with urine samples from healthy controls (n = 368, age ranging from 1 month to 25 years), and from Imssouane region children (n = 350, age ranging from 6 months to 24 month). Precision, linearity, recovery, limits, and stability were tested. RESULTS: Urinary GAGs reference values are age and ethnicity dependent. The validation parameters established displayed great precision and accuracy leading to recoveries according to internationally accepted values for bioanalytical methods. Urinary GAGs were stable for a maximum of 7 weeks at 40 °C. Screening of Imssouane children resulted in the detection of a 6-month-old child, diagnosed with MPS I. CONCLUSIONS: Our results demonstrate the usefulness of quantifying glycosaminoglycans for early screening of MPS.

Disease and subtype specific signatures enable precise diagnosis of the mucopolysaccharidoses.

PURPOSE: Expanding treatments for the mucopolysaccharidoses-a family of genetic disorders-place unprecedented demands for accurate, timely diagnosis because best outcomes are seen with early initiation of appropriate therapies. Here we sought to improve the diagnostic odyssey by measuring specific glycosaminoglycan fragments with terminal residues complicit with the genetic defect resulting in precise diagnosis rather than the usual first-line, ambiguous total glycosaminoglycan determinations that return poor diagnostic yield. METHODS: A derivatizing reagent was added to urine aliquots (0.5 µmol creatinine) before separation of the glycosaminoglycan fragments by liquid chromatography and quantification with electrospray ionization-tandem mass spectrometry using multiple reaction monitoring for 10 targeted fragments plus the internal standard. RESULTS: All 93 mucopolysaccharidosis patients were correctly identified as 1 of 10 subtypes from a total of 723 de-identified subjects-blinded to diagnosis-based on the presence of specific "signature" glycosaminoglycan fragments. Employing reference intervals calculated from 630 unaffected urines, with 99% confidence intervals, provided a laboratory test with 100% specificity and sensitivity. CONCLUSION: This novel urine assay allows diagnosis of 10 mucopolysaccharidosis subtypes in a single test. The precise quantification of unique glycosaminoglycan fragments also enables longitudinal biochemical monitoring following therapeutic interventions.

A selective screening program for the early detection of mucopolysaccharidosis: Results of the FIND project - a 2-year follow-up study.

The mucopolysaccharidoses (MPSs) are underdiagnosed but they are evaluated in few newborn screening programs, probably due to the many challenges remaining, such as the identification of late-onset phenotypes. Systematic screening at the onset of clinical symptoms could help to early identify patients who may benefit from specific treatments. The aim of this prospective study was to assess a novel selective screening program, the FIND project, targeting patients aged 0 to 16 years with clinical manifestations of MPS. The project was designed to increase awareness of these diseases among pediatricians and allow early diagnosis.From July 2014 to June 2016, glycosaminoglycan (GAG) levels normalized to creatinine levels were determined in urine-impregnated analytical paper submitted by pediatricians who had patients with clinical signs and/or symptoms compatible with MPS. When high GAG concentrations were detected, a new liquid urine sample was requested to confirm and identify the GAG present. When a specific form of MPS was suspected, enzyme activity was analyzed using blood-impregnated paper to determine MPS type (I, IIIB, IIIC, IVA, IVB, VI, or VII). Age-specific reference values for GAG were previously established using 145 urine samples from healthy children.GAG levels were normal in 147 (81.7%) of the 180 initial samples received. A liquid sample was requested for the other 33 cases (18.3%); GAG levels were normal in 13 of these and slightly elevated in 12, although the electrophoresis study showed no evidence of MPS. Elevated levels with corresponding low enzymatic activity were confirmed in 8 cases. The mean time from onset of clinical symptoms to detection of MPS was 22 months, and just 2 cases were detected at the beginning of the project were detected with 35 and 71 months of evolution of clinical symptoms. Our screening strategy for MPS had a sensitivity of 100%, a specificity of 85%, and a positive predictive value of 24%.The FIND project is a useful and cost-effective screening method for increasing awareness of MPS among pediatricians and enabling the detection of MPS at onset of clinical symptoms.

Screening for mucopolysaccharidoses in the Turkish population: Analytical and clinical performance of an age-range specific, dye-based, urinary glycosaminoglycan assay.

Comprehensive analytical and diagnostic performance of urinary quantitative GAG analysis with dimethylmethylene blue (DMB) and the age-specific reference ranges were determined in Turkish population, which has a high incidence of MPSs. Precision, linearity, recovery and accuracy/trueness, limits, stability, and effect of interferents were tested according to CLSI guideline. Clinical performance was evaluated with ROC analyses including 45 MPS patients. Intra-day and inter-day precisions were <5% and <11% (CV), respectively. LoD was 9.12mg/L and LoQ was 23.3mg/L. The highest reference values for urinary GAG excretion were determined in an age-specific manner. In the 2-13years age cohort, a cut-off of 89.86mg/g creatinine resulted in 98.07% sensitivity and 93.33% specificity. Proteinuria and hematuria interfered with analysis in some instances. Neither leukocyturia nor pH changes affected the assay. Stability analysis indicated that freezing urine samples for transfer is unnecessary. Of the 45 MPS patient samples evaluated, only three tested negative including MPS II, IVA and VI. Despite limitations due to low levels of urinary GAG excretion in some cases, urinary GAG analysis with DMB with its technical simplicity, low cost, and precise quantitative results, is a valuable screening method, particularly in populations with a high rate of MPSs.

Glycan-based biomarkers for mucopolysaccharidoses.

The mucopolysaccharidoses (MPS) result from attenuation or loss of enzyme activities required for lysosomal degradation of the glycosaminoglycans, hyaluronan, heparan sulfate, chondroitin/dermatan sulfate, and keratan sulfate. This review provides a summary of glycan biomarkers that have been used to characterize animal models of MPS, for diagnosis of patients, and for monitoring therapy based on hematopoietic stem cell transplantation and enzyme replacement therapy. Recent advances have focused on the non-reducing terminus of the glycosaminoglycans that accumulate as biomarkers, using a combination of enzymatic digestion with bacterial enzymes followed by quantitative liquid chromatography/mass spectrometry. These new methods provide a simple, rapid diagnostic strategy that can be applied to samples of urine, blood, cerebrospinal fluid, cultured cells and dried blood spots from newborn infants. Analysis of the non-reducing end glycans provides a method for monitoring enzyme replacement and substrate reduction therapies and serves as a discovery tool for uncovering novel biomarkers and new forms of mucopolysaccharidoses.

Quantitation of urinary glycosaminoglycans using dimethylene blue as a screening technique for the diagnosis of mucopolysaccharidoses: an evaluation.

BACKGROUND: The mucopolysaccharidoses (MPSs) are a group of inherited disorders due to defects in lysosomal enzymes catalysing the breakdown of glycosaminoglycans (GAGs). Usually they are detected using techniques to separate the accumulating GAGs in urine. However more recently an improved dye-based method measuring total urine GAG concentrations has become available. We have evaluated this method in the context of its application as a general screening technique in a routine metabolic laboratory. METHOD: An automated method for the quantitation of urinary GAGs using dimethylene blue was developed on a Cobas Fara analyser and evaluated in terms of its specificity, sensitivity and value in the diagnosis of the MPSs. The test was applied to 6156 anonymized urine samples received for tests for inherited metabolic diseases and to 121 samples from 85 patients with a variety of proven MPSs. RESULTS: A substantial number of samples from unaffected individuals gave abnormal results while a significant number of samples from patients with MPSs gave results within normal limits. CONCLUSION: In the context of our laboratory, it was not appropriate for use as a screening technique when applied to all specimens received for metabolic tests or as a first-line screening test for samples where mucopolysaccharide analysis was requested. However it was retained and used in parallel with cellulose acetate electrophoresis to aid interpretation.

Two-dimensional NMR spectroscopy of urinary glycosaminoglycans from patients with different mucopolysaccharidoses.

Patients with different types of mucopolysaccharidoses (MPS) lack specific lysosomal enzymes, which leads to tissue accumulation and urinary excretion of glycosaminoglycans (GAGs). Since little is known about the molecular composition of the excreted GAG fragments, we used two-dimensional [1H,13C]-correlation nuclear magnetic resonance (NMR) spectroscopy for a detailed analysis of the urinary GAGs of patients with MPS types I, II, IIIA, IVA and VI. The method revealed that the molecular structures of the excreted GAGs, i.e. heparan sulfate (HS), dermatan sulfate (DS), chondroitin sulfate (CS), and keratan sulfate (KS) are clearly distinct for the different MPS types. The chain terminal residues that are the normal substrates for the defective enzymes constitute characteristic sets of signals for each MPS type. The GAG chains show variations in carbohydrate composition and sulfation patterns that can be related to the different MPS types and clinical features. For example, two patients with MPS IIIA (M. Sanfilippo) with signs of CNS degeneration but only mild somatic features excrete a highly sulfated variant of HS, resembling HS in porcine brain, whereas a patient with MPS I (M. Scheie) and two patients with MPS II (M. Hunter), who present primarily with coarse facial features, joint contractures and skeletal deformities excrete a different type of HS with lower sulfation. In another case study, a patient with MPS IVA (M. Morquio), who presented mainly with skeletal dysplasia, excreted not only excessive amounts of KS but also a highly sulfated CS variant, resembling CS in articular cartilage. The high-resolution NMR analysis of urinary GAGs presented here for the first time provides a solid basis for future studies with a larger number of patients to further explore pathogenesis and course of the MPS diseases.

Determination of monosaccharides and disaccharides in mucopolysaccharidoses patients by electrospray ionisation mass spectrometry.

The mucopolysaccharidoses are a group of lysosomal storage disorders characterised by the storage of glycosaminoglycans. With the exception of Hunters syndrome (MPS II), which is X-linked, they are autosomal recessively inherited resulting in a defect in any one of 10 lysosomal enzymes needed to catabolise glycosaminoglycans. The type and size of the glycosaminoglycans stored in lysosomes are determined by the particular enzyme deficiency. These glycosaminoglycan elevations are subsequently observed in tissue, circulation, and urine. A method has been developed for the derivatisation and quantification of sulfated N-acetylhexosamine-containing mono- and disaccharides from patient samples by electrospray ionisation tandem mass spectrometry. Urine from most mucopolysaccharidoses types had significant increases in di- and monosulfated N-acetylhexosamines (GalNAc4,6S, GalNAc6S, GalNAc4S, or GlcNAc6S) and monosulfated N-acetylhexosamine-uronic acid disaccharides (GalNAc6S-UA, GalNAc4S-UA, or GlcNAc6S-UA). Analysis of plasma and dried blood spots on filter paper collected from mucopolysaccharidoses patients showed elevations of total monosulfated N-acetylhexosamines but less than that seen in urine. Urine samples from bone marrow transplant recipients, mucopolysaccharidosis IVA and mucopolysaccharidosis VI patients, showed decreases in HexNAcS, HexNAcS(2)/GalNAc4,6S, and HexNAcS-UA post-transplant. This decrease correlated with clinical improvement to levels comparable with those identified in patients with less severe phenotypes. These metabolic markers therefore have potential applications in diagnosis, phenotype prediction and monitoring of current and future therapies, particularly for the mucopolysaccharidosis IIID, IVA, VI, and multiple sulfatase deficiency. This paper reports a sensitive and simple method for the measurement of sulfated N-acetylhexosamines and sulfated disaccharides shown to be elevated in some mucopolysaccharidosis and multiple sulfatase deficient patients.

El laboratorio clínico en el abordaje diagnóstico de las mucopolisacaridosis: presentación de un caso / Mucopolysaccharidoses diagnosis and clinic laboratory

Las mucopolisacaridosis son un grupo de alteraciones genéticas clasificadas dentro de los "Errores Innatos del Metabolismo" (EIM). Estos EIM son informados por varios autores con una incidencia baja (de 1:5000 a 1:320000 nacidos vivos). Sin embargo pueden ser más frecuentes de lo comunicado en la literatura, sobre todo cuando se tienen presentes como causa de enfermedad y son aplicados los estudios de escrutinio neonatal actualmente accesibles. Se detallan algunos de los análisis de laboratorio utilizados para su diagnóstico, y se describe brevemente un caso de mucopolisacaridosis infantil, que resalta la importancia del diagnóstico precoz mediante la aplicación de métodos sencillos. Se sugiere implementar y aplicar estos procedimientos en todo laboratorio clínico, para apoyar el diagnóstico oportuno de dichos desórdenes genéticos

Estudio bioquímico de un paciente con enfermedad lisosomal heredometabólica / Biochemical study of a patient with lysosomal inherited metabolic disease

Se reporta el caso de una niña de 18 meses de edad que presenta características fenotípicas de tipo "hurleroide, a la cual se le realizó la determinación de las enzimas relacionadas con el metabolismo de los mucopolisacáridos (MPS), así como la caracterización cinética de la enzima denominada alfa-L-iduronidasa, cuya deficiencia condujo al diagnóstico de dicha enfermedad

A screening method for mucopolysaccharidoses with increased urinary excretion of sulfated N-acetylhexosamines.

Patients with mucopolysaccharidosis have been reported to excrete elevated amounts of sulfated N-acetylhexosamines in their urine. Based on this finding, a new and simple colorimetric screening test for these disorders is presented. Chromatography of whole urine on Dowex AG 1-X8, from each of 23 normal controls, 5 patients with mucopolysaccharidosis and one patient with multiple sulfatase deficiency, was used to separate the sulfated hexosamines. The fractions eluted with 2M NaCl were analyzed according to the method of Reissig. Patients with Sanfilippo syndrome, type A, Sanfilippo syndrome, type D, Maroteaux-Lamy syndrome, Morquio syndrome, type A, and multiple sulfatase deficiency were clearly distinguished from normal controls. The procedure appeared most sensitive for Sanfilippo syndrome, type D, and multiple sulfatase deficiency, each of which involves deficient activity of N-acetylglucosamine 6-sulfate sulfatase.

Age-related concentrations of glycosaminoglycans in Random urine: a contribution to the lavoratorial detection of mucopolysaccharidoses

As mucopolissacridoses (MPS) säo rotineiramente detectadas pela dosagem de glicosaminoglicanos (GAG), usualmente realizadas em urina de 24 horas. Como os pacientes suspeitos säo geralmente crianças sem controle miccional e a excreçäo de GAG varia com a idade, nós estudamos os valores de GAG em amostras ocasionais de urina de 78 crianças normais e de 16 pacientes com MPS de 6 meses a 12 anos de idade. Este trabalho teve objetivos (a) estabelecer valores normais para os índices urinários GAG/volume (GAG/L e GAG/cratinina (GAG/creat), relacionados com a idade, e (b) testar a utilidade desses índices na identificaçäo de pacientes com MPS. Nos dois índices a concentraçäo de GAG diminui com a idade. Os índices GAG/L e GAG/CREAT discriminaram todos os pacientes, com uma exceçäo em cada índice, em pacientes diferentes. Nossos resultados indicam que amostras ocasionais de urina säo adequadas para a dosagem de GAG desde que os índices de GAG/L e GAG/CREAT sejam avaliados simultanemante e que a idade do paciente seja considerada na interpretaçäo dos resultados, especialmente em crianças pequenas

Selective urinary screening for mucopolysaccharidoses.

Two methods for analysis of urinary glycosaminoglycans (GAGs) have been modified and improved for efficient screening of mucopolysaccharidoses. Urinary excretion of GAGs was estimated by spectrophometric measurement of alcian blue complex formation and was used in conjunction with qualitative analysis by thin layer chromatography. After normal variation in GAG excretion was established using 120 urine samples, these screening methods were applied to a total of 2057 urine samples over 4 years. Six patients with abnormal urinary GAG excretion had a mean of 50.5 +/- 20.6 mg GAG/mmol creatinine compared to 3.4 +/- 2.9 for age-matched controls. Qualitative analysis by thin layer chromatography using alternating solvent systems identified the GAGs excreted in excess and facilitated selection of specific enzyme assays for final confirmation. Six cases were diagnosed prospectively and demonstrate these quantitative and qualitative methods to be economical, efficient, and suitable for clinical use.

[Mass-screening for acid mucopolysaccharidosis using urine samples by modified HCl-albumin turbidity method].

To establish a method for screening acid mucopolysaccharidosis (AMPS), the HCl-albumin turbidity method was reexamined and modified for semiquantitative analysis. Mucopolysaccharide samples and HCl-albumin in buffer solution were incubated at 37 degrees C for 30 minutes, and the optical density at 600 nm (OD 600) of the reaction mixtures were measured. When the same amount of the samples were tested, OD 600 of dermatan sulfate and hyaluronic acid were higher than that of keratan sulfate. When the cut-off point of OD 600 was set at 0.100 based on the values obtained from randomly selected subjects, only 3 of 328 urine samples (0.9%) showed value higher than the cut off value. The values in five patients with AMPS were all above 0.800 without overlapping those of the control subjects. These results suggest that mass-screening for AMPS using urine sample is possible by the partially modified HCl-albumin turbidity method with the cut-off point of OD 600 set at 0.100.

Diagnostic test for mucopolysaccharidosis. II. Rapid quantification of glycosaminoglycan in urine samples collected on a paper matrix.

The direct 1,9-dimethylmethylene blue (DMB) method for quantifying sulfated glycosaminoglycan (GAG) in urine (Clin Chem 1989; 35:374-9) has been adapted to a convenient means for sample collection and transport as a test to identify individuals with mucopolysaccharidosis (MPS) storage diseases. Results correlated moderately well (r = 0.85) with those of a commonly used, but more laborious, quantitative method. In studying factors to maximize differentiation of pathological from normal values, we found that GAG excretion (expressed as milligrams GAG per gram creatinine) fits a logarithmic function with respect to age and varies markedly below age five years. This must be considered in developing normative values and forming diagnoses. Of 112 separate urine specimens obtained from 41 MPS patients representing the major MPS diseases, glycosaminoglycan excretion by all exceeded that for age-matched normal individuals. The convenience of this method allowed us to establish the first normative values for three-week-old infants (n = 435) found to have a mean glycosaminoglycan excretion of 179 (SD 86.3) mg of GAG per gram of creatinine. This method improves the diagnostic capability for those MPS diseases that have been particularly difficult to identify (Sanfilippo's syndrome and Morquio's syndrome), and may also provide a test for other disorders with previously unrecognized abnormal excretion of glycosaminoglycan (e.g., mucolipidosis and acromesomelic dysplasia). Most importantly, this MPS diagnostic test is unique in its suitability for mass screening of newborn infants.

Diagnostic test for mucopolysaccharidosis. I. Direct method for quantifying excessive urinary glycosaminoglycan excretion.

This direct method for quantifying excessive urinary glycosaminoglycan excretion exploits the specific binding of 1,9-dimethylmethylene blue (DMB). The procedure obviates cumbersome and labor-intensive procedures for separating glycosaminoglycans from other constituents of urine. Pediatric pharmaceutical formulations (except heparin), in concentrations expected in urine, do not interfere with spectrophotometry, nor does protein. Results can be expressed in terms of urinary creatinine; thus the test is applicable to very small urine specimens (0.1 mL), such as those obtainable from neonates. In a pilot study, results of the direct DMB test for 48 urine specimens agreed with the clinical diagnosis, and quantitative measurements correlated moderately (r = 0.76) with results of a commonly used procedure (carbazole-borate reactivity after precipitation with cetylpyridinium chloride). The present method was also used to assess metabolic correction in a patient with Hurler's syndrome after treatment by bone-marrow transplantation. This quantitative method surmounts the major technical problems of developing mass screening programs for infants, thus offering the potential for earlier diagnosis and treatment of mucopolysaccharidosis diseases.

A ten-year follow-up study of urinary screening for inherited metabolic disorders.

Urine samples of 200,000 sucklings in 6th-8th month after delivery was analyzed by means of chromatographic screening test for amino acids, sugars and muccopolysacharides. In 1.4% of them persistent and in 51.6% transitory hyperaminoacidurias, in 0.15% persistent and 12.5% transitory melliturias and in 0.05% alcaptonuria were detected. No one case of muccopolysacharidosis was detected. 239 persistent metabolic disorders were detected. These disorders were not possible to detect by means of an obligatory screening test performed on 5th-6th day after delivery from capillary blood. All positive detected cases were verified by detailed metabolic investigation (thin layer, liquid and gas-chromatography).