Fabry pedigree analysis: A successful program for targeted genetic approach.

BACKGROUND: Fabry disease (FD) is an X-linked disorder of glycosphingolipid catabolism caused by a deficiency of the lysosomal enzyme alpha-galactosidase A (GLA). FD is still an underdiagnosed disorder worldwide. Moreover, there is delay between symptom onset and Fabry diagnosis of at least 10 years. Family screening offers an important benefit for detection of new patients. The aim of this work is to present the approach along with the results of a targeted genetic strategy for pedigree analysis for FD in Argentina. METHODS: By this strategy as soon as a new index Fabry patient is diagnosed, the pedigree group contacts the physician and a meeting is arranged with the physician and the family to build the family tree. RESULTS: Pedigree analysis was carried out for full in 31 families. In the work period, we have tested 1,462 relatives, and 501 were diagnosed FD. The proportion of positive detection was 33%. CONCLUSION: The targeted family screening approach is successful to detect undiagnosed Fabry patients. By this approach, the highest ratio index to pedigree ever reported for FD pedigree analysis of 1:15 was obtained.

Efficacy of pentosan polysulfate in in vitro models of lysosomal storage disorders: Fabry and Gaucher Disease.

Gaucher and Fabry diseases are the most prevalent sphingolipidoses. Chronic inflammation is activated in those disorders, which could play a role in pathogenesis. Significant degrees of amelioration occur in patients upon introduction of specific therapies; however, restoration to complete health status is not always achieved. The idea of an adjunctive therapy that targets inflammation may be a suitable option for patients. PPS is a mixture of semisynthetic sulfated polyanions that have been shown to have anti-inflammatory effects in mucopolysaccharidosis type I and II patients and animal models of type I, IIIA and VI. We hypothesized PPS could be a useful adjunctive therapy to inflammation for Gaucher and Fabry diseases. The objective of this work is to analyze the in vitro effect of PPS on inflammatory cytokines in cellular models of Gaucher and Fabry diseases, and to study its effect in Gaucher disease associated in vitro bone alterations. Cultures of peripheral blood mononuclear cells from Fabry and Gaucher patients were exposed to PPS. The secretion of proinflammatory cytokines was significantly reduced. Peripheral blood cells exposed to PPS from Gaucher patients revealed a reduced tendency to differentiate to osteoclasts. Osteoblasts and osteocytes cell lines were incubated with an inhibitor of glucocerebrosidase, and conditioned media was harvested in order to analyze if those cells secrete factors that induce osteoclastogenesis. Conditioned media from this cell cultures exposed to PPS produced lower numbers of osteoclasts. We could demonstrate PPS is an effective molecule to reduce the production of proinflammatory cytokines in in vitro models of Fabry and Gaucher diseases. Moreover, it was effective at ameliorating bone alterations of in vitro models of Gaucher disease. These results serve as preclinical supportive data to start clinical trials in human patients to analyze the effect of PPS as a potential adjunctive therapy for Fabry and Gaucher diseases.

Apical left ventricular hypertrophy and mid-ventricular obstruction in fabry disease.

We report the case of a rare cardiac presentation of Fabry disease. Although concentric left ventricular hypertrophy is a major cardiac finding in Fabry disease, there is no case report of dynamic obstruction at mid-left ventricular level. We describe a 59-year-old-woman suffering from a severe form of Fabry disease, mimicking an apical hypertrophic cardiomyopathy with mid-ventricular obstruction. Differentiation of Fabry disease from hypertrophic cardiomyopathy is crucial given the therapeutic and prognostic differences. Fabry disease should always be suspected in an adult, independently of the pattern of left ventricular hypertrophy.

The Continuous Challenge of Diagnosing patients with Fabry disease in Argentina: Genotype, Experiences, Anecdotes, and New Learnings

Abstract The lysosomal storage disorder Fabry disease (FD) is caused by pathogenic mutations in the α-galactosidase A gene, localized in X chromosome. Deficient enzymatic activity of the product of this gene, the lysosomal hydrolase α-galactosidase A, leads to accumulation of its substrate globotriaosylceramide. Diagnosis of FD starts with clinical suspicion followed by confirmatory laboratory testing. The aim of this work is to report the 14 years' experience and learnings in the diagnosis of patients with Fabry disease in Argentina from a specialized lysosomal diseases diagnosis laboratory and to report the genotype characterization of the 25 families from Argentina with FD detected by us.

Uncoupling of osteoblast-osteoclast regulation in a chemical murine model of Gaucher disease.

Gaucher disease (GD) is caused by mutations in the GBA gene that confer a deficient level of activity of glucocerebrosidase (GCase). This deficiency leads to accumulation of the glycolipid glucocerebroside in the lysosomes of cells of monocyte/macrophage system. Type I GD is the mildest form and is characterized by the absence of neuronopathic affection. Bone compromise in Gaucher disease patients is the most disabling aspect of the disease. However, pathophysiological aspects of skeletal alterations are still poorly understood. The homeostasis of bone tissue is maintained by the balanced processes of bone resorption by osteoclasts and formation by osteoblasts. We decided to test whether bone resorption and/or bone formation could be altered by the use of a chemical in vitro murine model of Gaucher disease. We used two sources of cells from monocyte/macrophages lineage isolated from normal mice, splenocytes (S) and peritoneal macrophages (PM), and were exposed to CBE, the inhibitor of GCase (S-CBE and PM-CBE, respectively). Addition of both conditioned media (CM) from S-CBE and PM-CBE induced the differentiation of osteoclasts precursors from bone marrow to mature and functional osteoclasts. TNF-α could be one of the factors responsible for this effect. On the other hand, addition of CM to an osteoblast cell culture resulted in a reduction in expression of alkaline phosphatase and mineralization process. In conclusion, these results suggest implication of changes in both bone formation and bone resorption and are consistent with the idea that both sides of the homeostatic balance are affected in GD.

Two-dimensional speckle tracking echocardiography for early detection of myocardial damage in young patients with Fabry disease.

Fabry disease (FD) is characterized by left ventricular hypertrophy (LVH). Conventional echocardiography is not sensitive enough to perform the preclinical diagnosis To assess whether longitudinal myocardial strain of the left ventricle (LV), using speckle tracking, is useful to detect early myocardial involvement in FD. Forty-four patients with FD who were diagnosed with genetic testing were prospectively included and were compared to a sex-matched control group. They were divided into three groups: 22 with LVH (Group I), 22 without LVH (Group II), and 22 healthy volunteers (Group III). LV longitudinal strain was measured from the apical views. An ANOVA test was used for multiple comparisons for variables with a normal distribution, and a Kruskal-Wallis test was used for variables with non-Gaussian distribution. Longitudinal LV strain was different in the three groups: it was ≥-15% in at least one segment in all Group I patients, in 50% of patients of Group II and in no patient of Group III. Seventy percent of the segments with abnormal strain in Group II were located in the basal regions (32/46). These findings show that the presence of at least one strain value ≥-15% demonstrates subclinical myocardial dysfunction in patients with preclinical FD. Longitudinal myocardial LV strain measured with speckle tracking is a useful tool to detect early myocardial involvement in young patients with FD. This information allows the detection and treatment of myocardial dysfunction at an early stage, which is of high clinical importance.

Fabry disease peripheral blood immune cells release inflammatory cytokines: role of globotriaosylceramide.

Fabry disease is an X-linked lysosomal disorder (LD) due to deficiency of the enzyme α-galactosidase A (αGal), which leads to the accumulation of neutral glycosphingolipids, mainly globotriaosylceramide (Gb3). Several mechanisms contribute to the diverse physiopathological alterations observed in this disease, and it has been suggested that an underlying proinflammatory state could play a significant role. The aim of this study is to investigate the presence of a proinflammatory state in the different subsets of peripheral blood mononuclear cells (PBMC) and to understand the mechanisms that contribute to its onset and perpetuation. We have shown that cultured PBMC from Fabry patients present a higher proinflammatory cytokine expression and production. Moreover, we determined that among PBMC, dendritic cells and monocytes present a basal proinflammatory cytokine production profile, which is further exacerbated with an inflammatory stimulus. Finally we established that normal, monocyte-derived dendritic cells and macrophages display the same proinflammatory profile when cultured in the presence of Gb3 and an inhibitor of αGal. Furthermore, this effect can be abolished using a TLR4 blocking antibody, indicating that TLR4 is necessary in the process. In summary, our results demonstrate the presence of a proinflammatory state involving two key subsets of innate immunity, and provide direct evidence of Gb3 having a proinflammatory role, likely mediated by TLR4, a finding that could help in the understanding of the underlying causes of the inflammatory pathogenesis of Fabry disease.

Induction of osteoclastogenesis in an in vitro model of Gaucher disease is mediated by T cells via TNF-α.

Gaucher disease is a lysosomal storage disorder caused by deficiency of glucocerebrosidase enzymatic activity leading to accumulation of its substrate glucocerebrosidase mainly in macrophages. Skeletal disorder of Gaucher disease is the major cause of morbidity and is highly refractory to enzyme replacement therapy. However, pathological mechanisms of bone alterations in Gaucher disease are still poorly understood. We hypothesized that cellular alteration in Gaucher disease produces a proinflammatory milieu leading to bone destruction through enhancement of monocyte differentiation to osteoclasts and osteoclasts resorption activity. Against this background we decided to investigate in an in vitro chemical model of Gaucher disease, the capacity of secreted soluble mediators to induce osteoclastogenesis, and the mechanism responsible for this phenomena. We demonstrated that soluble factors produced by CBE-treated PBMC induced differentiation of osteoclasts precursors into mature and active osteoclasts that express chitotriosidase and secrete proinflammatory cytokines. We also showed a role of TNF-α in promoting osteoclastogenesis in Gaucher disease chemical model. To analyze the biological relevance of T cells in osteoclastogenesis of Gaucher disease, we investigated this process in T cell-depleted PBMC cultures. The findings suggest that T cells play a role in osteoclast formation in Gaucher disease. In conclusion, our data suggests that in vitro GCASE deficiency, along with concomitant glucosylceramide accumulation, generates a state of osteoclastogenesis mediated in part by pro-resorptive cytokines, especially TNF-α. Moreover, T cells are involved in osteoclastogenesis in Gaucher disease chemical model.

Higher apoptotic state in Fabry disease peripheral blood mononuclear cells.: effect of globotriaosylceramide.

Fabry disease is an X-linked lysosomal storage disorder (LSD) due to deficiency of the enzyme α-galactosidase A, resulting in intracellular deposition of globotriaosylceramide (Gb3). Accumulation of Gb3 is probably related to tissue and organ dysfunctions. Diverse pathological mechanisms are elicited in LSDs, giving together the phenotypic expression of each disease. The purpose of the present study is to investigate if apoptosis could play a role in Fabry disease pathogenesis and to understand the mechanisms involved in the proapoptotic state. We have demonstrated that Fabry disease peripheral blood mononuclear cells display a higher apoptotic state, which is reduced by enzyme replacement therapy (ERT), and is mediated, at least in part, by activation of the intrinsic pathway of caspases. We could rule out the implication of "unfolded protein response-ER stress" in this apoptotic process. To further confirm the suggestion that Gb3 is associated to apoptotic cell death, we treated normal cells with Gb3 at concentrations found in Fabry patients. Addition of Gb3 resulted in a dose-dependent induction of apoptosis involving the intrinsic pathway. In summary, PBMC from Fabry patients display a higher apoptotic state, which could be mainly related to elevated Gb3.

Myocardial alterations in the murine model of fabry disease can be reversed by enzyme replacement therapy.

BACKGROUND: Fabry disease results from deficiency of alpha-galactosidase A (AGA), causing lysosomal storage of globotriaosylceramide in heart and other tissues. Since 2003, enzymatic replacement therapy with recombinant AGA agalsidase alfa (R-AGA) was approved for clinical use. METHODS: We evaluated whether, in mice knocked out for AGA (FM, n = 31), the myocardium was altered with respect to the wild-type mice (WT, n = 25) and whether alterations were reversed in FM treated with intravenous R-AGA, 0.5 mg/kg every other week during 2 months (FM-AGA, n = 12). RESULTS: Left ventricular (LV) contractility was depressed in FM, evaluated by LV ΔP/Δt (FM = 2832 ± 85 mm Hg/s, WT = 3179 ± 119 mm Hg/s; P < 0.05), papillary muscle contraction (FM = 39.8 ± 17.3 mg, WT = 67.5 ± 15.7 mg; P < 0.05), or shortening fraction measured by M-mode echocardiography (FM = 30% ± 6%, WT = 47% ± 2%; P < 0.05). LV stiffness (arrested hearts) decreased in FM (FM = 35.57 ± 3.5 mm Hg/20 µl; WT = 68.86 ± 6.12 mm Hg/20 µl; P < 0.05). FM myocytes showed augmented size, disorganized architecture, and intracytoplasmic vacuolization. Alterations reverted in FM-AGA: LV ΔP/Δt = 3281 ± 456 mm Hg/s and LV stiffness = 58.83 ± 2.15 mm Hg/20 µl, with normalization of myocyte architecture. No reversion was detected with AGA solvent. CONCLUSIONS: The FM represent a mild, early stage of the disease, since myocardial alterations are not prominent and appear in nonhypertrophic hearts. Reversion of alterations in the FM-AGA suggests that enzymatic replacement therapy can be useful when administered in early stages of this disease.

Fabry disease: treatment and diagnosis.

Fabry disease is an X-linked lysosomal disorder that results from a deficiency of the lysosomal enzyme alpha-galactosidase A leading to accumulation of glycolipids, mainly globotriaosylceramide in the cells from different tissues. Classical Fabry disease affects various organs. Clinical manifestations start at early age and include angiokeratoma, acroparesthesia, hypohydrosis, heat/exercise intolerance, gastrointestinal pain, diarrhea, and fever. The main complications of Fabry disease are more prominent after the age of 30 when kidney, heart, and/or cerebrovascular disorders appear. Most of the heterozygous females are symptomatic. Enzyme replacement therapy (ERT) is the only specific treatment for Fabry disease. The beneficial effect of ERT on different organs/systems has been extensively evaluated. Quality of life of patients receiving ERT is improved. Enzyme replacement stabilizes or slows the decline in renal function and reduces left ventricular hypertrophy. Fabry disease may be underdiagnosed because of nonspecific and multiorgan symptoms. Different screening strategies have been carried out in different at-risk populations in order to detect undiagnosed Fabry patients. An increasing knowledge about Fabry disease within the medical community increases the chances of patients to receive a timely diagnosis and, consequently, to access the appropriate therapy.

An easy and sensitive method for determination of globotriaosylceramide (Gb3) from urinary sediment: utility for Fabry disease diagnosis and treatment monitoring.

BACKGROUND: Fabry disease is an X-linked disorder that results from the deficiency of the lysosomal enzyme alpha-galactosidase A. The defect leads to the accumulation of globotriaosylceramide (Gb3). The detection of Gb3 accumulated in different tissues may help in the diagnosis and enzyme replacement therapy monitoring. For this reason, we developed a simple method available to clinical laboratories to measure this analyte. METHODS: Gb3 excretion was determined by the incubation of urine sediment glycolipids from Fabry patients with agalsidase alpha and subsequent determination of galactose produced. RESULTS: The amount of urinary Gb3 in Fabry hemizygotes was significantly higher (p = 0.00001) than the amount in normal controls. Patients undergoing enzyme replacement therapy with agalsidase alpha showed a significantly lower content of Gb3 in urine sediment. This method showed a good recovery and comparability with a previously validated method. CONCLUSIONS: We developed an easy method for quantification of Gb3 in urine samples from Fabry patients, by the use of the specific recombinant enzyme for this glycolipid, that does not require complex infrastructure. Urinary Gb3 as measured by this enzymatic method could be useful for the diagnosis and monitoring of treatment in Fabry patients.

Immunofluorescence detection of globotriaosylceramide deposits in conjunctival biopsies of Fabry disease patients.

BACKGROUND: Fabry disease is an X-linked disorder due to a deficiency of alpha-galactosidase A and leads to the accumulation of globotriaosylceramide (Gb3) in various cells. The detection of Gb3 deposits may help in the diagnosis. To date, no immunofluorescence-specific detection of Gb3 in conjunctival biopsies has been reported. The aim of this work was to detect Gb3 accumulation in conjunctival biopsies from Fabry patients by immunofluorescence. METHODS: Conjunctival biopsies taken from Fabry males and females, before and after enzyme replacement therapy, and normal controls were processed for immunofluorescence with a monoclonal antibody specific for Gb3. RESULTS: Positive green immunofluorescence was observed in all biopsies from Fabry patients before enzyme replacement therapy. After 6 months of treatment, immunofluorescence in blood vessels was not observed. CONCLUSIONS: Immunofluorescence detection of Gb3 in conjunctival biopsies may be a reliable method for the diagnosis of Fabry disease in family members, and to evaluate effectiveness of enzyme replacement therapy.