Regis Rolim Guidobono

Reliable detection of mucopolysacchariduria in dried-urine filter paper samples.

BACKGROUND The mucopolysaccharidoses (MPS) are inherited metabolic disorders with bone, joint, and visceral abnormalities, leading to multi-organ dysfunction and, sometimes, neurological manifestations. These diseases are caused by storage of glycosaminoglycans (GAGs) and other complex molecules in tissues, among other pathogenic mechanisms. Definitive diagnosis of the affected individual is mainly based on the identification of the specific enzyme deficiency. New therapies are available or are in development for these pathologies, and early diagnosis seems to be important for the therapy outcomes. Almost all MPS patients have increased levels of GAGs in urine being their evaluation usually the first step in the screening of these conditions. Test on urine may be challenging as transportation of liquid urine samples in appropriate conditions for long distances, especially across international borders, could be difficult. METHODS With the aim of overcoming the difficulties related to the use of liquid samples, we extended and validated previous studies about colorimetric determination of GAGs in dried-urine filter paper (DUFP) samples. RESULTS In the conditions we described, there are no differences in the concentration of GAGs between urine and DUFP samples. Untreated patients with MPS and normal controls were well discriminated using any of the samples. CONCLUSIONS Dried-urine filter paper is a suitable sample for the colorimetric quantitation of GAGs, and that its incorporation as an additional tool for screening of MPS should be considered by reference laboratories.

Identification of a novel missense mutation in Brazilian patient with a severe form of mucopolysaccharidosis type IVA.

Mucopolysaccharidosis type 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). We report molecular characterization of a patient who presents the new missense mutation p.C165Y in homozygosis. Bioinformatics analysis predicted this mutation as being probably pathogenic. To evaluate the possibility that this alteration was a polymorphism we tested 100 alleles and all the results were negative. These findings together with the observation that this alteration is not present in controls, suggest that it is a disease-causing mutation, which was correlated with the severe phenotype observed in our patient. We conclude that molecular analysis of the GALNS gene, in addition to enzyme assays, is important for diagnosis and contributes to the better understanding of the relationship between genotype and phenotype, which is important as enzyme replacement therapy (ERT) will soon become available and treatment decisions will have to be take in such cases.

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 newborns with abnormal results in a newborn screening program for four lysosomal storage diseases in Brazil

Lysosomal storage diseases (LSDs) are genetic disorders, clinically heterogeneous, mainly caused by defects in genes encoding lysosomal enzymes that degrade macromolecules. Several LSDs already have specific therapies that may improve clinical outcomes, especially if introduced early in life. With this aim, screening methods have been established and newborn screening (NBS) for some LSDs has been developed. Such programs should include additional procedures for the confirmation (or not) of the cases that had an abnormal result in the initial screening. We present here the methods and results of the additional investigation performed in four babies with positive initial screening results in a program of NBS for LSDs performed by a private laboratory in over 10,000 newborns in Brazil. The suspicion in these cases was of Mucopolysaccharidosis I - MPS I (in two babies), Pompe disease and Gaucher disease (one baby each). One case of pseudodeficiency for MPS I, 1 carrier for MPS I, 1 case of pseudodeficiency for Pompe disease and 1 carrier for Gaucher disease were identified. This report illustrates the challenges that may be encountered by NBS programs for LSDs, and the need of a comprehensive protocol for the rapid and precise investigation of the babies who have an abnormal screening result.