RESUMO
Eleven patients from Yakutia with a new lysosomal disease assumed then as mucopolysaccharidosis-plus syndrome (MPS-PS) were reported by Gurinova et al. in 2014. Up to now, a total number of 39 patients have been reported; in all of them, the c.1492C>T (p.Arg498Trp) variant of the VPS33A gene was detected. Here, we describe the first Polish MPS-PS patient with a novel homozygous c.599G>C (p.Arg200Pro) VPS33A variant presenting over 12 years of follow-up with some novel clinical features, including fetal ascites (resolved spontaneously), recurrent joint effusion and peripheral edemas, normal growth, and visceral obesity. Functional analyses revealed a slight presence of chondroitin sulphate (only) in urine glycosaminoglycan electrophoresis, presence of sialooligosaccharides in urine by thin-layer chromatography, and normal results of lysosomal enzymes activity and lysosphingolipids concentration in dried blood spot. The comparison with other MPS-PS described cases was also provided. The presented description of the natural history of MPS-PS in our patient may broaden the spectrum of phenotypes in this disease.
Assuntos
Mucopolissacaridoses , Proteínas de Transporte Vesicular , Sulfatos de Condroitina/urina , Glicosaminoglicanos/urina , Humanos , Mucopolissacaridoses/sangue , Mucopolissacaridoses/diagnóstico , Mucopolissacaridoses/genética , Mucopolissacaridoses/urina , Mutação , Polônia , Esfingolipídeos/sangue , Proteínas de Transporte Vesicular/genéticaRESUMO
The mucopolysaccharidoses (MPSs) are a class of inborn errors of metabolism caused by deficiency of each of the enzymes involved in the lysosomal degradation of mucopolysaccharides. Newborn screening panels worldwide have been recently expanded to include one or more MPS disorders, as treatments are available and are most efficacious if initiated early in life. Here we report the first multiplex assay of 10 enzymatic activities in dried blood spots and fibroblast lysates that allow newborn screening and diagnosis of all MPS disorders except the ultrarare MPS-IX. The assay consists of incubation of enzyme-specific substrates with dried blood spot punches or fibroblast lysate followed by quantification of enzymatic products using liquid chromatography-tandem mass spectrometry (LC-MS/MS) together with internal standards. Assay of all MPS enzymes using fluorimetric or other methods has not been possible. The steps of the LC-MS/MS assay are sufficiently simple and rapid to be used in newborn screening and diagnostic laboratories. Assays showed acceptable precision, and enzymatic activities measured in confirmed MPS samples are well below the reference range.
Assuntos
Teste em Amostras de Sangue Seco/métodos , Fibroblastos/metabolismo , Mucopolissacaridoses/diagnóstico , Espectrometria de Massas em Tandem/métodos , Fibroblastos/citologia , Humanos , Mucopolissacaridoses/sangueRESUMO
Several studies have been published on the frequency of the mucopolysaccharidoses (MPS) in different countries. The objective of the present study was to estimate the birth prevalence (BP) of MPS in Brazil. MPS diagnosis registered at MPS-Brazil Network and in Instituto Vidas Raras were reviewed. BP was estimated by (a) the number of registered patients born between 1994 and 2015 was divided by the number of live births (LBs), and (b) a sample of 1,000 healthy individuals was tested for the most frequent variant in IDUA gene in MPS I (p.Trp402Ter) to estimate the frequency of heterozygosity and homozygosity. (a) The BP based on total number of LBs was (cases per 100,000 LBs): MPS overall: 1.25; MPS I: 0.24; MPS II: 0.37; MPS III: 0.21; MPS IV: 0.14; MPS VI: 0.28; MPS VII: 0.02. (b) The overall frequency of p.Trp402Ter was 0.002. Considering the frequency of heterozygotes for the p.Trp402Ter IDUA variant in the RS state, the frequency of this variant among MPS I patients and the relative frequency of the different MPSs, we estimated the birth prevalence of MPS in total and of each MPS type, as follows: MPS overall: 4.62; MPS I: 0.95; MPS II: 1.32; MPS III: 0.56; MPS IV: 0.57; MPS VI: 1.02; MPS VII: 0.05. This study provided original data about BP and relative frequency of the MPS types, in Brazil, based on the frequency of the commonest IDUA pathogenic variant and in the records of two large patient databases.
Assuntos
Iduronidase/genética , Mucopolissacaridoses/genética , Brasil/epidemiologia , Feminino , Humanos , Iduronidase/sangue , Nascido Vivo , Masculino , Mucopolissacaridoses/sangue , Mucopolissacaridoses/epidemiologia , Mucopolissacaridoses/patologia , Mucopolissacaridose I/sangue , Mucopolissacaridose I/epidemiologia , Mucopolissacaridose I/genética , Mucopolissacaridose II/sangue , Mucopolissacaridose II/epidemiologia , Mucopolissacaridose II/genética , Mucopolissacaridose III/sangue , Mucopolissacaridose III/epidemiologia , Mucopolissacaridose III/genética , Mucopolissacaridose VI/sangue , Mucopolissacaridose VI/epidemiologia , Mucopolissacaridose VI/genética , Mutação/genéticaRESUMO
OBJECTIVE: To test, in a newborn screening (NBS) laboratory, the performance of liquid chromatography-tandem mass spectrometry (LC-MS/MS) to assay 5 enzymatic activities in dried blood spots (DBS) for NBS of 5 lysosomal storage diseases (mucopolysaccharidosis [MPS]-II, MPS-IIIB, MPS-IVA, MPS-VI, and MPS-VII). STUDY DESIGN: Three mm punches from de-identified DBS were obtained from the Washington NBS laboratory and submitted to the 5-plex LC-MS/MS assay. Screen cut-offs were established by analyzing the enzymatic activity in patients confirmed to have the MPS disorder. DNA sequencing of the relevant gene was performed on a second DBS punch for all samples with enzyme activity below 10% of the mean daily activity. RESULTS: (1) For MPS-II, 18 below cut-off samples, 1 pathogenic genotype, and 2 "high risk" genotypes; (2) For MPS-IIIB, no below cut-off samples; (3) For MPS-IVA, 8 below cut-off samples, 4 non-pathogenic genotypes, 4 genotypes unobtainable; (4) For MPS-VI, 4 below cut-off samples and no high-risk genotypes; (5) For MPS-VII, 1 below cut-off sample confirmed by genotype and clinical report to be affected. CONCLUSIONS: These results establish that the number of initial screen positive samples is low and manageable. Thus, population newborn screening for these conditions is feasible in a state newborn screening laboratory.
Assuntos
Mucopolissacaridoses/sangue , Mucopolissacaridoses/diagnóstico , Triagem Neonatal , Cromatografia Líquida , Teste em Amostras de Sangue Seco/estatística & dados numéricos , Humanos , Recém-Nascido , Mucopolissacaridoses/enzimologia , Projetos Piloto , Espectrometria de Massas em TandemRESUMO
Identifying diseases displaying chronic low plasma Coenzyme Q10 (CoQ) values may be important to prevent possible cardiovascular dysfunction. The aim of this study was to retrospectively evaluate plasma CoQ concentrations in a large cohort of pediatric and young adult patients. We evaluated plasma CoQ values in 597 individuals (age range 1 month to 43 years, average 11 years), studied during the period 2005-2016. Patients were classified into 6 different groups: control group of healthy participants, phenylketonuric patients (PKU), patients with mucopolysaccharidoses (MPS), patients with other inborn errors of metabolism (IEM), patients with neurogenetic diseases, and individuals with neurological diseases with no genetic diagnosis. Plasma total CoQ was measured by reverse-phase high-performance liquid chromatography with electrochemical detection and ultraviolet detection at 275 nm. ANOVA with Bonferroni correction showed that plasma CoQ values were significantly lower in the PKU and MPS groups than in controls and neurological patients. The IEM group showed intermediate values that were not significantly different from those of the controls. In PKU patients, the Chi-Square test showed a significant association between having low plasma CoQ values and being classic PKU patients. The percentage of neurogenetic and other neurological patients with low CoQ values was low (below 8%). In conclusión, plasma CoQ monitoring in selected groups of patients with different IEM (especially in PKU and MPS patients, but also in IEM under protein-restricted diets) seems advisable to prevent the possibility of a chronic blood CoQ suboptimal status in such groups of patients.
Assuntos
Erros Inatos do Metabolismo/genética , Mucopolissacaridoses/genética , Doenças do Sistema Nervoso/sangue , Fenilcetonúrias/genética , Ubiquinona/análogos & derivados , Adolescente , Adulto , Estudos de Casos e Controles , Criança , Pré-Escolar , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Erros Inatos do Metabolismo/sangue , Mucopolissacaridoses/sangue , Mutação , Doenças do Sistema Nervoso/genética , Fenilcetonúrias/sangue , Estudos Retrospectivos , Análise de Sequência de DNA , Ubiquinona/sangue , Adulto JovemRESUMO
Dried blood spot (DBS) technology is a cheap and easy method largely applied in newborn screening. Mucopolysaccharidoses (MPS) are characterized by the deficit of enzymes that degrade glycosaminoglycans (GAGs) characterized by progressive worsening of the conditions. For a possible early diagnosis of MPS, we developed a method of uronic acid (UA)-GAGs determination in DBS of 600 healthy newborns and from a small group of MPS subjects matched for age. Spotted blood UA-GAGs of the normal newborns are composed of 67.2% chondroitin sulfate (CS), 28.6% heparan sulfate (HS) and 4.4% hyaluronic acid with a CS/HS ratio of 2.35 and a total GAGs content of 0.43 µg/DBS. A chemical evaluation of CS and HS structure was performed by measuring their disaccharide composition, sulfation and the overall charge density. The DBS of four different MPS types presented an increase of total or single UA-GAGs content and/or modifications of the CS and HS disaccharide composition as well as chemical signature also related to the MPS enzymatic defect. The modifications of the UA-GAGs composition, parameters and structure of healthy newborns determined in DBS would be useful for a possible early diagnosis of various MPS types.
Assuntos
Teste em Amostras de Sangue Seco , Glicosaminoglicanos/sangue , Glicosaminoglicanos/química , Mucopolissacaridoses/sangue , Mucopolissacaridoses/diagnóstico , Configuração de Carboidratos , Humanos , Recém-NascidoRESUMO
To explore the correlation between glycosaminoglycan (GAG) levels and mucopolysaccharidosis (MPS) type, we have evaluated the GAG levels in blood of MPS II, III, IVA, and IVB and urine of MPS IVA, IVB, and VI by tandem mass spectrometry. Dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS; mono-sulfated KS, di-sulfated KS), and the ratio of di-sulfated KS in total KS were measured. Patients with untreated MPS II had higher levels of DS and HS in blood while untreated MPS III had higher levels of HS in blood than age-matched controls. Untreated MPS IVA had higher levels of KS in blood and urine than age-matched controls. The ratio of blood di-sulfated KS/total KS in untreated MPS IVA was constant and higher than that in controls for children up to 10â¯years of age. The ratio of urine di-sulfated KS/total KS in untreated MPS IVA was also higher than that in age-matched controls, but the ratio in untreated MPS IVB was lower than controls. ERT reduced blood DS and HS in MPS II, and urine KS in MPS IVA patients, although GAGs levels remained higher than the observed in age-matched controls. ERT did not change blood KS levels in MPS IVA. MPS VI under ERT still had an elevation of urine DS level compared to age-matched controls. There was a positive correlation between blood and urine KS in untreated MPS IVA patients but not in MPS IVA patients treated with ERT. Blood and urine KS levels were secondarily elevated in MPS II and VI, respectively. Overall, measurement of GAG levels in blood and urine is useful for diagnosis of MPS, while urine KS is not a useful biomarker for monitoring therapeutic efficacy in MPS IVA.
Assuntos
Glicosaminoglicanos/sangue , Glicosaminoglicanos/urina , Mucopolissacaridoses/sangue , Mucopolissacaridoses/urina , Adolescente , Adulto , Biomarcadores/sangue , Biomarcadores/urina , Criança , Pré-Escolar , Dermatan Sulfato/sangue , Dermatan Sulfato/urina , Feminino , Glicosaminoglicanos/isolamento & purificação , Heparitina Sulfato/sangue , Heparitina Sulfato/urina , Humanos , Sulfato de Queratano/sangue , Sulfato de Queratano/urina , Masculino , Mucopolissacaridoses/classificação , Mucopolissacaridoses/patologia , Mucopolissacaridose II/sangue , Mucopolissacaridose II/patologia , Mucopolissacaridose II/urina , Mucopolissacaridose III/sangue , Mucopolissacaridose III/patologia , Mucopolissacaridose III/urina , Mucopolissacaridose IV/sangue , Mucopolissacaridose IV/patologia , Mucopolissacaridose IV/urina , Mucopolissacaridose VI/sangue , Mucopolissacaridose VI/patologia , Mucopolissacaridose VI/urina , Espectrometria de Massas em Tandem , Adulto JovemRESUMO
BACKGROUND: We expanded the use of tandem mass spectrometry combined with liquid chromatography (LC-MS/MS) for multiplex newborn screening of seven lysosomal enzymes in dried blood spots (DBS). The new assays are for enzymes responsible for the mucopolysaccharidoses (MPS-I, -II, -IIIB, -IVA, -VI, and -VII) and type 2 neuronal ceroid lipofuscinosis (LINCL). METHODS: New substrates were prepared and characterized for tripeptidyl peptidase 1 (TPP1), α-N-acetylglucosaminidase (NAGLU), and lysosomal ß-glucuronidase (GUSB). These assays were combined with previously developed assays to provide a multiplex LC-MS/MS assay of 7 lysosomal storage diseases. Multiple reaction monitoring of ion dissociations for enzyme products and deuterium-labeled internal standards was used to quantify the enzyme activities. RESULTS: Deidentified DBS samples from 62 nonaffected newborns were analyzed to simultaneously determine (run time 2 min per DBS) the activities of TPP1, NAGLU, and GUSB, along with those for α-iduronidase (IDUA), iduronate-2-sulfatase (I2S), N-acetylgalactosamine-6-sulfatase (GALNS), and N-acetylgalactosamine-4-sulfatase (ARSB). The activities measured in the 7-plex format showed assay response-to-blank-activity ratios (analytical ranges) of 102-909 that clearly separated healthy infants from affected children. CONCLUSIONS: The new multiplex assay provides a robust comprehensive newborn screening assay for the mucopolysaccharidoses. The method has been expanded to include additional lysosomal storage diseases.
Assuntos
Teste em Amostras de Sangue Seco/métodos , Doenças por Armazenamento dos Lisossomos/enzimologia , Mucopolissacaridoses/metabolismo , Triagem Neonatal/métodos , Lipofuscinoses Ceroides Neuronais/enzimologia , Cromatografia Líquida , Humanos , Recém-Nascido , Doenças por Armazenamento dos Lisossomos/sangue , Doenças por Armazenamento dos Lisossomos/diagnóstico , Mucopolissacaridoses/sangue , Lipofuscinoses Ceroides Neuronais/sangue , Lipofuscinoses Ceroides Neuronais/diagnóstico , Espectrometria de Massas em Tandem , Tripeptidil-Peptidase 1RESUMO
BACKGROUND: Mucopolysaccharidoses (MPS) are a group of inborn errors of metabolism that are progressive and usually result in irreversible skeletal, visceral, and/or brain damage, highlighting a need for early diagnosis. METHODS: This pilot study analyzed 2862 dried blood spots (DBS) from newborns and 14 DBS from newborn patients with MPS (MPS I, n = 7; MPS II, n = 2; MPS III, n = 5). Disaccharides were produced from polymer GAGs by digestion with chondroitinase B, heparitinase, and keratanase II. Heparan sulfate (0S, NS), dermatan sulfate (DS) and mono- and di-sulfated KS were measured by liquid chromatography tandem mass spectrometry (LC-MS/MS). Median absolute deviation (MAD) was used to determine cutoffs to distinguish patients from controls. Cutoffs were defined as median + 7× MAD from general newborns. RESULTS: The cutoffs were as follows: HS-0S > 90 ng/mL; HS-NS > 23 ng/mL, DS > 88 ng/mL; mono-sulfated KS > 445 ng/mL; di-sulfated KS > 89 ng/mL and ratio di-KS in total KS > 32 %. All MPS I and II samples were above the cutoffs for HS-0S, HS-NS, and DS, and all MPS III samples were above cutoffs for HS-0S and HS-NS. The rate of false positives for MPS I and II was 0.03 % based on a combination of HS-0S, HS-NS, and DS, and for MPS III was 0.9 % based upon a combination of HS-0S and HS-NS. CONCLUSIONS: Combination of levels of two or more different GAGs improves separation of MPS patients from unaffected controls, indicating that GAG measurements are potentially valuable biomarkers for newborn screening for MPS.
Assuntos
Glicosaminoglicanos/metabolismo , Mucopolissacaridoses/diagnóstico , Acetilglucosaminidase/sangue , Acetilglucosaminidase/metabolismo , Condroitinases e Condroitina Liases/sangue , Condroitinases e Condroitina Liases/metabolismo , Cromatografia Líquida/métodos , Dermatan Sulfato/sangue , Dermatan Sulfato/metabolismo , Dissacarídeos/sangue , Dissacarídeos/metabolismo , Glicosaminoglicanos/sangue , Heparitina Sulfato/sangue , Heparitina Sulfato/metabolismo , Humanos , Recém-Nascido , Mucopolissacaridoses/sangue , Mucopolissacaridoses/metabolismo , Triagem Neonatal/métodos , Projetos Piloto , Polissacarídeo-Liases/sangue , Polissacarídeo-Liases/metabolismo , Espectrometria de Massas em Tandem/métodosRESUMO
BACKGROUND: Children and adults with the lysosomal storage diseases mucopolysaccharidosis (MPS) types I, II and VI live shortened lives permeated by chronic pain and physical disability. Current treatments do not alleviate these problems. Thus there is a critical need to understand the mechanism of chronic pain and disability in MPS in order to improve the way we treat patients. A potential target is inflammation. HYPOTHESIS: We hypothesized that excessive inflammation mediated by the tumor necrosis factor-α (TNF-α) inflammatory pathway is the fundamental cause of much of the chronic pain and physical disability in MPS. METHODS: 55 patients with MPS I, II, or VI were enrolled over the course of a 5-year prospective longitudinal natural history study and evaluated annually for 2-5years. 51 healthy controls were enrolled in a separate cross-sectional study of bone and energy metabolism. TNF-α was measured by ELISA. Pain and physical disability were measured by the Children's Health Questionnaire - Parent Form 50 (CHQ-PF50). Differences in log-transformed TNF-α levels and associations with CHQ domains were evaluated using a linear mixed effects model with random intercept. RESULTS: TNF-α levels were measured in 48 MPS (age: 5-17years; 35% female) and 51 controls (age: 8-17years; 53% female). Among MPS, 22 (46%) were treated with hematopoietic cell transplantation (HCT) alone, 24 (50%) with enzyme replacement therapy (ERT) alone, and 2 (4%) with both HCT and ERT. TNF-α levels are higher in MPS compared to healthy controls (p<0.001). Higher TNF-α levels are associated with increased pain and decreased physical function, social limitations due to physical health, and physical summary score (all p<0.05). TNF-α levels were not significantly associated with the general health score. TNF-α levels did not change significantly over time in MPS. CONCLUSIONS: Higher TNF-α levels are implicated in the pain and decreased physical function present in individuals with MPS despite treatment with ERT and/or HCT, suggesting that TNF-a inhibition could potentially be a useful adjunctive therapy. Further investigation into the role of TNF-α inhibition in MPS to decrease pain and improve physical function is indicated.
Assuntos
Pessoas com Deficiência , Mucopolissacaridoses/sangue , Mucopolissacaridoses/complicações , Dor/etiologia , Fator de Necrose Tumoral alfa/sangue , Adolescente , Criança , Pré-Escolar , Estudos Transversais , Progressão da Doença , Feminino , Nível de Saúde , Humanos , Masculino , Mucopolissacaridoses/diagnóstico , Mucopolissacaridoses/terapia , Medição da Dor , Avaliação de Resultados da Assistência ao Paciente , Estudos ProspectivosRESUMO
BACKGROUND: There is interest in newborn screening and diagnosis of lysosomal storage diseases because of the development of treatment options that improve clinical outcome. Assays of lysosomal enzymes with high analytical range (ratio of assay response from the enzymatic reaction divided by the assay response due to nonenzymatic processes) are desirable because they are predicted to lead to a lower rate of false positives in population screening and to more accurate diagnoses. METHODS: We designed new tandem mass spectrometry (MS/MS) assays that give the largest analytical ranges reported to date for the use of dried blood spots (DBS) for detection of mucopolysaccharidoses type II (MPS-II), MPS-IVA, and MPS-VI. For comparison, we carried out fluorometric assays of 6 lysosomal enzymes using 4-methylumbelliferyl (4MU)-substrate conjugates. RESULTS: The MS/MS assays for MPS-II, -IVA, and -VI displayed analytical ranges that are 1-2 orders of magnitude higher than those for the corresponding fluorometric assays. The relatively small analytical ranges of the 4MU assays are due to the intrinsic fluorescence of the 4MU substrates, which cause high background in the assay response. CONCLUSIONS: These highly reproducible MS/MS assays for MPS-II, -IVA, and -VI can support multiplex newborn screening of these lysosomal storage diseases. MS/MS assays of lysosomal enzymes outperform 4MU fluorometric assays in terms of analytical range. Ongoing pilot studies will allow us to gauge the impact of the increased analytical range on newborn screening performance.
Assuntos
Teste em Amostras de Sangue Seco/métodos , Mucopolissacaridoses/diagnóstico , Triagem Neonatal/métodos , Espectrometria de Massas em Tandem/métodos , Fluorometria , Humanos , Recém-Nascido , Mucopolissacaridoses/sangue , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Mucopolysaccharidoses (MPS) are caused by deficiency of one of a group of specific lysosomal enzymes, resulting in excessive accumulation of glycosaminoglycans (GAGs). We previously developed GAG assay methods using liquid chromatography tandem mass spectrometry (LC-MS/MS); however, it takes 4-5 min per sample for analysis. For the large numbers of samples in a screening program, a more rapid process is desirable. The automated high-throughput mass spectrometry (HT-MS/MS) system (RapidFire) integrates a solid phase extraction robot to concentrate and desalt samples prior to direction into the MS/MS without chromatographic separation; thereby allowing each sample to be processed within 10s (enabling screening of more than one million samples per year). The aim of this study was to develop a higher throughput system to assay heparan sulfate (HS) using HT-MS/MS, and to compare its reproducibility, sensitivity and specificity with conventional LC-MS/MS. HS levels were measured in the blood (plasma and serum) from control subjects and patients with MPS II, III, or IV and in dried blood spots (DBS) from newborn controls and patients with MPS I, II, or III. Results obtained from HT-MS/MS showed 1) that there was a strong correlation of levels of disaccharides derived from HS in the blood, between those calculated using conventional LC-MS/MS and HT-MS/MS, 2) that levels of HS in the blood were significantly elevated in patients with MPS II and III, but not in MPS IVA, 3) that the level of HS in patients with a severe form of MPS II was higher than that in an attenuated form, 4) that reduction of blood HS level was observed in MPS II patients treated with enzyme replacement therapy or hematopoietic stem cell transplantation, and 5) that levels of HS in newborn DBS were elevated in patients with MPS I, II or III, compared to those of control newborns. In conclusion, HT-MS/MS provides much higher throughput than LC-MS/MS-based methods with similar sensitivity and specificity in an HS assay, indicating that HT-MS/MS may be feasible for diagnosis, monitoring, and newborn screening of MPS.
Assuntos
Heparitina Sulfato/sangue , Ensaios de Triagem em Larga Escala , Espectrometria de Massas , Mucopolissacaridoses/sangue , Mucopolissacaridoses/diagnóstico , Triagem Neonatal , Adolescente , Adulto , Estudos de Casos e Controles , Criança , Pré-Escolar , Cromatografia Líquida , Teste em Amostras de Sangue Seco/métodos , Teste em Amostras de Sangue Seco/normas , Glicosaminoglicanos/sangue , Humanos , Lactente , Recém-Nascido , Triagem Neonatal/métodos , Triagem Neonatal/normas , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem , Adulto JovemRESUMO
We aimed to investigate the diagnostic utility of serum DPP-IV enzyme activity, urinary GAG/Cre ratio, chitotriosidase activity, total adenosine deaminase (ADA) and ADA-1 isoenzyme activity in the diagnosis of MPS. 31 MPS patients which were previously diagnosed by clinical and enzymatic analysis and 31 healthy controls matched with age and gender were included in this study. Serum DPP-IV enzyme activity, urinary GAG/Cre ratio, total ADA and ADA-1 isoenzyme activity were significantly higher in patients than in controls (p<0.001, p<0.001, p=0.038 and p=0.006, respectively). There were significant correlations between serum DPP-IV enzyme activity and urinary GAG/Cre ratios, ADA-1 activity, ADA-1/total ADA (r=0.498, p<0.001; r=0.348, p=0.006; r=0.270, p=0.034, respectively). Area under ROC curve for DPP-IV enzyme activity was 0.988, p<0.001 and for urinary GAG/Cre ratio was 0.986, p<0.001. DPP-IV enzyme activity and urinary GAG/Cre ratio were the most significant parameters according to the univariate logistic regression analysis (p=0.001 and p<0.001, respectively). The measurement of serum DPP-IV enzyme activity can be used complementary to the urinary GAG/Cre ratio for first-line MPS screening, since it is more less prone to age and hydration related interferences.
Assuntos
Dipeptidil Peptidase 4/sangue , Mucopolissacaridoses/sangue , Mucopolissacaridoses/diagnóstico , Adenosina Desaminase/sangue , Adolescente , Criança , Creatinina/urina , Diagnóstico Precoce , Feminino , Glicosaminoglicanos/urina , Hexosaminidases/sangue , Humanos , Isoenzimas/sangue , Masculino , Mucopolissacaridoses/enzimologia , Reprodutibilidade dos TestesRESUMO
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.
Assuntos
Glicosaminoglicanos/química , Mucopolissacaridoses/diagnóstico , Animais , Biomarcadores/química , Cromatografia Líquida , Modelos Animais de Doenças , Teste em Amostras de Sangue Seco , Ensaios Enzimáticos , Terapia de Reposição de Enzimas , Glicosaminoglicanos/sangue , Glicosaminoglicanos/líquido cefalorraquidiano , Glicosaminoglicanos/urina , Transplante de Células-Tronco Hematopoéticas , Humanos , Imunoensaio , Recém-Nascido , Espectrometria de Massas , Mucopolissacaridoses/sangue , Mucopolissacaridoses/líquido cefalorraquidiano , Mucopolissacaridoses/terapia , Mucopolissacaridoses/urina , OxirreduçãoRESUMO
Mucopolysaccharidoses (MPS) are caused by deficiency of lysosomal enzyme activities needed to degrade glycosaminoglycans (GAGs), which are long unbranched polysaccharides consisting of repeating disaccharides. GAGs include: chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS), and hyaluronan. Their catabolism may be blocked singly or in combination depending on the specific enzyme deficiency. There are 11 known enzyme deficiencies, resulting in seven distinct forms of MPS with a collective incidence of higher than 1 in 25,000 live births. Accumulation of undegraded metabolites in lysosomes gives rise to distinct clinical syndromes. Generally, the clinical conditions progress if untreated, leading to developmental delay, systemic skeletal deformities, and early death. MPS disorders are potentially treatable with enzyme replacement therapy or hematopoietic stem cell transplantation. For maximum benefit of available therapies, early detection and intervention are critical. We recently developed a novel high-throughput multiplex method to assay DS, HS, and KS simultaneously in blood samples by using high performance liquid chromatography/tandem mass spectrometry for MPS. The overall performance metrics of HS and DS values on MPS I, II, and VII patients vs. healthy controls at newborns were as follows using a given set of cut-off values: sensitivity, 100%; specificity, 98.5-99.4%; positive predictive value, 54.5-75%; false positive rate, 0.62-1.54%; and false negative rate, 0%. These findings show that the combined measurements of these three GAGs are sensitive and specific for detecting all types of MPS with acceptable false negative/positive rates. In addition, this method will also be used for monitoring therapeutic efficacy. We review the history of GAG assay and application to diagnosis for MPS.
Assuntos
Testes Genéticos , Glicosaminoglicanos/sangue , Mucopolissacaridoses/sangue , Mucopolissacaridoses/diagnóstico , Sulfatos de Condroitina/sangue , Cromatografia Líquida de Alta Pressão , Dermatan Sulfato/sangue , Glicosaminoglicanos/genética , Heparitina Sulfato/sangue , Humanos , Ácido Hialurônico/sangue , Sulfato de Queratano/sangue , Mucopolissacaridoses/genética , Mucopolissacaridoses/patologia , Espectrometria de Massas em TandemRESUMO
The evaluation of plasmatic galactosaminoglycans, dermatan sulfate (DS) and chondroitin sulfate (CS) can be helpful in the early identification of MPS patients, also considering that primary storage of one type of GAG can lead to secondary accumulation of other lysosomal substrates. We explore the possibility to determine plasmatic DS and CS in numerous healthy pediatric (and sometimes adult) subjects depending on age and in patients affected by various forms of MPS. A highly sensitive HPLC separation and fluorescence detection was applied for plasma/serum DS and CS determination after a specific enzymatic treatment able to release their constituent disaccharides. DS and CS content decrease significantly with age in controls having high values in the first year (~8 µg/mL). A highly significant decrease was observed for 1-5-year-old (â¼-33%) and 5-10-year-old (â¼-65%) healthy subgroups. No further decrease was determined showing a stabilization after 5 years of age. MPS I Scheie and Hurler patients showed rather similar DS and CS content significantly higher than controls matched for age. Similarly, MPS II, III and IV subjects all presented significantly higher plasmatic DS and CS content compared to healthy subjects matched for age. The same trend was determined for the only patient affected by MPS VI. Plasmatic DS and CS analyzed by the present procedure may be a useful diagnostic and screening marker for various forms of MPS.
Assuntos
Sulfatos de Condroitina/sangue , Dermatan Sulfato/sangue , Mucopolissacaridoses/sangue , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Mucopolissacaridoses/diagnóstico , Polissacarídeos/sangueRESUMO
Mucopolysaccharidoses (MPSs) are complex lysosomal storage disorders that result in the accumulation of glycosaminoglycans (GAGs) in urine, blood, and tissues. Lysosomal enzymes responsible for GAG degradation are defective in MPSs. GAGs including chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), and keratan sulfate (KS) are disease-specific biomarkers for MPSs. This unit describes a stable isotope dilution-tandem mass spectrometric method for quantifying CS, DS, and HS in urine samples. The GAGs are methanolyzed to uronic or iduronic acid-N-acetylhexosamine or iduronic acid-N-sulfo-glucosamine dimers and mixed with internal standards derived from deuteriomethanolysis of GAG standards. Specific dimers derived from HS, DS, and CS are separated by ultra-performance liquid chromatography (UPLC) and analyzed by electrospray ionization tandem mass spectrometry (MS/MS) using selected reaction monitoring for each targeted GAG product and its corresponding internal standard. This new GAG assay is useful for identifying patients with MPS types I, II, III, VI, and VII.
Assuntos
Cromatografia Líquida/métodos , Glicosaminoglicanos/urina , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Biomarcadores/urina , Sulfatos de Condroitina/urina , Dermatan Sulfato/urina , Glicosaminoglicanos/química , Heparitina Sulfato/urina , Humanos , Sulfato de Queratano/urina , Mucopolissacaridoses/sangue , Mucopolissacaridoses/urina , Controle de QualidadeRESUMO
Mucopolysaccharidoses (MPS) are a group of lysosomal storage diseases caused by mutations in lysosomal enzymes involved in degradation of glycosaminoglycans (GAGs). Patients with MPS grow poorly and become physically disabled due to systemic bone disease. While many of the major skeletal effects in mouse models for MPS have been described, no detailed analysis that compares GAGs levels and characteristics of bone by micro-CT has been done. The aims of this study were to assess severity of bone dysplasia among four MPS mouse models (MPS I, IIIA, IVA and VII), to determine the relationship between severity of bone dysplasia and serum keratan sulfate (KS) and heparan sulfate (HS) levels in those models, and to explore the mechanism of KS elevation in MPS I, IIIA, and VII mouse models. Clinically, MPS VII mice had the most severe bone pathology; however, MPS I and IVA mice also showed skeletal pathology. MPS I and VII mice showed severe bone dysplasia, higher bone mineral density, narrowed spinal canal, and shorter sclerotic bones by micro-CT and radiographs. Serum KS and HS levels were elevated in MPS I, IIIA, and VII mice. Severity of skeletal disease displayed by micro-CT, radiographs and histopathology correlated with the level of KS elevation. We showed that elevated HS levels in MPS mouse models could inhibit N-acetylgalactosamine-6-sulfate sulfatase enzyme. These studies suggest that KS could be released from chondrocytes affected by accumulation of other GAGs and that KS could be useful as a biomarker for severity of bone dysplasia in MPS disorders.
Assuntos
Doenças do Desenvolvimento Ósseo/metabolismo , Doenças do Desenvolvimento Ósseo/patologia , Glicosaminoglicanos/metabolismo , Mucopolissacaridoses/metabolismo , Mucopolissacaridoses/patologia , Animais , Biomarcadores/sangue , Densidade Óssea/fisiologia , Doenças do Desenvolvimento Ósseo/sangue , Doenças do Desenvolvimento Ósseo/diagnóstico por imagem , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/patologia , Condrócitos/diagnóstico por imagem , Condrócitos/patologia , Modelos Animais de Doenças , Feminino , Heparitina Sulfato/sangue , Humanos , Sulfato de Queratano/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucopolissacaridoses/sangue , Canal Medular/diagnóstico por imagem , Canal Medular/patologia , Microtomografia por Raio-X/métodosRESUMO
BACKGROUND: Although coronary artery pathology is a prominent feature of mucopolysaccharidosis (MPS), it may be underestimated by coronary angiography because of its diffuse nature. It is also generally assumed that cardiovascular risk is increased in MPS and reduced following hematopoietic stem cell transplantation (HSCT) or enzyme replacement therapy (ERT), but this has never been formally evaluated. Non-invasive methods of assessing vascular endothelial function may provide a measure of cardiovascular risk in MPS. We evaluated endothelial function, using digital reactive hyperemia, in youth with MPS and in healthy controls. METHODS: Digital reactive hyperemic index (RHI) was measured in 12 children and adolescents (age 10.3 ± 3.9 years old; 11 boys) with treated MPS and nine age- and gender-matched (11.4 ± 4.0; 8 boys) healthy controls. An independent t-test was used to compare RHI between individuals with MPS and controls. RESULTS: Children and adolescents with MPS (MPS type II: N = 5; type I: N = 4; type VI: N = 3) whether treated by HSCT (N = 4) or ERT (N = 8) had significantly lower RHI compared to controls (MPS 1.22 ± 0.19 vs. controls 1.46 ± 0.32, p < 0.05). CONCLUSION: These preliminary findings suggest that children and adolescents with treated MPS have significantly poorer endothelial function when compared to healthy controls. Further investigation into the utility of endothelial function for risk stratification and the long term implications of reduced endothelial function in MPS is warranted.
Assuntos
Endotélio Vascular/fisiopatologia , Mucopolissacaridoses/fisiopatologia , Adolescente , Doenças Cardiovasculares/fisiopatologia , Criança , Estudos Transversais , Terapia de Reposição de Enzimas/métodos , Feminino , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Hiperemia/fisiopatologia , Masculino , Mucopolissacaridoses/sangue , Mucopolissacaridoses/terapia , Fatores de RiscoRESUMO
INTRODUCTION: Mucopolysaccharidoses (MPSs) are a group of lysosomal storage disorders (LSDs) caused by a defect in the degradation of glycosaminoglycans (GAGs). The accumulation of GAGs in MPS patients results in extensive, severe and progressive disease. Disease modifying therapy is available for three of the MPSs and is being developed for the other types. Early initiation of treatment, before the onset of irreversible tissue damage, clearly provides a favorable disease outcome. However, early diagnosis is difficult due to the rarity of these disorders in combination with the wide variety of clinical symptoms. Newborn screening (NBS) is probably the optimal approach, and several screening techniques for different MPSs have been studied. Here we describe a relatively simple and sensitive method to measure levels of dermatan and heparan sulfate derived disaccharides in dried blood spots (DBS) with HPLC-MS/MS, and show that this reliably separates MPS I, II and MPS III newborns from controls and heterozygotes. METHODS: Newborn DBS of 11 MPS I, 1 MPS II, and 6 MPS III patients, with phenotypes ranging from severe to relatively attenuated, were collected and levels of dermatan and heparan sulfate derived disaccharides in these DBS were compared with levels in DBS of newborn MPS I and MPS III heterozygotes and controls. RESULTS: The levels of dermatan and heparan sulfate derived disaccharides were clearly elevated in all newborn DBS of MPS I, II and III patients when compared to controls. In contrast, DBS of MPS I and III heterozygotes showed similar disaccharide levels when compared to control DBS. CONCLUSIONS: Our study demonstrates that measurement of heparan and dermatan sulfate derived disaccharides in DBS may be suitable for NBS for MPS I, II and MPS III. We hypothesize that this same approach will also detect MPS VI, and VII patients, as heparan sulfate and/or dermatan sulfate is also the primary storage products in these disorders.