RESUMO
BACKGROUND: Lysosomal storage diseases (LSD) are a group of genetic conditions which could present a vast spectrum of abnormalities that may include skeletal abnormalities, organ dysfunction, neuronal involvement, and tissue accumulation of complex molecules, among other manifestations. Definitive diagnosis of LSD is generally obtained by specific enzyme assays performed in leukocytes, fibroblasts, or more recently, dried-blood filter paper (DBFP) samples. METHODS: We recently introduced dried-leukocytes filter paper (DLFP) as an alternative source of enzyme to assay heparan sulfamidase and galactocerebrosidase activities, which could not be measured in DBFP samples using fluorometric methods. We present a new fluorometric methods on DLFP samples, for evaluation of α-glucosidase (GAA), ß-glucosidase (GBA), and N-acetylgalactosamine-6-sulfatase (GALNS) activities, key enzyme assays for the identification of patients with Pompe disease (PD), Gaucher disease (GD), and Morquio A disease (MD), respectively. RESULTS: We show a clear discrimination between confirmed PD, GD, and MD patients and healthy controls. CONCLUSIONS: We conclude that the assays of GAA, GBA, and GALNS on DLFP are reliable and useful methods for the identification of PD, GD, and MD diseases, respectively. As sample preparation is feasible in standard biochemical laboratories and transportation is very simple, it could enable patients living in remote areas to be investigated, diagnosed and eventually treated with the specific therapies available for these diseases.
Assuntos
Ensaios Enzimáticos/métodos , Doença de Gaucher/diagnóstico , Doença de Depósito de Glicogênio Tipo II/diagnóstico , Leucócitos/enzimologia , Mucopolissacaridose IV/diagnóstico , Fitas Reagentes/análise , Estudos de Casos e Controles , Condroitina Sulfatases/metabolismo , Dessecação , Ensaios Enzimáticos/instrumentação , Doença de Gaucher/sangue , Doença de Depósito de Glicogênio Tipo II/sangue , Humanos , Leucócitos/patologia , Mucopolissacaridose IV/sangue , Papel , alfa-Glucosidases/metabolismo , beta-Glucosidase/metabolismoRESUMO
BACKGROUND AND OBJECTIVES: The aim of this study was to evaluate genotoxicity and mutagenicity in peripheral blood and buccal mucosal cells in mucopolysaccharidosis (MPS) I, II or VI patients. METHODS: A total of 12 patients with MPS type I, II and VI attended at the Institute of Genetics and Inborn Errors of Metabolism treated with enzyme replacement therapy (ERT) and 10 healthy control volunteers were included in this study. Mechanically exfoliated cells from cheek mucosa (left and right side) were used to micronucleus test and single cell gel (comet) assay in peripheral blood cells. RESULTS: The results of this study detected the presence of genetic damage in peripheral blood for all individuals with MPS treated with ERT, regardless of type of MPS as depicted by tail moment results. In addition, an increased number of micronucleated cells were found in buccal cells of MPS type II patients. It was also observed an increase of other nuclear alterations closely related to cytotoxicity as depicted by the frequency of pyknosis, karyolysis and karyorrhexis in buccal mucosa cells of MPS VI patients (p < 0.05). CONCLUSION: Taken together, such results demonstrate that metabolic alterations induced by the enzymatic deficiency characteristic of MPS associated with ERT therapy can induce genotoxicity and mutagenicity in peripheral blood and buccal mucosa cells, respectively. This effect appears to be more pronounced to MPS II.
Assuntos
Núcleo Celular/patologia , Cromatina/patologia , Dano ao DNA , Fragmentação do DNA , Mucopolissacaridose II/patologia , Mucopolissacaridose IV/patologia , Mucopolissacaridose I/patologia , Adolescente , Adulto , Células Sanguíneas/patologia , Brasil , Forma do Núcleo Celular , Criança , Pré-Escolar , Análise Citogenética , Terapia de Reposição de Enzimas , Feminino , Humanos , Masculino , Mucosa Bucal/patologia , Mucopolissacaridose I/sangue , Mucopolissacaridose I/genética , Mucopolissacaridose I/terapia , Mucopolissacaridose II/sangue , Mucopolissacaridose II/genética , Mucopolissacaridose II/terapia , Mucopolissacaridose IV/sangue , Mucopolissacaridose IV/genética , Mucopolissacaridose IV/terapia , Adulto JovemRESUMO
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.