Newborn screening for lysosomal disorders in Brazil: A pilot study using customized fluorimetric assays.

Lysosomal storage disorders (LSDs) are a group of genetic disorders characterized by deficiency of specific lysosomal enzymes. In general, patients are clinically normal at birth, and progressively develop severe signs and symptoms. Diagnosis is usually made several years after onset of manifestations, preventing patients to have the benefits of the early treatment. Newborn screening programs are being considered for LSDs to allow early diagnosis and treatment. The present study evaluated the feasibility of a customized screening approach based on modified fluorometric assays with reduced amounts of reagents, substrates and samples for: mucopolysaccharidosis (MPS) type I (MPS I), MPS VI, Fabry, Gaucher, and Pompe diseases. We also evaluated the advantages of including blood chitotriosidase and urinary glycosaminoglycans in the protocol. By the measurement of the specific disease-associated enzymes (plus blood chitotriosidase and urinary glycosaminoglycans) we analyzed 834 de-identified DBS of unselected newborns. No positive case was detected, and the false-positive rates were low. Taking into consideration the limitations of this methodology, we believe that, after defining proper cutoffs, it could be a viable alternative to provide NBS for LSDs by laboratories that may not be able to afford the commercial methods available.

Oxidative and nitrative stress and pro-inflammatory cytokines in Mucopolysaccharidosis type II patients: effect of long-term enzyme replacement therapy and relation with glycosaminoglycan accumulation.

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disease caused by a deficient activity of iduronate-2-sulfatase, leading to abnormal accumulation of glycosaminoglycans (GAG). The main treatment for MPS II is enzyme replacement therapy (ERT). Previous studies described potential benefits of six months of ERT against oxidative stress in patients. Thus, the aim of this study was to investigate oxidative, nitrative and inflammatory biomarkers in MPS II patients submitted to long term ERT. It were analyzed urine and blood samples from patients on ERT (mean time: 5.2years) and healthy controls. Patients presented increased levels of lipid peroxidation, assessed by urinary 15-F2t-isoprostane and plasmatic thiobarbituric acid-reactive substances. Concerning to protein damage, urinary di-tyrosine (di-Tyr) was increased in patients; however, sulfhydryl and carbonyl groups in plasma were not altered. It were also verified increased levels of urinary nitrate+nitrite and plasmatic nitric oxide (NO) in MPS II patients. Pro-inflammatory cytokines IL-1ß and TNF-α were increased in treated patients. GAG levels were correlated to di-Tyr and nitrate+nitrite. Furthermore, IL-1ß was positively correlated with TNF-α and NO. Contrastingly, we did not observed alterations in erythrocyte superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase activities, in reduced glutathione content and in the plasmatic antioxidant capacity. Although some parameters were still altered in MPS II patients, these results may suggest a protective role of long-term ERT against oxidative stress, especially upon oxidative damage to protein and enzymatic and non-enzymatic defenses. Moreover, the redox imbalance observed in treated patients seems to be GAG- and pro-inflammatory cytokine-related.

Elevation of glycosaminoglycans in the amniotic fluid of a fetus with mucopolysaccharidosis VII.

OBJECTIVE: The aim of this study was to quantify glycosaminoglycans (GAGs) in amniotic fluid (AF) from an MPS VII fetus compared with age-matched fetuses obtained from normal pregnancies. METHOD: Disaccharides were measured by liquid chromatography tandem mass spectrometry, compared to age-matched controls. Enzyme assay was performed in AF supernatant or cultured amniocytes. GUSB was analyzed by next generation sequencing using Ion Torrent Personal Genome Machine with a customized panel. RESULTS: No activity of ß-glucuronidase was detected in fetal cells. The pregnancy was spontaneously terminated in the third trimester. Genetic studies identified a homozygous mutation of p.N379D (c.1135A > G) in the GUSB gene. Liquid chromatography tandem mass spectrometry showed that chondroitin sulfate, dermatan sulfate, heparan sulfate, and keratan sulfate levels were markedly increased in the MPS VII AF, compared to those in age-matched control AF (dermatan sulfate, heparan sulfate, and chondroitin-6-sulfate more than 10 × than age-matched controls; chondroitin-4-sulfate and keratan sulfate more than 3 times higher). CONCLUSION: This is the first report of specific GAG analysis in AF from an MPS VII fetus, indicating that GAG elevation in AF occurs by 21 weeks of gestation and could be an additional tool for prenatal diagnosis of MPS VII and potentially other MPS types. © 2017 John Wiley & Sons, Ltd.

Oxidative stress and inflammation in mucopolysaccharidosis type IVA patients treated with enzyme replacement therapy.

Mucopolysaccharidosis type IVA (MPS IVA) is an inborn error of glycosaminoglycan (GAG) catabolism due to the deficient activity of N-acetylgalactosamine-6-sulfate sulfatase that leads to accumulation of the keratan sulfate and chondroitin 6-sulfate in body fluids and in lysosomes. The pathophysiology of this lysosomal storage disorder is not completely understood. The aim of this study was to investigate oxidative stress parameters, pro-inflammatory cytokine and GAG levels in MPS IVA patients. We analyzed urine and blood samples from patients under ERT (n=17) and healthy age-matched controls (n=10-15). Patients presented a reduction of antioxidant defense levels, assessed by a decrease in glutathione content and by an increase in superoxide dismutase activity in erythrocytes. Concerning lipid and protein damage, it was verified increased urine isoprostanes and di-tyrosine levels and decreased plasma sulfhydryl groups in MPS IVA patients compared to controls. MPS IVA patients showed higher DNA damage than control group and this damage had an oxidative origin in both pyrimidine and purine bases. Interleukin 6 was increased in patients and presented an inverse correlation with GSH levels, showing a possible link between inflammation and oxidative stress in MPS IVA disease. The data presented suggest that pro-inflammatory and pro-oxidant states occur in MPS IVA patients even under ERT. Taking these results into account, supplementation of antioxidants in combination with ERT can be a tentative therapeutic approach with the purpose of improving the patient's quality of life. To the best of our knowledge, this is the first study relating MPS IVA patients with oxidative stress.

Experience of the NPC Brazil Network with a Comprehensive Program for the Screening and Diagnosis of Niemann-Pick Disease Type C.

Niemann-Pick disease type C (NPC) is a lysosomal disorder caused by impaired cholesterol metabolism. Levels of lysosphingomyelin 509 (LysoSM509) have been shown elevated in dried blood spots (DBS) of NPC and acid sphingomyelinase deficiency patients. In this study, we report our experience using a two-tier approach (1st tier is the quantification of lysoSM509 by ultra-performance liquid chromatography tandem mass spectrometry followed by the 2nd tier with next-generation sequencing of the NPC1 and NPC2 genes). DBS samples from 450 suspected patients were received by the NPC Brazil network. Of these, 33 samples had elevated levels of lysoSM509, and in 25 of them, variants classified as pathogenic, likely pathogenic, or of unknown significance were identified in the NPC1 or NPC2 genes by next-generation sequencing. The quantification of lysoSM509 in DBS as a first-tier test for the diagnosis of NPC followed by molecular analysis of the NPC1 and NPC2 genes almost doubled the detection rate when compared to the performance of chitotriosidase activity as a first-tier biomarker, and it could likely be increased with the addition of a third tier with MLPA of the two genes involved. This strategy seems suitable for the neonatal screening (NBS) of NPC if this disease is eventually adopted by NBS programs.

Quantification of lysosphingomyelin and lysosphingomyelin-509 for the screening of acid sphingomyelinase deficiency.

BACKGROUND: Acid sphingomyelinase deficiency (ASMD) is a lysosomal disorder caused by deficiency of acid sphingomyelinase (ASM) leading to the accumulation of sphingomyelin (SM) in a variety of cell types. Lysosphingomyelin (LysoSM) is the de-acetylated form of SM and it has been shown as a biomarker for ASMD in tissues, plasma, and dried blood spots (DBS) and lysosphingomyelin-509 (LysoSM509) is the carboxylated analogue of LysoSM. High levels of Lysosphingomyelin 509 (LysoSM509) have also been shown in ASMD patients. In this study, we report the utility of the quantification of LysoSM and LysoSM509 in DBS of patients from Latin America with ASMD by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). RESULTS: DBS samples from 14 ASMD patients were compared with 15 controls, and 44 general newborns. All patients had their diagnosis confirmed by the quantification of ASM and the measurement of the activity of chitotriosidase. All patients had significantly higher levels of lysoSM and lysoSM509 compared to controls and general newborns. CONCLUSIONS: The quantification of lysosphingolipids in DBS is a valuable tool for the diagnosis of ASMD patients and lysoSM can be useful in the differential diagnosis with NPC. This method is also valuable in the ASMD newborn screening process.

Mucopolysaccharidosis type VI (Maroteaux-Lamy syndrome): assessment of joint mobility and grip and pinch strength.

OBJECTIVE: To describe the profile of joint mobility and grip and pinch strength of MPS VI patients and to correlate this with urinary excretion of glycosaminoglycans (GAGs), ARSB activity, and the distance covered in a 6-minute walking test (6MWT). METHODS: This was an observational study of 28 patients with MPS VI, who had not undergone specific treatment. All patients were assessed for amplitude of joint mobility (shoulder, elbow, and knee), grip and pinch strength and urinary GAG excretion and also performed the 6MWT. RESULTS: Shoulder flexion exhibited the greatest limitation, with no correlation with age, followed by knee extension and elbow flexion, both of which were correlated inversely with age. Hand grip strength was compromised in all patients, and pinch strength exhibited a positive correlation with age. CONCLUSIONS: The fact that restricted shoulder flexion was not correlated with age suggests that this finding is present early on in MPS VI and that it constitutes an important clinical sign that should arouse diagnostic suspicion of this disease. The amplitude of knee extension and elbow flexion, in turn, are possible markers of disease progression since they have a negative correlation with age. Further studies are needed to confirm these hypotheses.

Comparison between the biochemical properties of plasma chitotriosidase from normal individuals and from patients with Gaucher disease, GM1-gangliosidosis, Krabbe disease and heterozygotes for Gaucher disease.

OBJECTIVES: The aim of the present work was to establish the range of chitotriosidase (CT) activity in normal individuals (controls), patients with Gaucher disease (GD), GM1-gangliosidosis (GM1), Krabbe disease (KD) and heterozygotes for Gaucher disease (HG). The kinetics of the enzyme in the five groups was also investigated. DESIGN AND METHODS: Plasma CT activity, as well as Km, Vmax, optimum pH and thermal stability of the enzyme was determined in plasma of controls, GM1, KD, GD and HG subjects. RESULTS: CT activity in GD, GM1 and KD patients was, respectively, around 600-fold, 15-fold and 12-fold greater than in normal individuals. There was no significant difference between CT activity in the HG and the control group. We also demonstrated that all CT kinetic parameters evaluated (optimum pH, Km, Vmax, thermal stability) in plasma of GD, KD and GM1 patients were significantly different from those of normal individuals. Regarding to thermal stability, our results show that CT activity in the control group was more stable than in the other groups. CONCLUSIONS: Based on the differences found in the biochemical parameters studied, we presume that the parameters analyzed may be useful in the diagnosis of the Lysosomal Storage Diseases.

A clinical study of 77 patients with mucopolysaccharidosis type II.

AIM: This study aims to assess the clinical features of 77 South American patients (73 Brazilian) with mucopolysaccharidosis type II (MPS II). METHODS: Details of the patients and their disease manifestations were obtained from a review of medical records, interviews with the patients and/or their families, and physical examination of the patients. RESULTS: Mean birth weight was 3360 g, median age at onset of symptoms was 18 months and median age at diagnosis was 6 years. For the whole sample (median age, 8.2 years; range, 2.8-53.0 years), neurological degeneration, typical pebbly skin lesions, seizures and extensive dermal melanocytosis were found in 23.3, 13.0, 13.0 and 1.3% of the cases, respectively. The most frequently reported echocardiogram abnormality was mitral valve regurgitation. Refraction errors were the most common ophthalmological manifestation. The following characteristics were found to be associated with the severe form of MPS II: earlier age at biochemical diagnosis, higher levels of urinary glycosaminoglycans, language development delay, behavioural disturbances, poor school performance and mental retardation. CONCLUSION: Our results suggest that there is a considerable delay between the onset of signs and symptoms and the diagnosis of MPS II in Brazil (and probably in South America as well), and that many complications of this disease are underdiagnosed and undertreated. Therefore, the implementation of programmes aiming to increase the awareness of the disease, the availability of biochemical diagnostic tests and the provision of better support to affected patients is urgently needed.

White matter lesions in Fabry disease before and after enzyme replacement therapy: a 2-year follow-up.

PURPOSE: To report the clinical and neuroimaging, central nervous system (CNS) findings of patients with Fabry disease (FD) during 24 months of enzyme replacement therapy (ERT) with agalsidase-alpha. METHOD: Eight patients were included. Six completed 24 months of ERT. Clinical and magnetic resonance imaging (MRI) data were obtained at 0, 12 and 24 months of ERT. White matter lesions (WML) were evaluated as well as their relation to age, symptoms and neurological examination (CNS score). RESULTS: MRI was stable in 3 patients. WML and CNS score worsened in one patient, fluctuated in another, and improved in the sixth patient. In the whole series, there were 15 WML at baseline, and 19 at the 24th month. In two years, 4 lesions disappeared, whereas 8 appeared. CONCLUSION: A widespread pattern of silent WML in FD was seen. In two years, some WML appeared, and some disappeared. If these phenomena were related to the natural history, remains to be demonstrated.

Biochemical characterization of chitotriosidase enzyme: comparison between normal individuals and patients with Gaucher and with Niemann-Pick diseases.

OBJECTIVES: The aim of the present study was to establish the range of chitotriosidase (CT) activity in normal individuals, patients with Gaucher disease (GD) and Niemann-Pick disease (NPD), types A or B. The kinetics of CT in these three groups was also investigated. DESIGN AND METHODS: CT activity, as well as Km, Vmax, optimum pH, and thermal stability of the enzyme were determined in the plasma of control, GD, and NPD subjects. RESULTS: CT activity in GD and NPD patients was, respectively, around 600-fold and 30-fold greater than in normal individuals. We observed significant differences in optimum pH, Vmax, and thermal stability between the various groups. Km was different in normal individuals relative to GD and NPD patients. However, there was no significant difference between Km values in patients with GD and with NPD. CONCLUSIONS: Based on the differences found in the biochemical parameters studied, our results may be important to help the identification of patients not only with GD but also with NPD.

Practical and reliable enzyme test for the detection of mucopolysaccharidosis IVA (Morquio Syndrome type A) in dried blood samples.

BACKGROUND: Mucopolysaccharidosis IVA (MPS IVA), or Morquio Syndrome type A, is an autosomal recessive disease caused by deficiency of the lysosomal enzyme N-acetylgalactosamine-6-sulfatase (GALNS), resulting in excessive lysosomal storage of keratan sulfate in many tissues and organs. This accumulation causes a severe skeletal dysplasia with short stature, and affects the eye, heart and other organs, with many signs and symptoms. Morquio A syndrome is estimated to occur in 1 in 200,000 to 300,000 live births. Clinical trials with enzyme replacement therapy for this disease are in progress, and it is probable that the treatment, when available, would be more effective if started early. We describe an innovative fluorometric method for the assay of GALNS in dried blood spots (DBS). METHODS: We used dried blood spots (DBS) as the enzyme source and compared it with leukocytes samples, having studied 25 MPS IVA patients and 54 healthy controls. We optimized the assay conditions, including incubation time and stability of DBS samples. To eppendorf type tubes containing a 3-mm diameter blood spot we added elution liquid and substrate solution. After 2 different incubations at 37°C, the amount of hydrolyzed product was compared with a calibrator to allow the quantification of the enzyme activity. Results in DBS were compared to the ones obtained in leukocytes using the standard technique. RESULTS: The fluorescent methodology was validated in our laboratory and the assay was found sensitive and specific, allowing reliable detection of MPS IVA patients. The use of DBS simplifies the collection and transport steps, and is especially useful for testing patients from more remote areas of large countries, and when samples need to cross country borders. CONCLUSION: This assay could be easily incorporated into the protocol of reference laboratories and play a role in the screening for MPS IVA, contributing to earlier detection of affected patients.

Application of a comprehensive protocol for the identification of Gaucher disease in Brazil.

Gaucher disease (GD) is a sphingolipidosis caused by a genetic defect that leads to glucocerebrosidase (beta-glucosidase) deficiency. Between January 1982 and October 2003, 1,081 blood samples from patients suspected of having GD were referred for biochemical analysis. The activities of the enzymes beta-glucosidase (beta-glu) and chitotriosidase (CT) were measured in these samples. Among the 412 diagnosed cases of GD (38.1%), the great majority were GD type 1. The Brazilian regions with the greatest concentration of these patients were the Southeast, South, and Northeast. The mean age of patients at diagnosis was 19 years. The activity of beta-glu in patients with GD was, on average, 10.7% of that of normal individuals. CT was, on average, 269 times more elevated in this group of patients. Among the 669 cases with no confirmation of GD, there were patients with Niemann-Pick disease types A, B, or C (44 cases), possible heterozygotes for GD (59 cases), patients with other lysosomal storage diseases (LSDs) (19 cases) or with other inborn errors of metabolism (3 cases). In 508 cases, no metabolic disorder was found. This study shows that the biochemical protocol employed was effective for the detection of GD, a disease that is reasonably frequent in Brazil.

Application of a protocol for the detection of disorders of sialic acid metabolism to 124 high-risk Brazilian patients.

Lysosomal storage disorders (LSD) present great clinical variability. Included in this group are sialic acid metabolism disorders (SAMD). In the present study, we describe the application of a 3-step protocol for the diagnosis of SAMD, including (1). oligosaccharide and sialyloligosaccharide chromatography; (2). quantitative determination of sialic acid; and (3). measurement of neuraminidase activity. Application of our protocol to 124 individuals at risk for SAMD led to the diagnosis of five affected patients, two with type I sialidosis, one with type II sialidosis, and two with galactosialidosis. Due to its simplicity and efficiency, we propose the use of this protocol for the diagnostic evaluation of patients with suspected SAMD, which could be specially useful to non-specialized laboratories and to services located in developing countries.

Investigation of lysosomal storage diseases in nonimmune hydrops fetalis.

OBJECTIVE: To investigate lysosomal storage diseases (LSD) in cases of nonimmune hydrops fetalis (NIHF). METHODS: Thirty-three cases of NIHF were investigated, 28 in the prenatal period and 5 in hydropic newborns. In addition to a general investigation for NIHF, specific enzymatic analyses for the detection of LSD were performed. RESULTS: In our sample, we detected five patients (15%) with LSD, each patient having one of the following diseases: mucolipidosis, Niemann-Pick disease, galactosialidosis, sialidosis and mucopolysaccharidosis type IV A. CONCLUSION: Although LSDs are rare disorders as a group, they should be considered as a possible cause of NIHF, even in the absence of consanguinity or of a previous family history. By excluding the more frequent causes of NIHF, an LSD investigation assists in clarifying the etiology of many hydropic cases, making more appropriate genetic counseling for parents possible.

Mucopolissacaridose tipo VI (síndrome de Maroteaux-Lamy): avaliação da mobilidade articular e das forças de garra e de pinça / Mucopolysaccharidosis type VI (Maroteaux-Lamy syndrome): assessment of joint mobility and grip and pinch strength

OBJETIVO: Descrever o perfil de mobilidade articular e das forças de garra e de pinça de indivíduos com MPS VI, além de sua correlação com a excreção urinária de glicosaminoglicanos, atividade da ARSB e distância percorrida no teste de caminhada de 6 minutos. MÉTODOS: Estudo observacional de 28 pacientes com MPS VI, não submetidos a tratamento específico. Todos os pacientes foram avaliados em relação à amplitude da mobilidade articular, forças de garra e de pinça, excreção urinária de glicosaminoglicanos, atividade da ARSB e teste de caminhada de 6 minutos. RESULTADOS: Demonstrou-se maior comprometimento de flexão de ombro, sem correlação com a idade, e da extensão de joelho e flexão de cotovelo, estas últimas correlacionadas negativamente com a idade. A força de garra mostrou-se comprometida em todos os pacientes, e a força de pinça apresentou correlação positiva com idade. CONCLUSÕES: A restrição da flexão de ombro, sem correlação com a idade, sugere que este achado esteja presente precocemente na MPS VI e se constitua em sinal clínico importante para suspeita diagnóstica desta doença. A amplitude da extensão de joelho e da flexão de cotovelo, por sua vez, por apresentarem correlação negativa com a idade, são possíveis marcadores da evolução da doença. Estudos adicionais são necessários para confirmação dessas hipóteses.

OBJECTIVE: To describe the profile of joint mobility and grip and pinch strength of MPS VI patients and to correlate this with urinary excretion of glycosaminoglycans (GAGs), ARSB activity, and the distance covered in a 6-minute walking test (6MWT). METHODS: This was an observational study of 28 patients with MPS VI, who had not undergone specific treatment. All patients were assessed for amplitude of joint mobility (shoulder, elbow, and knee), grip and pinch strength and urinary GAG excretion and also performed the 6MWT. RESULTS: Shoulder flexion exhibited the greatest limitation, with no correlation with age, followed by knee extension and elbow flexion, both of which were correlated inversely with age. Hand grip strength was compromised in all patients, and pinch strength exhibited a positive correlation with age. CONCLUSIONS: The fact that restricted shoulder flexion was not correlated with age suggests that this finding is present early on in MPS VI and that it constitutes an important clinical sign that should arouse diagnostic suspicion of this disease. The amplitude of knee extension and elbow flexion, in turn, are possible markers of disease progression since they have a negative correlation with age. Further studies are needed to confirm these hypotheses.

White matter lesions in Fabry disease before and after enzyme replacement therapy: a 2-year follow-up / Lesões da substância branca na doença de Fabry antes e depois da terapia de reposição enzimática: um seguimento de 2 anos

PURPOSE: To report the clinical and neuroimaging, central nervous system (CNS) findings of patients with Fabry disease (FD) during 24 months of enzyme replacement therapy (ERT) with agalsidase-alpha. METHOD: Eight patients were included. Six completed 24 months of ERT. Clinical and magnetic resonance imaging (MRI) data were obtained at 0, 12 and 24 months of ERT. White matter lesions (WML) were evaluated as well as their relation to age, symptoms and neurological examination (CNS score). RESULTS: MRI was stable in 3 patients. WML and CNS score worsened in one patient, fluctuated in another, and improved in the sixth patient. In the whole series, there were 15 WML at baseline, and 19 at the 24th month. In two years, 4 lesions disappeared, whereas 8 appeared. CONCLUSION: A widespread pattern of silent WML in FD was seen. In two years, some WML appeared, and some disappeared. If these phenomena were related to the natural history, remains to be demonstrated.

OBJETIVO: Relatar os achados neurológicos e de imagem do sistema nervoso central (SNC), observados durante 24 meses de tratamento de reposição enzimática (ERT) com agalsidase-alfa, em pacientes com a doença de Fabry (FD). MÉTODO: 8 pacientes foram incluídos; 6 completaram 24 meses de ERT. Os dados foram obtidos aos 0, 12 e 24 meses de ERT. Lesões de substância branca (WML) foram avaliadas assim como sua relação com a idade e o exame neurológico (escore SNC). RESULTADOS: Os achados de ressonância nuclear magnética foram estáveis em 3 pacientes. As WML e o escore SNC pioraram em um caso; flutuaram em um outro caso; e melhoraram no sexto paciente. No todo, havia 15 WML antes da ERT e 19 WML depois de 24 meses de ERT. Em dois anos, 4 lesões desapareceram e 8 novas surgiram. CONCLUSÕES: Viu-se um padrão difuso de WML assintomáticas, na FD. Em dois anos, algumas WML surgiram, enquanto outras desapareceram. Resta demonstrar se esses fenômenos fazem parte da história natural da doença.

Early infantile form of galactosialidosis in a female baby with a prenatal diagnosis of fetal ascites - first case in Brazil

Apresentamos o primeiro caso de galactosialidose do tipo infantil precoce identificado entre a população brasileira, uma grave e rara doença de depósito lisossomal, com apenas 12 casos claramente descritos mundialmente. Estudos clínicos, patológicos e bioquímicos realizados foram consistentes com os dados já publicados na literatura científica. Detectamos a doença em uma menina de 7 meses de idade, com diagnóstico de ascite no período pré-natal e evolução compatível com doença de depósito, através da cromatografia em camada fina para oligossacarídeos, que é parte integrante do programa de triagem para erros inatos do metabolismo (EIM) em crianças de alto risco, realizado no Estado do Rio de Janeiro.