Vanessa Gonçalves Pereira

Reference values for lysosomal enzymes activities using dried blood spots samples - a Brazilian experience

BackgroundLysosomal storage diseases (LSD) are inherited disorders caused by deficiency of lysosomal enzymes in which early diagnosis is essential to provide timely treatment. This study reports interval values for the activity of lysosomal enzymes that are deficient in Mucopolysaccharidosis type I, Fabry, Gaucher and Pompe disease, using dried blood spots on filter paper (DBS) samples in a Brazilian population.ResultsReference activity values were obtained from healthy volunteers samples for alpha-galactosidase A (4.57 ± 1.37 umol/L/h), beta-glucosidase (3.06 ± 0.99 umol/L/h), alpha-glucosidase (ratio: 13.19 ± 4.26; % inhibition: 70.66 ± 7.60), alpha-iduronidase (3.45 ± 1.21 umol/L/h) and beta-galactosidase (14.09 ± 4.36 umol/L/h).ConclusionReference values of five lysosomal enzymes were determined for a Brazilian population sample. However, as our results differ from other laboratories, it highlights the importance of establishing specific reference values for each center.

Evidence of lysosomal membrane permeabilization in mucopolysaccharidosis type I: Rupture of calcium and proton homeostasis

Mucopolysaccharidosis type I (MPS I) is caused by a deficiency of α‐iduronidase (IDUA), which leads to intralysosomal accumulation of glysosaminoglycans. Patients with MPS I present a wide range of clinical manifestations, but the mechanisms by which these alterations occur are still not fully understood. Genotype–phenotype correlations have not been well established for MPS I; hence, it is likely that secondary and tertiary alterations in cellular metabolism and signaling may contribute to the physiopathology of the disease. The aim of this study was to analyze Ca2+ and H+ homeostasis, lysosomal leakage of cysteine proteases, and apoptosis in a murine model of MPS I. After exposition to specific drugs, cells from Idua−/− mice were shown to release more Ca2+ from the lysosomes and endoplasmic reticulum than Idua+/+ control mice, suggesting a higher intraorganelle store of this ion. A lower content of H+ in the lysosomes and in the cytosol was found in cells from Idua−/− mice, suggesting an alteration of pH homeostasis. In addition, Idua−/− cells presented a higher activity of cysteine proteases in the cytosol and an increased rate of apoptotic cells when compared to the control group, indicating that lysosomal membrane permeabilization might occur in this model. Altogether, our results suggest that secondary alterations—as changes in Ca2+ and H+ homeostasis and lysosomal membrane permeabilization—may contribute for cellular damage and death in the physiopathology of MPS I. J. Cell. Physiol. 223: 335–342, 2010.

Evaluation of oxidative stress markers and cardiovascular risk factors in Fabry Disease patients

Fabry Disease, an X-linked inborn error of metabolism, is characterized by progressive renal insufficiency, with cardio and cerebrovascular involvement. Homocysteine (Hcy) is considered a risk factor for vascular diseases, but the mechanisms by which it produces cardiovascular damage are still poorly understood. Regarding the vascular involvement in FD patients, the analysis of factors related to thromboembolic events could be useful to improving our understanding of the disease. The aim of this study was to evaluate plasma Hcy and other parameters involved in the methionine cycle, as well as oxidative stress markers. The sample consisted of a group of 10 male FD patients and a control group of 8 healthy individuals, paired by age. Venous blood was collected for Hcy determination, molecular analysis, identification of thiobarbituric acid reactive substances, total glutathione and antioxidant enzymes activity, as well as vitamins quantification. Comparative analysis of FD patients versus the control group indicated hyperhomocysteinemia in 8 of the 10 FD patients, as well as a significant increase in overall glutathione levels and catalase activity. It is inferred that FD patients, apart from activation of the antioxidant system, present increased levels of plasma Hcy, although this is probably unrelated to common alterations in the methionine cycle.

Evaluation of α-iduronidase in dried blood spots is an accurate tool for mucopolysaccharidosis I diagnosis.

Background: Mucopolysaccharidosis type I (MPS I) is caused by a deficiency of the α‐L‐iduronidase (IDUA), which leads to the accumulation of glycosaminoglycans in lysosomes. MPS I patients present a spectrum ranging from a severe to an attenuated phenotype. Once clinical suspicion is present, diagnosis of MPS I can be performed by enzyme activity determination and/or molecular analysis. The aim of this study was to establish a reference interval value to IDUA activity using a dried blood spots (DBS) assay and to evaluate whether this assay could be a secure tool to diagnose MPS I patients. Results: IDUA activity range on HV DBS samples were 1.40–7.78 µmol/l blood/hr. Regarding the validation group, 11 of the 36 individuals clinically suspected of MPS I had the diagnosis confirmed by DBS and reference assay (leukocytes). When we considered the new proposed cutoff value of 1.5 µmol/l blood/hr, the sensitivity, specificity, and predictive values were 100%. Conclusions: Our results strongly suggest that the determination of IDUA activity using a DBS assay is a secure tool for MPS I diagnosis. However, it is extremely important to assure that all recommendations for collection, transport, and storage are correctly followed to guarantee the quality of the samples. J. Clin. Lab. Anal. 25:251–254, 2011.

Mesenchymal stem cells do not prevent antibody responses against human α-L-iduronidase when used to treat mucopolysaccharidosis type I.

Mucopolysaccharidosis type I (MPSI) is an autosomal recessive disease that leads to systemic lysosomal storage, which is caused by the absence of α-L-iduronidase (IDUA). Enzyme replacement therapy is recognized as the best therapeutic option for MPSI; however, high titers of anti-IDUA antibody have frequently been observed. Due to the immunosuppressant properties of MSC, we hypothesized that MSC modified with the IDUA gene would be able to produce IDUA for a long period of time. Sleeping Beauty transposon vectors were used to modify MSC because these are basically less-immunogenic plasmids. For cell transplantation, 4×106 MSC-KO-IDUA cells (MSC from KO mice modified with IDUA) were injected into the peritoneum of KO-mice three times over intervals of more than one month. The total IDUA activities from MSC-KO-IDUA before cell transplantation were 9.6, 120 and 179 U for the first, second and third injections, respectively. Only after the second cell transplantation, more than one unit of IDUA activity was detected in the blood of 3 mice for 2 days. After the third cell transplantation, a high titer of anti-IDUA antibody was detected in all of the treated mice. Anti-IDUA antibody response was also detected in C57Bl/6 mice treated with MSC-WT-IDUA. The antibody titers were high and comparable to mice that were immunized by electroporation. MSC-transplanted mice had high levels of TNF-alpha and infiltrates in the renal glomeruli. The spreading of the transplanted MSC into the peritoneum of other organs was confirmed after injection of 111In-labeled MSC. In conclusion, the antibody response against IDUA could not be avoided by MSC. On the contrary, these cells worked as an adjuvant that favored IDUA immunization. Therefore, the humoral immunosuppressant property of MSC is questionable and indicates the danger of using MSC as a source for the production of exogenous proteins to treat monogenic diseases.

Genomic instability in blood cells from murine model of mucopolysaccharidosis type I

Mucopolysaccharidosis type I (MPS I) is caused by a deficiency of alfa-iduronidase (IDUA), which leads to intralysosomal accumulation of glycosaminoglycans. Some studies have revealed that oxidative stress plays an important role in MPS I. However, the mechanisms by which these alterations occur are still not fully understood. The aim of this study was to analyze genomic instability in blood cells from murine model of MPS I by single cell gel (comet) assay and micronucleus test. The results pointed out genetic damage in blood cells as depicted by the single cell gel (comet) assay results. By contrast, no increase of micronucleated cells were found in mouse blood cells when compared to negative control. Taken together, our results suggest that IDUA deficiency induces genomic damage in blood cells. Certainly, this finding offers new insights into the mechanisms underlying the relation between IDUA deficiency and clinical manifestations that can occur in MPS I patients.

Treatment of adult MPSI mouse brains with IDUA-expressing mesenchymal stem cells decreases GAG deposition and improves exploratory behavior

BackgroundMucopolysaccharidosis type I (MPSI) is caused by a deficiency in alpha-L iduronidase (IDUA), which leads to lysosomal accumulation of the glycosaminoglycans (GAGs) dermatan and heparan sulfate. While the currently available therapies have good systemic effects, they only minimally affect the neurodegenerative process. Based on the neuroprotective and tissue regenerative properties of mesenchymal stem cells (MSCs), we hypothesized that the administration of MSCs transduced with a murine leukemia virus (MLV) vector expressing IDUA to IDUA KO mouse brains could reduce GAG deposition in the brain and, as a result, improve neurofunctionality, as measured by exploratory activity.MethodsMSCs infected with an MLV vector encoding IDUA were injected into the left ventricle of the brain of 12- or 25-month-old IDUA KO mice. The behavior of the treated mice in the elevated plus maze and open field tests was observed for 1 to 2 months. Following these observations, the brains were removed for biochemical and histological analyses.ResultsAfter 1 or 2 months of observation, the presence of the transgene in the brain tissue of almost all of the treated mice was confirmed using PCR, and a significant reduction in GAG deposition was observed. This reduction was directly reflected in an improvement in exploratory activity in the open field and the elevated plus maze tests. Despite these behavioral improvements and the reduction in GAG deposition, IDUA activity was undetectable in these samples. Overall, these results indicate that while the initial level of IDUA was not sustainable for a month, it was enough to reduce and maintain low GAG deposition and improve the exploratory activity for months.ConclusionsThese data show that gene therapy, via the direct injection of IDUA-expressing MSCs into the brain, is an effective way to treat neurodegeneration in MPSI mice.

The Underexploited Role of Non-Coding RNAs in Lysosomal Storage Diseases

Non-coding RNAs (ncRNAs) are a functional class of RNA involved in the regulation of several cellular processes which may modulate disease onset, progression, and prognosis. Lysosomal storage diseases (LSD) are a group of rare disorders caused by mutations of genes encoding specific hydrolases or non-enzymatic proteins, characterized by a wide spectrum of manifestations. The alteration of ncRNA levels is well established in several human diseases such as cancer and auto-immune disorders; however, there is a lack of information focused on the role of ncRNA in rare diseases. Recent reports related to changes in ncRNA expression and its consequences on LSD physiopathology show us the importance to keep advancing in this field. This article will summarize recent findings and provide key points for further studies on LSD and ncRNA association.

α- L-iduronidase gene-based therapy using the phiC31 system to treat mucopolysaccharidose type I mice.

Mucopolysaccharidose type I (MPSI) is a lysosomal monogenic disease caused by mutations in the gene for α‐ l‐iduronidase (IDUA). MPSI patients need a constant supply of IDUA to alleviate progression of the disease. IDUA gene transfer using integrative vectors might provide a definitive solution and support advancement to clinical trials, although studies have not yet been satisfactory. To achieve a stable IDUA gene expression in vivo, phiC31 was tested in the present study.

Differential expression of microRNAs from miR-17 family in the cerebellum of mucopolysaccharidosis type I mice

Mucopolysaccharidosis type I (MPS I) is caused by deficiency of α-l-iduronidase, involved in degradation of glycosaminoglycans. Clinical manifestations are widely variable and patients with severe phenotype present developmental delay and cognitive decline, among other systemic alterations. MPS I patients present secondary accumulation of gangliosides in neuronal cells, besides accumulation of undegraded glycosaminoglycans. Reduction of Neu1 expression has been previously observed in the cerebellum of MPS I mice; to be active, neuraminidase 1 forms the lysosomal multienzyme complex (LMC) with two other proteins, β-galactosidase and protective protein/cathepsin A, involved in stepwise degradation of gangliosides in the lysosomes. In this study, we evaluated relative expression of LMC genes and six possible regulators of their expression, microRNAs (miRNAs) from miR-17 family, which are predicted to target at least two LMC components, in the cerebellum of MPS I mice by real-time PCR. Neu1 was significantly underexpressed in MPS I mice cerebellum, whereas expression of other LMC genes was similar to controls. miR-20b and miR-106b were differentially expressed in MPS I mice, suggesting that they may be involved in the reduction of Neu1 expression; miR-20b-5p was overexpressed while miR-20b-3p and miR-106b-5p were underexpressed. The ratio between miR-20b-3p and miR-20b-5p was also altered in cerebellum of MPS I mice. Confirmation of binding predictions and analysis of the direct role of these miRNAs in the regulation of Neu1 expression could bring important information regarding LMC function. Since miRNAs from miR-17 family are involved in regulation of diverse biological processes, our results also point to new pathogenic cascades to be investigated in MPS I.