Cristina Brinckmann Oliveira Netto

Globotriaosylceramide is correlated with oxidative stress and inflammation in Fabry patients treated with enzyme replacement therapy

Fabry disease is an X-linked inborn error of glycosphingolipid catabolism due to deficient activity of α-galactosidase A that leads to accumulation of the enzyme substrates, mainly globotriaosylceramide (Gb3), in body fluids and lysosomes of many cell types. Some pathophysiology hypotheses are intimately linked to reactive species production and inflammation, but until this moment there is no in vivo study about it. Hence, the aim of this study was to investigate oxidative stress parameters, pro-inflammatory cytokines and Gb3 levels in Fabry patients under treatment with enzyme replacement therapy (ERT) and finally to establish a possible relation between them. We analyzed urine and blood samples of patients under ERT (n=14) and healthy age-matched controls (n=14). Patients presented decreased levels of antioxidant defenses, assessed by reduced glutathione (GSH), glutathione peroxidase (GPx) activity and increased superoxide dismutase/catalase (SOD/CAT) ratio in erythrocytes. Concerning to the damage to biomolecules (lipids and proteins), we found that plasma levels of malondialdehyde (MDA) and protein carbonyl groups and di-tyrosine (di-Tyr) in urine were increased in patients. The pro-inflammatory cytokines IL-6 and TNF-α were also increased in patients. Urinary Gb3 levels were positively correlated with the plasma levels of IL-6, carbonyl groups and MDA. IL-6 levels were directly correlated with di-Tyr and inversely correlated with GPx activity. This data suggest that pro-inflammatory and pro-oxidant states occur, are correlated and seem to be induced by Gb3 in Fabry patients.

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.

Fabry Disease in Hemodialysis Patients in Southern Brazil: Prevalence Study and Clinical Report

Background. Fabry disease (FD) is a lysosomal storage disorder caused by a deficiency of α-Galactosidase A (α-Gal A). Fabry nephropathy typically progresses throughout the fifth decade to end-stage renal disease (ESRD), requiring hemodialysis and/or kidney transplantation. Objective. To estimate the prevalence of FD among ESRD males on hemodialysis treatment in Rio Grande do Sul, the southernmost state of Brazil. Methods. Screening for α-Gal A activity was performed by a dried blood spot (normal reference value: >1.5 nmoles/hour/mL). Positive screening results were confirmed by plasma α-Gal A activity assay (reference value: >3.3 nmoles/hour/mL). Results. Five hundred fifty-eight male patients on hemodialysis were evaluated. Of these, only two had low α-Gal A activity and were diagnosed with Fabry disease (0.36%). One of these, age 42, had left ventricular hypertrophy and renal manifestations of Fabry disease without the classic symptoms. The other, age 46, had the classical manifestations of angiokeratomas, acroparesthesias, hypohidrosis, and ocular opacities. Conclusions. Although the prevalence of Fabry disease was very low in our study (0.36%), routine screening of male hemodialysis patients would enable earlier identification of many other affected relatives in their families who might benefit from specific clinical treatment.

Improvement of sympathetic skin responses under enzyme replacement therapy in Fabry disease

SummaryAim To report the effect of enzyme replacement therapy (ERT) in sympathetic skin responses (SSR) of patients with Fabry disease. Patients and methods Seven male patients were included in an open-label protocol using agalsidase-alfa, continued at regular intervals. Five patients completed 24 months of ERT and two of them completed 18 months. Two main measurements were performed at baseline, as well as 1 and 2 years after ERT: (1) a standard neurological examination (NE), with a detailed evaluation of the sensory perception of light touch, pinprick, cold, hot, and vibratory stimuli; (2) the SSR amplitudes. Results Although there were no significant differences between NE in this time period, all patients reported general improvement in their subjective reports of acroparaesthesia and sweating. Before starting ERT, the SSR amplitudes were either too small (3/7 patients) or absent (4/7 patients): the average (range) amplitude of 122 μV (0 through 492) was statistically smaller than that found in a control group, i.e. 1453.6 μV (619.7–2754) (p<0.0001, t-test). Mean ± SD SSR amplitude increased to 1088± 690 μV in the second year of ERT, reaching the range found in a normal control group (p=0.004). Conclusion ERT improved SSR continuously in Fabry patients in 2 years of observation. Although the mechanism of the SSR improvement is unknown, this response to ERT can be clinically significant if it reflects a normalization in sweating.

Clinical aspects of neuropathic lysosomal storage disorders

The purpose of this review is to describe neurological phenotypes associated with lysosomal storage diseases (LSDs), focusing on features arising from primary neuronal involvement. Clinical presentation, progression and genetic data, are discussed in detail in Part 2, the electronic material. Main features are summarized in Part 1. Insights gained from several observational studies are discussed. Prospective studies of the natural history of most neuronopathic LSDs have been hampered by the rarity of these conditions and the short survival of affected patients. Increasingly, longitudinal observations relating to neurological manifestations are being reported. Better clinical studies are necessary, including repeated measurements of disease progression to facilitate the development of sensitive scoring systems and appropriate counseling of affected individuals and their families. Ideally, clinical studies should involve a large cohort. As treatment becomes available, knowledge of disease expression and factors that influence the phenotype may enable critical assessment of therapeutic outcomes. It is hoped that increased familiarity with the clinical expression of individual LSDs will allow early diagnosis, so families at risk are given options to consider during future pregnancies. Early diagnosis also permits the introduction of timely intervention, to favoring improved outcome in cases that are potentially treatable.

Kidney Function and 24-Hour Proteinuria in Patients with Fabry Disease during 36 Months of Agalsidase Alfa Enzyme Replacement Therapy: A Brazilian Experience

Background. Prior to the introduction of enzyme replacement therapy (ERT), management of Fabry disease (FD) consisted of symptomatic and palliative measures. ERT has been available for several years using recombinant human agalsidase alfa, an analogue of alpha-galactosidase A (GALA). However, the limitations of ERT in improving kidney function have not been established. This study evaluates the safety and therapeutic effect of agalsidase alfa replacement in terms of kidney function and reduction in 24-hour proteinuria. Methods. During the period between January 1, 2002, and August 1, 2005, nine Fabry patients (7 male, 2 female) were treated according to protocol, receiving 0.2 mg/kg agalsidase alfa IV every two weeks. Kidney function was evaluated by measuring the glomerular filtration rate (GFR) using chromium ethylene diamine tetra-acetate clearance (51Cr-EDTA mL/min/ 1.73 m2) at baseline, 12, 24, and 36 months. 24-hour proteinuria was measured at baseline, 3, 6, 12, 18, 24, and 36 months of ERT. Kidney disease was classified according to National Kidney Foundation Disease Outcome Quality Initiative (NKF/DOQI) Advisory Board criteria, which define stage I chronic kidney disease (CKD) as GFR ≥ 90mL/min/1.73 m2, stage II as 60–89 mL/min/1.73m2, stage III as 30–59 mL/min/1.73 m2, stage IV as 15–29 mL/min/1.73m2, and stage V as < 15 mL/min/1.73m2. Results. Six patients completed 36 months of therapy, 2 patients completed 18 months, and 1 patient completed 12 months. Mean patient age at baseline was 34.6 ± 11.3 years. During the study period, kidney function remained stable in patients with stages I, II, or III CKD. One patient, who entered the study with stage IV CKD, progressed to end-stage chronic kidney disease, beginning hemodialysis after 7 months and receiving a kidney transplant after 12 months of ERT. Proteinuria also remained stable in the group of patients with pathologic proteinuria. The use of agalsidase alfa was well tolerated in 99.5% of the infusions administered. Conclusion. Over the course of 36 months of ERT, there was no change in kidney function and 24-hour proteinuria. This suggests that agalsidase alfa may slow or halt the progression of kidney disease when used before extensive kidney damage occurs. No significant side effects were observed with ERT during the course of the study.

DNA damage in Fabry patients: An investigation of oxidative damage and repair.

Fabry disease (FD) is a lysosomal storage disorder associated with loss of activity of the enzyme α-galactosidase A. In addition to accumulation of α-galactosidase A substrates, other mechanisms may be involved in FD pathophysiology, such as inflammation and oxidative stress. Higher levels of oxidative damage to proteins and lipids in Fabry patients were previously reported. However, DNA damage by oxidative species in FD has not yet been studied. We investigated basal DNA damage, oxidative DNA damage, DNA repair capacity, and reactive species generation in Fabry patients and controls. To measure oxidative damage to purines and pyrimidines, the alkaline version of the comet assay was used with two endonucleases, formamidopyrimidine DNA-glycosylase (FPG) and endonuclease III (EndoIII). To evaluate DNA repair, a challenge assay with hydrogen peroxide was performed. Patients presented significantly higher levels of basal DNA damage and oxidative damage to purines. Oxidative DNA damage was induced in both DNA bases by H2O2 in patients. Fabry patients presented efficient DNA repair in both assays (with and without endonucleases) as well as significantly higher levels of oxidative species (measured by dichlorofluorescein content). Even if DNA repair be induced in Fabry patients (as a consequence of continuous exposure to oxidative species), the repair is not sufficient to reduce DNA damage to control levels.

Effects of imiglucerase on the growth and metabolism of Gaucher disease type I patients: a systematic review.

BackgroundGaucher disease (GD) type I is the most common type of GD. Its main clinical manifestations are hepatosplenomegaly as well as bone and hematological abnormalities. The objective of the present study was to perform a literature review on the growth and metabolism of GD type I patients.MethodsWe searched Pubmed and Scielo.br databases with predetermined study limits: case series (n≥5), clinical trials, systematic reviews, and meta-analyses, and enzyme replacement therapy (ERT) with alglucerase or imiglucerase. The outcomes of interest were the following: growth and development, weight, height, malnutrition, overweight, obesity, basal metabolism, hypermetabolism, insulin resistance, and diabetes. A total of 175 articles were found, of which 28 met the inclusion criteria; these articles were grouped into three central themes: 1) growth of children and adolescents before and after ERT; 2) metabolic changes that remained during ERT; and 3) changes in metabolic status resulting from the treatment.Results and discussionThe articles included in the present literature review are very heterogeneous, which hinders the analysis of data. They indicated that GD patients usually show low weight and height before ERT, which are improved with treatment in children and adolescents. Studies evaluating the energy metabolism by indirect calorimetry have indicated that the disease is associated with hypermetabolism. In adults, some changes in energy metabolism remain on ERT, and alterations, such as insulin resistance, seem to be associated with the treatment. It is not clear which are the required doses of imiglucerase for obtaining an adequate cost-effective relation, as well as the advisable therapeutic measures to avoid possible long-term adverse effects related to ERT.ConclusionsERT tends to normalise the growth of children and adolescents with GD type I, it seems to cause a partial response in relation to some metabolic changes associated with the disease, and it can causes metabolic changes such as weight gain in adult patients. Therefore, additional research is necessary.

Increased Oxidative Damage in Carriers of the Germline TP53 p.R337H Mutation

Germline mutations in TP53 are the underlying defect of Li-Fraumeni Syndrome (LFS) and Li-Fraumeni-like (LFL) Syndrome, autosomal dominant disorders characterized by predisposition to multiple early onset cancers. In Brazil, a variant form of LFS/LFL is commonly detected because of the high prevalence of a founder mutation at codon 337 in TP53 (p.R337H). The p53 protein exerts multiple roles in the regulation of oxidative metabolism and cellular anti-oxidant defense systems. Herein, we analyzed the redox parameters in blood samples from p.R337H mutation carriers (C, n = 17) and non-carriers (NC, n = 17). We identified a significant increase in erythrocyte GPx activity and in plasma carbonyl content,an indicator of protein oxidative damage, in mutation carriers compared to non-carriers (P = 0.048 and P = 0.035, respectively). Mutation carriers also showed a four-fold increase in plasma malondialdehyde levels, indicating increased lipid peroxidation (NC = 40.20±0.71, C = 160.5±0.88, P<0.0001). Finally, carriers showed increased total antioxidant status but a decrease in plasma ascorbic acid content. The observed imbalance could be associated with deregulated cell bioenergetics and/or with increased inflammatory stress, two effects that may result from loss of wild-type p53 function. These findings provide the first evidence that oxidative damage occurs in carriers of a germline TP53 mutation, and these may have important implications regarding our understanding of the mechanisms responsible for germline TP53 p.R337H mutation-associated carcinogenesis.

Prevalence of Hispanic BRCA1 and BRCA2 mutations among hereditary breast and ovarian cancer patients from Brazil reveals differences among Latin American populations.

Germline mutations in BRCA1 or BRCA2 (BRCA) are responsible for 5-15% of breast (BC) and ovarian cancers (OC), predisposing to the development of early onset and often multiple primary tumors. Since mutation carriers can benefit from risk-reducing interventions, the identification of individuals with hereditary breast and ovarian cancer (HBOC) syndrome has a significant clinical impact. We assessed whether a panel assay for recurrent Hispanic BRCA mutations (HISPANEL) has an adequate breadth of coverage to be suitable as a cost effective screening tool for HBOC in a cohort of patients from Southern Brazil. A multiplex, PCR-based panel was used to genotype 232 unrelated patients for 114 germline BRCA mutations, finding deleterious mutations in 3.5% of them. This mutation prevalence is within the range detected by the HISPANEL among BC patients unselected for family history in other Latin American settings. The HISPANEL would have accounted for 27% of the BRCA mutations detected by complete sequencing in a comparison cohort (n = 193). This prevalence may be region-specific since significant differences in population structure exist in Brazil. Comprehensive analysis of BRCA in a larger set of HBOC patients from different Brazilian regions is warranted, and the results could inform customization of the HISPANEL as an affordable mutation screening tool.