Genetic variants of unknown significance in alpha-galactosidase A: Cellular delineation from Fabry disease.

Fabry disease (FD) is an X-linked multiorgan disorder caused by variants in the alpha-galactosidase A gene (GLA). Depending on the variant, disease phenotypes range from benign to life-threatening. More than 1000 GLA variants are known, but a link between genotype and phenotype in FD has not yet been established for all. p.A143T, p.D313Y, and p.S126G are frequent examples of variants of unknown significance (VUS). We have investigated the potential pathogenicity of these VUS combining clinical data with data obtained in human cellular in vitro systems. We have analyzed four different male subject-derived cell types for alpha-galactosidase A enzyme (GLA) activity and intracellular Gb3 load. Additionally, Gb3 load in skin tissue as well as clinical data were studied for correlates of disease manifestations. A reduction of GLA activity was observed in cells carrying p.A143T compared with controls (p < 0.05). In cells carrying the p.D313Y variant, a reduced GLA activity was found only in endothelial cells (p < 0.01) compared with controls. No pathological changes were observed in cells carrying the p.S126G variant. None of the VUS investigated caused intracellular Gb3 accumulation in any cell type. Our data of aberrant GLA activity in cells of p.A143T hemizygotes and overall normal cellular phenotypes in cells of p.D313Y and p.S126G hemizygotes contribute a basic science perspective to the clinically highly relevant discussion on VUS in GLA.

A phase III, open-label clinical trial evaluating pegunigalsidase alfa administered every 4 weeks in adults with Fabry disease previously treated with other enzyme replacement therapies.

Pegunigalsidase alfa, a PEGylated α-galactosidase A enzyme replacement therapy (ERT) for Fabry disease, has a longer plasma half-life than other ERTs administered intravenously every 2 weeks (E2W). BRIGHT (NCT03180840) was a phase III, open-label study in adults with Fabry disease, previously treated with agalsidase alfa or beta E2W for ≥3 years, who switched to 2 mg/kg pegunigalsidase alfa every 4 weeks (E4W) for 52 weeks. Primary objective assessed safety, including number of treatment-emergent adverse events (TEAEs). Thirty patients were enrolled (24 males); 23 previously received agalsidase beta. Pegunigalsidase alfa plasma concentrations remained above the lower limit of quantification throughout the 4-week dosing interval. Thirty-three of 182 TEAEs (in 9 patients) were considered treatment-related; all were mild/moderate. No patients developed de novo anti-drug antibodies (ADAs). In the efficacy analysis (n = 29), median (inter-quartile range) eGFR change from baseline over 52 weeks was -1.9 (-5.9; 1.8) mL/min/1.73 m2 (n = 28; males [n = 22]: -2.4 [-5.2; 3.2]; females [n = 6]: -0.7 [-9.2; 2.0]). Overall, median eGFR slope was -1.9 (-8.3; 1.9) mL/min/1.73 m2/year (ADA-negative [n = 20]: -1.2 [-6.4; 2.6]; ADA-positive [n = 9]: -8.4 [-11.6; -1.0]). Lyso-Gb3 concentrations were low and stable in females, with a slight increase in males (9/24 ADA-positive). The BRIGHT study results suggest that 2 mg/kg pegunigalsidase alfa E4W is tolerated well in stable adult patients with Fabry disease. Due to the low number of patients in this study, more research is needed to demonstrate the effects of pegunigalsidase alfa given E4W. Further evidence, outside of this clinical trial, should be factored in for physicians to prolong the biweekly ERT intervals to E4W. TAKE-HOME MESSAGE: Treatment with 2 mg/kg pegunigalsidase alfa every 4 weeks could offer a new treatment option for patients with Fabry disease.

Single-mutations at the galactose-binding site of enzymes GalK, GalU, and LgtC enable the efficient synthesis of UDP-6-azido-6-deoxy-d-galactose and azido-functionalized Gb3 analogs.

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, 4ßGal1, 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.

Investigation of bone mineral density and the changes by enzyme replacement therapy in patients with Fabry disease.

BACKGROUND: Fabry disease (FD) is an inherited disorder that causes organ dysfunction. However, only a few studies have reported on bone mineral density (BMD) in FD patients, and the relationship between BMD and clinical factors such as globotriaosylsphingosine (lyso-Gb3) remains unclear. Therefore, the current study sought to investigate BMD in FD patients, the relationship between BMD and lyso-Gb3, and the effects of enzyme replacement therapy (ERT) on changes in BMD and lyso-Gb3. METHODS: This single-center, observational study included 15 patients who visited our facility for FD between January 2008 and June 2021. We assessed BMD and clinical characteristics in study patients, including plasma lyso-Gb3 levels, and examined the relationship between BMD and plasma lyso-Gb3 levels, and changes in BMD after starting ERT. RESULTS: Male patients' BMD had reduced, whereas female patients' BMD was preserved. Male patients had significantly higher plasma lyso-Gb3 levels than female patients. Moreover, plasma lyso-Gb3 levels were found to be significantly related to the lumbar spine and femoral BMD. These were strongly linked with plasma lyso-Gb3 levels in male patients, whereas no strong link was observed in female patients. Furthermore, BMD significantly increased only in male patients although plasma lyso-Gb3 levels significantly decreased by ERT in all patients. CONCLUSION: BMD decreased possibly due to Gb3 accumulation, and ERT could increase BMD in male FD patients.

Characterization of cellular phenotypes in neurons derived from induced pluripotent stem cells of male patients with Fabry disease.

Fabry disease (FD) is an X-linked inherited lysosomal metabolism disorder in which globotriaosylceramide (Gb3) accumulates in various organs resulting from a deficiency in alpha-galactosidase A. The clinical features of FD include progressive impairments of the renal, cardiac, and peripheral nervous systems. In addition, patients with FD often develop neuropsychiatric symptoms, such as depression and dementia, which are believed to be induced by the cellular injury of cerebrovascular and partially neuronal cells due to Gb3 accumulation. Although the analysis of autopsy brain tissue from patients with FD showed no accumulation of Gb3, abnormal deposits of Gb3 were found in the neurons of several brain areas, including the hippocampus. Therefore, in this study, we generated induced pluripotent stem cells (iPSCs) from patients with FD and differentiated them into neuronal cells to investigate pathological and biological changes in the neurons of FD. Neural stem cells (NSCs) and neurons were successfully differentiated from the iPSCs we generated; however, cellular damage and morphological changes were not found in these cells. Immunostaining revealed no Gb3 accumulation in NSCs and neurons. Transmission electron microscopy did not reveal any zebra body-like structures or inclusion bodies, which are characteristic of FD. These results indicated that neuronal cells derived from FD-iPSCs exhibited normal morphology and no Gb3 accumulation. It is likely that more in vivo environment-like cultures are needed for iPSC-derived neurons to reproduce disease-specific features.

Novel lectin-based chimeric antigen receptors target Gb3-positive tumour cells.

The link between cancer and aberrant glycosylation has recently become evident. Glycans and their altered forms, known as tumour-associated carbohydrate antigens (TACAs), are diverse, complex and difficult to target therapeutically. Lectins are naturally occurring glycan-binding proteins  that offer a unique opportunity to recognise TACAs. T cells expressing chimeric antigen receptors (CARs) have proven to be a successful immunotherapy against leukaemias, but so far have shown limited success in solid tumours. We developed a panel of lectin-CARs that recognise the glycosphingolipid globotriaosylceramide (Gb3), which is overexpressed in various cancers, such as Burkitt's lymphoma, colorectal, breast and pancreatic. We have selected the following lectins: Shiga toxin's B-subunit from Shigella dysenteriae, LecA from Pseudomonas aeruginosa, and the engineered lectin Mitsuba from Mytilus galloprovincialis as antigen-binding domains and fused them to a well-known second-generation CAR. The Gb3-binding lectin-CARs have demonstrated target-specific cytotoxicity against Burkitt's lymphoma-derived cell lines as well as solid tumour cells from colorectal and triple-negative breast cancer. Our findings reveal the big potential of lectin-based CARs as therapeutical applications to target Gb3 and other TACAs expressed in haematological malignancies and solid tumours.

Mass Spectrometry Analysis of Globotriaosylsphingosine and Its Analogues in Dried Blood Spots.

Fabry disease (FD) is an X-linked lysosomal storage disorder where impaired α-galactosidase A enzyme activity leads to the intracellular accumulation of undegraded glycosphingolipids, including globotriaosylsphingosine (lyso-Gb3) and related analogues. Lyso-Gb3 and related analogues are useful biomarkers for screening and should be routinely monitored for longitudinal patient evaluation. In recent years, a growing interest has emerged in the analysis of FD biomarkers in dried blood spots (DBSs), considering the several advantages compared to venipuncture as a technique for collecting whole-blood specimens. The focus of this study was to devise and validate a UHPLC-MS/MS method for the analysis of lyso-Gb3 and related analogues in DBSs to facilitate sample collection and shipment to reference laboratories. The assay was devised in conventional DBS collection cards and in Capitainer®B blood collection devices using both capillary and venous blood specimens from 12 healthy controls and 20 patients affected with FD. The measured biomarker concentrations were similar in capillary and venous blood specimens. The hematocrit (Hct) did not affect the correlation between plasma and DBS measurements in our cohort (Hct range: 34.3-52.2%). This UHPLC-MS/MS method using DBS would facilitate high-risk screening and the follow-up and monitoring of patients affected with FD.

Human Gb3/CD77 synthase produces P1 glycotope-capped N-glycans, which mediate Shiga toxin 1 but not Shiga toxin 2 cell entry.

The human Gb3/CD77 synthase, encoded by the A4GALT gene, is an unusually promiscuous glycosyltransferase. It synthesizes the Galα1→4Gal linkage on two different glycosphingolipids (GSLs), producing globotriaosylceramide (Gb3, CD77, Pk) and the P1 antigen. Gb3 is the major receptor for Shiga toxins (Stxs) produced by enterohemorrhagic Escherichia coli. A single amino acid substitution (p.Q211E) ramps up the enzyme's promiscuity, rendering it able to attach Gal both to another Gal residue and to GalNAc, giving rise to NOR1 and NOR2 GSLs. Human Gb3/CD77 synthase was long believed to transfer Gal only to GSL acceptors, therefore its GSL products were, by default, considered the only human Stx receptors. Here, using soluble, recombinant human Gb3/CD77 synthase and p.Q211E mutein, we demonstrate that both enzymes can synthesize the P1 glycotope (terminal Galα1→4Galß1→4GlcNAc-R) on a complex type N-glycan and a synthetic N-glycoprotein (saposin D). Moreover, by transfection of CHO-Lec2 cells with vectors encoding human Gb3/CD77 synthase and its p.Q211E mutein, we demonstrate that both enzymes produce P1 glycotopes on N-glycoproteins, with the mutein exhibiting elevated activity. These P1-terminated N-glycoproteins are recognized by Stx1 but not Stx2 B subunits. Finally, cytotoxicity assays show that Stx1 can use P1 N-glycoproteins produced in CHO-Lec2 cells as functional receptors. We conclude that Stx1 can recognize and use P1 N-glycoproteins in addition to its canonical GSL receptors to enter and kill the cells, while Stx2 can use GSLs only. Collectively, these results may have important implications for our understanding of the Shiga toxin pathology.

Systemic mRNA Therapy for the Treatment of Fabry Disease: Preclinical Studies in Wild-Type Mice, Fabry Mouse Model, and Wild-Type Non-human Primates.

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.

One of the two N-glycans on the human Gb3/CD77 synthase is essential for its activity and allosterically regulates its function.

N-glycosylation is a posttranslational modification that influences many protein properties, such as bioactivity, folding or solubility. The same principles apply to key enzymes in glycosylation pathways, including glycosyltransferases, that also undergoing N-glycosylation, changes in which may affect their activity. Human Gb3/CD77 synthase (encoded by A4GALT) is a Golgi-resident glycosyltransferase, which catalyzes the synthesis of Galα1→4Gal disaccharide on glycosphingolipid- and glycoprotein-derived acceptors, creating Gb3 or P1 antigens and P1 glycotopes (Galα1→4Galß1→4GlcNAc-R), respectively. The molecules that contain Galα1→4Gal serve as receptors for pathogens and Shiga toxins, which are the major virulence factors of Shiga toxin-producing Escherichia coli (STEC). Human Gb3/CD77 synthase contains two N-glycosylation sites at positions N121 and N203. Using the recombinant soluble glycovariants of human Gb3/CD77 synthase with mutated N-glycosylation sequons expressed in HEK293E cells, we show that the glycovariants devoid of N-glycan at position N203 or simultaneously at N121 and N203 sites reveal no enzymatic activity. In contrast, the N-glycan at position N121 plays a negligible role, whereas the presence of both N-glycans is required for efficient secretion of the enzyme. Moreover, utilizing specific glycosidases, we have found that the fully N-glycosylated enzyme contains one complex and one hybrid/oligomannose N-glycan, while single mutants contain only the complex type. Finally, in silico analysis using the AlphaFold enzyme model showed that N-glycan attached to N203 sequon is located in a protein motif near the active site and may allosterically influence the activity. All these findings highlight the prerequisite role of N-glycosylation in human Gb3/CD77 synthase activity (N203 sequon) and solubility (both N121 and N203), with a particularly prominent role of N-glycan at position N203 in the regulation of enzyme activity.

Agalsidase-ß should be proposed as first line therapy in classic male Fabry patients with undetectable α-galactosidase A activity.

BACKGROUND: Fabry disease (FD) is a rare X-linked lysosomal storage disease caused by mutations in the α-galactosidase A (GLA) gene leading to deficiency of α-galactosidase A (α-gal A). This results in progressive multisystemic glycosphingolipid accumulation, especially globotriaosylceramide (Gb3) and globotriaosylsphingosine (Lyso-Gb3). Enzyme replacement therapy with two recombinant enzymes, agalsidase-α and -ß is approved for two different dosages. However, little is known about which enzyme is more effective in decreasing the metabolite load in male and female patients with the classic form of the disease. METHODS: In this prospective observational study, 14 consecutive adult Fabry patients (10 males) with a classic GLA-mutation, were switched from agalsidase-α to agalsidase-ß at the respective licensed doses. Lyso-Gb3 levels were measured before the switch and for a period of 12 months after the switch in dried blood spots by tandem mass spectrometry. RESULTS: Mean age at start of the switch was 36.7 ± 14 years. Plasma Lyso-Gb3 levels decreased from 27.2 ± 17.9 ng/mL before the switch to 16.8 ± 10.5 ng/mL after the switch (mean reduction of 30.1%; p = 0.004). The decrease was maximal in the subgroup of 7 male patients with no or very low residual enzyme activity (mean reduction of 40.4%). However, two females with high residual enzyme activity also showed a reduction >30% after the switch. In male patients, the reduction of plasma Lyso-Gb3 correlated negatively with the residual α-gal A activity: r = -0.803; p = 0.009. CONCLUSION: Agalsidase-ß at licensed dose is significantly more effective than agalsidase-α to reduce Lyso-Gb3 levels in classic Fabry patients, and should be used as first line therapy in classic males with no residual enzyme activity.

Glycan-Adhering Lectins and Experimental Evaluation of a Lectin FimH Inhibitor in Enterohemorrhagic Escherichia coli (EHEC) O157:H7 Strain EDL933.

In this study, we tried to develop a FimH inhibitor that inhibits adhesion of enterohemorrhagic Escherichia coli (EHEC) on the epithelium of human intestine during the initial stage of infections. Using a T7 phage display method with a reference strain, EHEC EDL933, FimH was selected as an adherent lectin to GM1a and Gb3 glycans. In order to detect the ligand binding domain (LBD) of FimH, we used a docking simulation and found three binding site sequences of FimH, i.e., P1, P2, and P3. Among Gb3 mimic peptides, P2 was found to have the strongest binding strength. Moreover, in vitro treatment with peptide P2 inhibited binding activity in a concentration-dependent manner. Furthermore, we conducted confirmation experiments through several strains isolated from patients in Korea, EHEC NCCP15736, NCCP15737, and NCCP15739. In addition, we analyzed the evolutionary characteristics of the predicted FimH lectin-like adhesins to construct a lectin-glycan interaction (LGI). We selected 70 recently differentiated strains from the phylogenetic tree of 2240 strains with Shiga toxin in their genome. We can infer EHEC strains dynamically evolved but FimH was conserved during the evolution time according to the phylogenetic tree. Furthermore, FimH could be a reliable candidate of drug target in terms of evolution. We examined how pathogen lectins interact with host glycans early in infection in EDL933 as well as several field strains and confirmed that glycan-like peptides worked as an initial infection inhibitor.

CARS Imaging Advances Early Diagnosis of Cardiac Manifestation of Fabry Disease.

Vibrational spectroscopy can detect characteristic biomolecular signatures and thus has the potential to support diagnostics. Fabry disease (FD) is a lipid disorder disease that leads to accumulations of globotriaosylceramide in different organs, including the heart, which is particularly critical for the patient's prognosis. Effective treatment options are available if initiated at early disease stages, but many patients are late- or under-diagnosed. Since Coherent anti-Stokes Raman (CARS) imaging has a high sensitivity for lipid/protein shifts, we applied CARS as a diagnostic tool to assess cardiac FD manifestation in an FD mouse model. CARS measurements combined with multivariate data analysis, including image preprocessing followed by image clustering and data-driven modeling, allowed for differentiation between FD and control groups. Indeed, CARS identified shifts of lipid/protein content between the two groups in cardiac tissue visually and by subsequent automated bioinformatic discrimination with a mean sensitivity of 90-96%. Of note, this genotype differentiation was successful at a very early time point during disease development when only kidneys are visibly affected by globotriaosylceramide depositions. Altogether, the sensitivity of CARS combined with multivariate analysis allows reliable diagnostic support of early FD organ manifestation and may thus improve diagnosis, prognosis, and possibly therapeutic monitoring of FD.

The binding mechanism of the virulence factor Streptococcus suis adhesin P subtype to globotetraosylceramide is associated with systemic disease.

Streptococcus suis is part of the pig commensal microbiome but strains can also be pathogenic, causing pneumonia and meningitis in pigs as well as zoonotic meningitis. According to genomic analysis, S. suis is divided into asymptomatic carriage, respiratory and systemic strains with distinct genomic signatures. Because the strategies to target pathogenic S. suis are limited, new therapeutic approaches are needed. The virulence factor S. suis adhesin P (SadP) recognizes the galabiose Galα1-4Gal-oligosaccharide. Based on its oligosaccharide fine specificity, SadP can be divided into subtypes PN and PO We show here that subtype PN is distributed in the systemic strains causing meningitis, whereas type PO is found in asymptomatic carriage and respiratory strains. Both types of SadP are shown to predominantly bind to pig lung globotriaosylceramide (Gb3). However, SadP adhesin from systemic subtype PN strains also binds to globotetraosylceramide (Gb4). Mutagenesis studies of the galabiose-binding domain of type PN SadP adhesin showed that the amino acid asparagine 285, which is replaced by an aspartate residue in type PO SadP, was required for binding to Gb4 and, strikingly, was also required for interaction with the glycomimetic inhibitor phenylurea-galabiose. Molecular dynamics simulations provided insight into the role of Asn-285 for Gb4 and phenylurea-galabiose binding, suggesting additional hydrogen bonding to terminal GalNAc of Gb4 and the urea group. Thus, the Asn-285-mediated molecular mechanism of type PN SadP binding to Gb4 could be used to selectively target S. suis in systemic disease without interfering with commensal strains, opening up new avenues for interventional strategies against this pathogen.

Blood group P1 antigen-bearing glycoproteins are functional but less efficient receptors of Shiga toxin than conventional glycolipid-based receptors.

Shiga toxin (STx) is a virulence factor produced by enterohemorrhagic Escherichia coli. STx is taken up by mammalian host cells by binding to the glycosphingolipid (GSL) globotriaosylceramide (Gb3; Galα1-4Galß1-4Glc-ceramide) and causes cell death after its retrograde membrane transport. However, the contribution of the hydrophobic portion of Gb3 (ceramide) to STx transport remains unclear. In pigeons, blood group P1 glycan antigens (Galα1-4Galß1-4GlcNAc-) are expressed on glycoproteins that are synthesized by α1,4-galactosyltransferase 2 (pA4GalT2). To examine whether these glycoproteins can also function as STx receptors, here we constructed glycan-remodeled HeLa cell variants lacking Gb3 expression but instead expressing pA4GalT2-synthesized P1 glycan antigens on glycoproteins. We compared STx binding and sensitivity of these variants with those of the parental, Gb3-expressing HeLa cells. The glycan-remodeled cells bound STx1 via N-glycans of glycoproteins and were sensitive to STx1 even without Gb3 expression, indicating that P1-containing glycoproteins also function as STx receptors. However, these variants were significantly less sensitive to STx than the parent cells. Fluorescence microscopy and correlative light EM revealed that the STx1 B subunit accumulates to lower levels in the Golgi apparatus after glycoprotein-mediated than after Gb3-mediated uptake but instead accumulates in vacuole-like structures probably derived from early endosomes. Furthermore, coexpression of Galα1-4Gal on both glycoproteins and GSLs reduced the sensitivity of cells to STx1 compared with those expressing Galα1-4Gal only on GSLs, probably because of competition for STx binding or internalization. We conclude that lipid-based receptors are much more effective in STx retrograde transport and mediate greater STx cytotoxicity than protein-based receptors.

Assessment of plasma lyso-Gb3 for clinical monitoring of treatment response in migalastat-treated patients with Fabry disease.

PURPOSE: To assess the utility of globotriaosylsphingosine (lyso-Gb3) for clinical monitoring of treatment response in patients with Fabry disease receiving migalastat. METHODS: A post hoc analysis evaluated data from 97 treatment-naive and enzyme replacement therapy (ERT)-experienced patients with migalastat-amenable GLA variants from FACETS (NCT00925301) and ATTRACT (NCT01218659) and subsequent open-label extension studies. The relationship between plasma lyso-Gb3 and measures of Fabry disease progression (left ventricular mass index [LVMi], estimated glomerular filtration rate [eGFR], and pain) and the relationship between lyso-Gb3 and incidence of Fabry-associated clinical events (FACEs) were assessed in both groups. The relationship between changes in lyso-Gb3 and kidney interstitial capillary (KIC) globotriaosylceramide (Gb3) inclusions was assessed in treatment-naive patients. RESULTS: No significant correlations were identified between changes in lyso-Gb3 and changes in LVMi, eGFR, or pain. Neither baseline lyso-Gb3 levels nor the rate of change in lyso-Gb3 levels during treatment predicted FACE occurrences in all patients or those receiving migalastat for ≥24 months. Changes in lyso-Gb3 correlated with changes in KIC Gb3 inclusions in treatment-naive patients. CONCLUSIONS: Although used as a pharmacodynamic biomarker in research and clinical studies, plasma lyso-Gb3 may not be a suitable biomarker for monitoring treatment response in migalastat-treated patients.

Accuracy diagnosis improvement of Fabry disease from dried blood spots: Enzyme activity, lyso-Gb3 accumulation and GLA gene sequencing.

The purpose of this study was to examine the applicability of the use of samples in dried blood spot (DBS) for the definitive diagnosis of Fabry disease (FD) in males and females and to compare the diagnostic role of α-galactosidase A activity (α-Gal A), levels of lyso-Gb3 and sequencing of the GLA gene in screening patients with suspected FD. Measurement of α-Gal A activity in suspected FD patients in DBS was made followed by lyso-Gb3 determination and GLA gene sequencing. Of the 2381 subjects analyzed, FD was confirmed in 24 patients. Thirteen different variants were considered like pathogenic, five of which had not been previously described (c.143A > G; c.455A > C; c.487G > T; c.554delA; c.1045_1046insA). None of the patients with normal enzyme activity had FD confirmation. The DBS measurement of α-Gal A was more sensitive than lyso-Gb3 levels in both men and women. Definitive diagnosis of FD from a single DBS is possible, allowing samples to be easily sent from anywhere to the reference laboratory.

Urinary mulberry bodies as a potential biomarker for early diagnosis and efficacy assessment of enzyme replacement therapy in Fabry nephropathy.

BACKGROUND: The inability of enzyme replacement therapy (ERT) to prevent progression of Fabry nephropathy (FN) in the presence of >1 g/day proteinuria underscores the necessity of identifying effective biomarkers for early diagnosis of FN preceding proteinuria. Here we attempted to identify biomarkers for early detection of FN. METHODS: Fifty-one Fabry disease (FD) patients were enrolled. Urinary mulberry bodies (uMBs) were immunostained for globotriaosylceramide (Gb3) and renal cell markers to determine their origin. The association between semiquantitative uMB excretion and the histological severity of podocyte vacuolation was investigated in seven patients using the vacuolated podocyte:glomerular average area ratio. The association between the semiquantitative estimate of uMB excretion and duration of ERT was analyzed. A longitudinal study was conducted to assess the effect of ERT on uMB excretion. RESULTS: Thirty-two patients (63%) had uMBs, while only 31% showed proteinuria. The uMBs were positive for Gb3, lysosomal-associated membrane protein 1 and podocalyxin, suggesting they were derived from lysosomes with Gb3 accumulation in podocytes. We observed more severe podocyte vacuolation with increased uMB excretion (P = 0.03 for trend); however, the same was not observed with increased proteinuria. The percentage of patients with substantial uMB excretion increased with shorter ERT duration (P = 0.018). Eighteen-month-long ERT reduced uMB excretion (P = 0.03) without affecting proteinuria. CONCLUSIONS: uMB excretion, implying ongoing podocyte injury, preceded proteinuria in most patients. Semiquantitative uMB estimates can serve as novel biomarkers for early FN diagnosis and for monitoring the efficacy of FD-specific therapies.

Pathology and pathogenic pathways in fabry nephropathy.

BACKGROUND: The pathophysiology of renal damage in Fabry nephropathy involves a complex biological mechanism. The intracellular deposition globotriaosylceramide (Gb3) is just the first step of the mechanism. The glycolipid deposition occurs in all renal cells (endothelial, epithelial and mesangial cells). It stimulates many biological processes, including cytokine release, epithelial-mesenchymal transdifferentiation, oxidative stress and the remodelling of vascular walls, resulting in subtle initial inflammation and eventually tissue fibrosis. It has been hypothesized that the processes activated by Gb3 deposition can subsequently progress independently of cellular deposition and that even Gb3 clearance by specific therapy cannot retard or stop these pathways. AIM: This review aims to gather the reported evidence of these cellular alterations and the resulting histological changes. Our approach is similar to a routine study of kidney biopsy. RESULTS: In the first part of the review, "histology" section, we describe the structures involved (glomeruli, vessels, tubules and interstitium) from a histological point of view. While in the second part, "pathogenesis" section, we present some interpretations about the implicated pathways based on the up-to-date available evidence.

Two Paralogous Gb3/CD77 Synthases in Birds Show Different Preferences for Their Glycoprotein and Glycosphingolipid Substrates.

Most glycosyltransferases show remarkable gross and fine substrate specificity, which is reflected in the old one enzyme-one linkage paradigm. While human Gb3/CD77 synthase is a glycosyltransferase that synthesizes the Galα1→4Gal moiety mainly on glycosphingolipids, its pigeon homolog prefers glycoproteins as acceptors. In this study, we characterized two Gb3/CD77 synthase paralogs found in pigeons (Columba livia). We evaluated their specificities in transfected human teratocarcinoma 2102Ep cells by flow cytofluorometry, Western blotting, high-performance thin-layer chromatography, mass spectrometry and metabolic labelling with 14C-galactose. We found that the previously described pigeon Gb3/CD77 synthase (called P) can use predominately glycoproteins as acceptors, while its paralog (called M), which we serendipitously discovered while conducting this study, efficiently synthesizes Galα1→4Gal caps on both glycoproteins and glycosphingolipids. These two paralogs may underlie the difference in expression profiles of Galα1→4Gal-terminated glycoconjugates between neoavians and mammals.