Kristiane Michelin-Tirelli

Parkinson's disease and the heterozygous state for glucocerebrosidase mutations among Brazilians

We have read with interest the publication by Spitz et al. [1] regarding their results on the association study of glucocerebrosidase mutations and Parkinson’s disease (PD) among Brazilian patients. Before them, several publications confirmed the association between PD and the heterozygote state for glucocerebrosidase (GBA) mutations in patients from Israel, USA, Canada, Norway, Venezuela, Taiwan (for a review see Refs. [1,2]), and now S~ao Paulo, Brazil. Table 1 summarizes these results. We also investigated the association between PD and GBA mutations in Brazilians by recruiting patients with PD in Porto Alegre, in the south of Brazil. PD was diagnosed according to the clinical criteria proposed by Gelb et al. [3]. Patients participated in the study, which was approved by the local and national ethics committees, after giving informed consent. A single neurologist (MS) examined all cases and performed a full neurological examination and applied the Hoehn and Yahr (HY) modified scale as well as the Unified PD rating scale (UPDRS). The study patients were unrelated. All patients had their GBA activity measured in their peripheral leukocytes using the standard 4-methylumbelliferyl-b-Dglycopyranoside assay. Activities below 10 nmol/h/mg were considered to be associated with an increased risk for the carrier state if they were confirmed in a second blood specimen. Common GBA mutations L444P, N370S, and IVS2þ1 were analyzed by PCR-RFLP using NciI, XhoI and HphI, respectively; while 84GG was analyzed by ARMS-PCR. The clinical data of the 62 (37 male) study patients are shown in Table 2. Mean SD age at examination was 50.14 10 yr.; mean SD age at onset was 41.4 10 yr. Patients were of Brazilian origin with mixed ethnic backgrounds, including Portuguese, Spanish, Italian, German, Amerindian and African ancestry. There were two (3.5%) heterozygotes for GD as follows: one carrying the N370S mutation and the other carrying the L444P mutation. Five patients, that is, the L444P carrier plus four additional patients without common mutations, had a leukocyte GBA activity below 10 nmol/h/mg of protein.

Novel Mutations in the Glucocerebrosidase Gene of Brazilian Patients with Gaucher Disease

Gaucher disease (GD) is an autosomal recessive disorder resulting from glucocerebrosidase (GC) deficiency due to mutations in the gene (GBA) coding for this enzyme. We have developed a strategy for analyzing the entire GBA coding region and applied this strategy to 48 unrelated Brazilian patients with GD. We used long-range PCR, genotyping based on the Taqman® assay, nested PCR, and direct DNA sequencing to define changes in the gene. We report here seven novel mutations that are likely to be harmful: S125N (c.491G>A), F213L (c.756T>G), P245T (c.850C>A), W378C (c.1251G>C), D399H (c.1312G>C), 982-983insTGC (c.980_982dupTGC), and IVS10+1G>T (c.1505+1G>T). The last alteration was found as a complex allele together with a L461P mutation. We also identified 24 different mutations previously reported by others. G377S was the third most frequent mutation among the patients included in this study, after N370S and L444P. Therefore, this mutation needs be included in preliminary screens of Brazilian GD patients. The identification of mutant GBA alleles is crucial for increasing knowledge of the GBA mutation spectrum and for better understanding of the molecular basis of GD.

Fabry disease: a new approach for the screening of females in high-risk groups.

OBJECTIVE Fabry disease (FD) is a rare X-linked inborn error of metabolism caused by deficient activity of lysosomal α-galactosidase A (α-GAL). Due to random X inactivation, α-GAL activity in heterozygous females ranges from very low to overlapping normal values. Determining this specific range and altering assays cutoffs could become a valuable tool for minimizing the need in DNA sequencing for screening of all potential carriers. Therefore, the aim of this study was to establish the range of enzyme in dried blood spots (DBS), plasma and leukocytes that suggests carrier status for FD. DESIGN AND METHODS α-GAL gene was sequenced in 453 women with clinical suspicion and/or positive family history of FD. This data was compared to the α-GAL activity measured in DBS (dried blood spots) and/or plasma and/or leukocytes. RESULTS About 12% of the samples had pathogenic mutations (c.30_32delG, c.718_719delAA, p.R118C, p.S126G, p.Y152X, p.A156D, p.C202Y, p.N215S, p.P259R, p.D264Y, p.V269M, p.R342Q and p.R356W). When compared to genotype, DBS was the least reliable biochemical test for screening, with very low specificity. Plasma and leukocyte activities presented high AUC in ROC curve analysis, both over 84%. When cutoffs were altered to identify all carriers, leukocyte specificity was higher than that of plasma (35.2% and 27.6%, respectively). Moderated correlation and agreement coefficients were found between them, which reinforces the need for using both data combined. CONCLUSION A combined approach involving plasma and leukocyte α-GAL activities, with distinct cutoffs for men and women, could represent a more accurate, faster and less expensive tool to screen women for FD in high-risk groups in middle- and low-income 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.

Quantification of glucosylceramide in plasma of Gaucher disease patients

Gaucher disease is a sphingolipidosis that leads to an accumulation of glucosylceramide. The objective of this study was to develop a methodology, based on the extraction, purification and quantification of glucosylceramide from blood plasma, for use in clinical research laboratories. Comparison of the glucosylceramide content in plasma from Gaucher disease patients, submitted to enzyme replacement therapy or otherwise, against that from normal individuals was also carried out. The glucosylceramide, separated from other glycosphingolipids by high performance thin layer chromatography (HPTLC) was chemically developed (CuSO4 / H3PO4) and the respective band confirmed by immunostaining (human anti-glucosylceramide antibody / peroxidase-conjugated secondary antibody). Chromatogram quantification by densitometry demonstrated that the glucosylceramide content in Gaucher disease patients was seventeen times higher than that in normal individuals, and seven times higher than that in patients on enzyme replacement therapy. The results obtained indicate that the methodology established can be used in complementary diagnosis and for treatment monitoring of Gaucher disease patients.

Relative frequency and estimated minimal frequency of Lysosomal Storage Diseases in Brazil: Report from a Reference Laboratory

Abstract Lysosomal storage diseases (LSDs) comprise a heterogeneous group of more than 50 genetic conditions of inborn errors of metabolism (IEM) caused by a defect in lysosomal function. Although there are screening tests for some of these conditions, diagnosis usually depends on specific enzyme assays, which are only available in a few laboratories around the world. A pioneer facility for the diagnosis of IEM and LSDs was established in the South of Brazil in 1982 and has served as a reference service since then. Over the past 34 years, samples from 72,797 patients were referred for investigation of IEM, and 3,211 were confirmed as having an LSD (4.41%, or 1 in 22), with 3,099 of these patients originating from Brazil. The rate of diagnosis has increased over time, in part due to the creation of diagnostic networks involving a large number of Brazilian services. These cases, referred from Brazilian regions, provide insight about the relative frequency of LSDs in the country. The large amount of data available allows for the estimation of the minimal frequency of specific LSDs in Brazil. The reported data could help to plan health care policies, as there are specific therapies available for most of the cases diagnosed.

Molecular and biochemical biomarkers for diagnosis and therapy monitorization of Niemann-Pick type C patients

Niemann‐Pick type C (NP‐C), one of 50 inherited lysosomal storage disorders, is caused by NPC protein impairment that leads to unesterified cholesterol accumulation in late endosomal/lysosomal compartments. The clinical manifestations of NP‐C include hepatosplenomegaly, neurological and psychiatric symptoms. Current diagnosis for NP‐C is based on observation of the accumulated cholesterol in fibroblasts of affected individuals, using an invasive and time expensive test, called Filipin staining. Lately, two metabolites that are markedly increased in NP‐C patients are arising as biomarkers for this disease screening: 7‐ketocholesterol and cholestane‐3β,5α,6β‐triol, both oxidized cholesterol products.

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

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.