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Fabry disease (FD) is a rare lysosomal disorder caused by α-galactosidase A deficiency, and it leads to the systemic deposition of globotriasylceramide. Demonstrations of the storage material in biopsies support this diagnosis. We report a histological and ultrastructural study of biopsies that were performed on 11 individuals from a family with the variant p.Gln279Arg in GLA, which is associated with the classical phenotype of Fabry disease. Intralysosomal deposits were found in all biopsies, corresponding to the skin, kidney, and endomyocardium in both sexes and at different ages. In nine of the skin biopsies, deposits were analysed by immunofluorescence and quantified at the ultrastructural level. Then, the findings were compared according to sex, genotype, and treatment. The quantification of the deposits in the skin biopsies revealed a broader involvement in men than in women. A significant clearance of the deposits was observed in one case after treatment. Tissue involvement was remarkable at diagnosis in all individuals. The findings from the skin biopsies were demonstrative of classic FD, thus supporting the diagnosis; repeated biopsy analyses suggested the benefit of early treatment. [ABSTRACT FROM AUTHOR]

Lysosomal accumulation of the glycosphingolipid globotriaosylceramide Gb3 is linked to the deficient activity of the α-galactosidase A in the Anderson–Fabry disease and an elevated level of deacylated Gb3 is a hallmark of this condition. Localization of Gb3 in the plasma membrane is critical for studying how the membrane organization and its dynamics are affected in this genetic disorder. Gb3 analogs containing a terminal 6-azido-functionalized galactose in its head group globotriose (αGal1, 4βGal1, and 4Glc) are attractive chemical reporters for bioimaging, as the azido-group may act as a chemical tag for bio-orthogonal click chemistry. We report here the production of azido-Gb3 analogs employing mutants of galactokinase, UTP–glucose-1-phosphate uridylyltransferase, and α-1,4-galactosyltransferase LgtC, which participate in the synthesis of the sugar motif globotriose. Variants of enzymes galactokinase/UTP–glucose-1-phosphate uridylyltransferase generate UDP-6-azido-6-deoxy- d -galactose, which is the galactosyl-donor used by LgtC for transferring the terminal galactose moiety to lactosyl-acceptors. Residues at the galactose-binding site of the 3 enzymes were modified to facilitate the accommodation of azido-functionalized substrates and variants outperforming the wild-type enzymes were characterized. Synthesis of 6-azido-6-deoxy- d -galactose-1-phosphate, UDP-6-azido-6-deoxy- d -galactose, and azido-Gb3 analogs by variants GalK-E37S, GalU-D133V, and LgtC-Q187S, respectively, is 3–6-fold that of their wild-type counterparts. Coupled reactions with these variants permit the production of the pricy, unnatural galactosyl-donor UDP-6-azido-6-deoxy- d -galactose with ~90% conversion yields, and products azido-globotriose and lyso-AzGb3 with substrate conversion of up to 70%. AzGb3 analogs could serve as precursors for the synthesis of other tagged glycosphingolipids of the globo-series. [ABSTRACT FROM AUTHOR]

[Display omitted] • Fabry Disease (FD) manifestations include organ damage, systemic inflammation, and fibrosis. • Lipidome changes possibly reflect systemic and cellular responses to FD. • Plasma ceramide ratios used in cardiovascular risk assessment were increased in FD. • Platelets accumulated lyso-Gb3, but not Gb3 in FD patients. • Identified lipids may serve as potential new screening and prognostic markers for FD. Fabry disease (FD) is an X-linked lysosomal storage disorder characterized by the progressive accumulation of globotriaosylceramide (Gb3) leading to systemic manifestations such as chronic kidney disease, cardiomyopathy, and stroke. There is still a need for novel markers for improved FD screening and prognosis. Moreover, the pathological mechanisms in FD, which also include systemic inflammation and fibrosis, are not yet fully understood. Plasma and platelets were obtained from 11 ERT (enzyme–replacement therapy)-treated symptomatic, 4 asymptomatic FD patients, and 13 healthy participants. A comprehensive targeted lipidomics analysis was conducted quantitating more than 550 lipid species. Sphingadiene (18:2;O2)-containing sphingolipid species, including Gb3 and galabiosylceramide (Ga2), were significantly increased in FD patients. Plasma levels of lyso-dihexosylceramides, sphingoid base 1-phosphates (S1P), and GM3 ganglioside were also altered in FD patients, as well as specific plasma ceramide ratios used in cardiovascular disease risk prediction. Gb3 did not increase in patients' platelets but displayed a high inter-individual variability in patients and healthy participants. Platelets accumulated, however, lyso-Gb3, acylcarnitines, C16:0-sphingolipids, and S1P. This study identified lipidome changes in plasma and platelets from FD patients, a possible involvement of platelets in FD, and potential new markers for screening and monitoring of this disease. [ABSTRACT FROM AUTHOR]

Background: Sporadic Parkinson's disease (PD) patients have lower α-galactosidase A (α-GAL A) enzymatic activity and Fabry disease (FD) patients potentially carry an increased risk of PD. Objective: Determination of PD prevalence in FD and clinical, biochemical and vascular neuroimaging description of FD pedigrees with concomitant PD. Methods: Clinical screening for PD in 229 FD patients belonging to 31 families, harbouring GLA gene mutation p.F113L, and subsequent pedigree analysis. Gender-stratified comparison of FD+/PD+ patients with their family members with FD but without PD (FD+/PD–) regarding Mainz scores, plasma & leukocytes α-GAL A enzymatic activity, urinary Gb3 and plasma Lyso-Gb3, vascular brain neuroimaging. Results: Prevalence of PD in FD was 1.3% (3/229) (3% in patients aged ≥50 years). Three FD patients, one female (73 years old) (P1) and two males (60 and 65 years old) (P2 and P3), three different pedigrees, presented akinetic-rigid PD, with weak response to levodopa (16% – 36%), and dopaminergic deficiency on 18F-DOPA PET. No pathogenic mutations were found in a PD gene panel. FD+/PD+ patients had worse clinical severity of FD (above upper 75% IQR in Mainz scores), and cortico-subcortical white matter/small vessel lesions. P3 patient was under enzyme therapy, started 1 year before PD diagnosis. P2-P3 patients had higher leucocyte α-GAL A activity (2,2-3 vs.1,0 (median)(nmol/h/mg)). Conclusion: We have shown a high prevalence of PD in a late-onset phenotype of FD, presenting high cerebrovascular burden and weak response to levodopa. Further studies will untangle how much of this PD phenotype is due to Gb3 deposition versus cerebrovascular lesions in the nigro-striatal network. [ABSTRACT FROM AUTHOR]

Fabry disease is an X-linked lysosomal storage disease caused by loss of alpha galactosidase A (α-Gal A) activity and is characterized by progressive accumulation of globotriaosylceramide and its analogs in all cells and tissues. Although enzyme replacement therapy (ERT) is considered standard of care, the long-term effects of ERT on renal and cardiac manifestations remain uncertain and thus novel therapies are desirable. We herein report preclinical studies evaluating systemic messenger RNA (mRNA) encoding human α-Gal A in wild-type (WT) mice, α-Gal A-deficient mice, and WT non-human primates (NHPs). The pharmacokinetics and distribution of h-α-Gal A mRNA encoded protein in WT mice demonstrated prolonged half-lives of α-Gal A in tissues and plasma. Single intravenous administration of h-α-Gal A mRNA to Gla -deficient mice showed dose-dependent protein activity and substrate reduction. Moreover, long duration (up to 6 weeks) of substrate reductions in tissues and plasma were observed after a single injection. Furthermore, repeat i.v. administration of h-α-Gal A mRNA showed a sustained pharmacodynamic response and efficacy in Fabry mice model. Lastly, multiple administrations to non-human primates confirmed safety and translatability. Taken together, these studies across species demonstrate preclinical proof-of-concept of systemic mRNA therapy for the treatment of Fabry disease and this approach may be useful for other lysosomal storage disorders. [ABSTRACT FROM AUTHOR]

Fabry disease is an X-linked lysosomal storage disorder caused by deficiency of α-galactosidase A, resulting in the accumulation of glycosphingolipids in various organs. Globotriaosylceramide (Gb3) and its isoforms and analogues have been identified and quantified as biomarkers of disease severity and treatment efficacy. The current study aimed to establish rapid methods for urinary Gb3 extraction and quantitation. Urine samples from 15 Fabry patients and 21 healthy control subjects were processed to extract Gb3 by mixing equal volumes of urine, methanol containing an internal standard, and chloroform followed by sonication and centrifugation. Thereafter, the lower phase was analyzed by MALDI-TOF MS and the relative peak areas of the internal standard and four major species of Gb3 determined. The results showed high reproducibility with intra- and inter-assay coefficients variation of 9.9% and 13.7%, respectively. The limit of detection was 0.15 ng/μL and the limit of quantitation was 0.30 ng/μL. Total urinary Gb3 levels in both genders of classic Fabry patients were significantly higher than in healthy controls ( p < 0.0001). Gb3 levels in Fabry males were higher than in Fabry females ( p = 0.08). We have established a novel assay for urinary total Gb3 that takes less than 15 min from start to finish. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

To avoid any irreversible organ damage in patients with Fabry disease (FD), therapeutic interventions should be undertaken at an early stage. Unfortunately, early detection of the disease based on clinical symptoms alone is complicated by the fact that all complications of FD are nonspecific and clinically indistinguishable from similar abnormalities that occur in common disorders. Although the diagnosis of FD based on the determination of α-GAL A activity and GLA gene analysis can be relatively easy in affected males with classical manifestations, in male individuals with attenuated phenotypes and in heterozygous female patients the residual enzyme activity can be significant. In these cases genetic analysis may also be inconclusive due to the difficulty in establishing whether some of the encountered abnormalities in the GLA gene are truly disease-causing mutations. Because of the limitations of the current detection methods for FD, attention is now paid to the identification of biomarkers that allow solid confirmation of the diagnosis as well as monitoring of disease manifestations and therapeutic management. [ABSTRACT FROM AUTHOR]

Background: Fabry disease is a hereditary genetic defect resulting in reduced activity of the enzyme α-galactosidase-A and the accumulation of globotriaosylceramide (Gb3) in body fluids and cells. Gb3 accumulation was especially reported for the vascular endothelium in several organs. Methods: Three Fabry disease patients were screened using a micro-RNA screen. An in vitro approach in human endothelial cells was used to determine miRNA regulation by Gb3. Results: In a micro-RNA screen of three Fabry patients undergoing enzyme replacement therapy, we found that miRNAs let-7a and let-7d were significantly increased after therapy. We demonstrate in vitro in endothelial cells that Gb3 induced activation of NF-κB and activated downstream targets. In addition, NF-κB activity directly reduced let-7a and let-7d miRNA expression as inhibiting NF-kB nuclear entry abolished the Gb3 effects. Conclusion: We suggest that let-7a and let-7d are potential markers for enzyme activity and inflammation in Fabry disease patients. [ABSTRACT FROM AUTHOR]

Fabry disease (FD) is a lysosomal storage disorder caused by deficient alpha-galactosidase A activity in the lysosome due to mutations in the GLA gene, resulting in gradual accumulation of globotriaosylceramide and other derivatives in different tissues. Substrate accumulation promotes different pathogenic mechanisms in which several mediators could be implicated, inducing multiorgan lesions, mainly in the kidney, heart and nervous system, resulting in clinical manifestations of the disease. Enzyme replacement therapy was shown to delay disease progression, mainly if initiated early. However, a diagnosis in the early stages represents a clinical challenge, especially in patients with a non-classic phenotype, which prompts the search for biomarkers that help detect and predict the evolution of the disease. We have reviewed the mediators involved in different pathogenic mechanisms that were studied as potential biomarkers and can be easily incorporated into clinical practice. Some accumulation biomarkers seem to be useful to detect non-classic forms of the disease and could even improve diagnosis of female patients. The combination of such biomarkers with some response biomarkers, may be useful for early detection of organ injury. The incorporation of some biomarkers into clinical practice may increase the capacity of detection compared to that currently obtained with the established diagnostic markers and provide more information on the progression and prognosis of the disease. [ABSTRACT FROM AUTHOR]

Fabry disease is a rare lysosomal storage disorder resulting from the lack of α-Gal A gene activity. Globotriaosylceramide (GB3, ceramide trihexoside) is a novel endogenous biomarker which predicts the incidence of Fabry disease. At the early stage efficacy/biomarker study, a rapid method to determine this biomarker in plasma and in all relevant tissues related to this disease simultaneously is required. However, the limited sample volume, as well as the various levels of GB3 in different matrices makes the GB3 quantitation very challenging. Hereby we developed a rapid method to identify GB3 in mouse plasma and various tissues. Preliminary stability tests were also performed in three different conditions: short-term, freeze-thaw, long-term. The calibration curve was well fitted over the concentration range of 0.042–10 μg/mL for GB3 in plasma and 0.082–20 μg/g for GB3 in various tissues. This method was successfully applied for the comparison of GB3 levels in Fabry model mice (B6;129-Glatm1Kul/J), which has not been performed previously to the best of our knowledge. [ABSTRACT FROM AUTHOR]