LysoGb3 quantification facilitates phenotypic categorization of Fabry disease patients: Insights gained by a novel MS/MS method.

BACKGROUND: Fabry disease (FD) is an X-linked lysosomal storage disease resulting from pathogenic variants in the GLA gene coding α-galactosidase A (AGAL) and cleaving terminal alpha-linked galactose. Globotriaosylceramide (Gb3) is the predominantly accumulated sphingolipid. Gb3, deacylated-Gb3 (lysoGb3), and methylated-Gb3 (metGb3) have been suggested as FD biomarkers. MATERIALS AND METHODS: We developed a novel LC-MS/MS method for assessing lysoGb3 levels in plasma and Gb3 and metGb3 in urine and tested 62 FD patients, 34 patients with GLA variants of unknown significance (VUS) and 59 healthy controls. AGAL activity in white blood cells (WBCs) and plasma was evaluated in parallel. RESULTS: In males, lysoGb3 concentrations in plasma separated classic and late-onset FD patients from each other and from individuals carrying GLA VUS and healthy controls. Calculating AGAL activity/plasmatic lysoGb3 ratio allowed to correctly categorize all females with classic and majority of patients with late-onset FD phenotypes. Correlation of AGAL activity in WBCS with lipid biomarkers identified threshold activity values under which the biomarkers' concentrations increase. CONCLUSION: We developed a novel simplified LC-MS/MS method for quantitation of plasma lysoGb3. AGAL activity/plasma lysoGb3 ratio was identified as the best predictor for FD. AGAL activity correlated with plasma lysoGb3 and corresponded to individual FD phenotypes.

Evaluating the Metabolic Basis of α-Gal A mRNA Therapy for Fabry Disease.

mRNA injection-based protein supplementation has emerged as a feasible treatment for Fabry disease. However, whether the introduction of LNP-encapsulated mRNA results in the alteration of metabolomics in an in vivo system remains largely unknown. In the present study, α-galactosidase A (α-Gal A) mRNA was generated and injected into the Fabry disease mouse model. The α-Gal A protein was successfully expressed. The level of globotriaosylsphingosine (Lyso-Gb3), a biomarker for Fabry disease, as well as pro-inflammatory cytokines such as nuclear factor kappa-B (NF-κB), interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α), were greatly decreased compared to the untreated control, indicating the therapeutic outcome of the mRNA drug. Metabolomics analysis found that the level of 20 metabolites was significantly altered in the plasma of mRNA-injected mice. These compounds are primarily enriched in the arachidonic acid metabolism, alanine, aspartate and glutamate metabolism, and glycolysis/gluconeogenesis pathways. Arachidonic acid and 5-hydroxyeicosatetraenoic acid (5-HETE), both of which are important components in the eicosanoid pathway and related to inflammation response, were significantly increased in the injected mice, possibly due to the presence of lipid nanoparticles. Moreover, mRNA can effectively alter the level of metabolites in the amino acid and energy metabolic pathways that are commonly found to be suppressed in Fabry disease. Taken together, the present study demonstrated that in addition to supplementing the deficient α-Gal A protein, the mRNA-based therapeutic agent can also affect levels of metabolites that may help in the recovery of metabolic homeostasis in the full body system.

Clinical analysis of 10 patients with Fabry disease / 中华内科杂志

We aimed to analyze the clinical data of 10 patients (6 male and 4 female) with Fabry disease (FD). The mean age of the patients was (28.80±9.27) years. Seven patients had classical FD and three had delayed onset FD. Among the 10 patients, six had skin involvement and cutaneous angiokeratoma; five had hypohidrosis or anhidrosis; nine had intermittent neuralgia; and three had supraorbital ridge protrusion, forehead bulge, and lip thickening. Five patients had proteinuria, including one with chronic kidney disease stage 3 and one with chronic kidney disease stage 5. Cardiac involvement occurred in three patients, two had myocardial hypertrophy and one had valvular insufficiency. The activity of galactosidase decreased in seven patients (2.80-1.55 μmol·L -1·h -1). Plasma deacetyl-GL-3 was elevated in all 10 patients(3.12-120.00 ng/ml). Three patients underwent renal biopsy, wherein two cases of focal segmental glomerulosclerosis and one of mesangial proliferative glomerulonephritis was found. A large number of myeloid and zebra bodies were found in the podocytes in three patients, including a small number of myeloid and zebra bodies in the renal tubular epithelial cells in one patient with occasional zebra bodies in the renal interstitium. Nine patients had GLA gene mutations. One patient was c.102T>A, a de novo mutation. Four patients were treated with agalsidase α injection (0.2 mg/kg, intravenous infusion every 2 weeks), and their prognosis was good. FD has various clinical manifestations and multi-system involvement, which requires multidisciplinary cooperation. Detection of galactosidase activity, plasma globotriaosylsphingosine, and GLA gene mutation can help for accurate diagnosis.

Effects of switching from agalsidase-α to agalsidase-ß on biomarkers, renal and cardiac parameters, and disease severity in fabry disease forming neutralizing antidrug antibodies: a case report.

Fabry disease is an X-linked hereditary disorder caused by deficient α-galactosidase A (GLA) activity. Patients with Fabry disease are often treated with enzyme replacement therapy (ERT). However, ERT often induces the formation of neutralizing antidrug antibodies (ADAs), which may impair the therapeutic efficacy. Here, we report the case of a 32-year-old man with Fabry disease and resultant neutralizing ADAs who was treated by switching from agalsidase-α to agalsidase-ß. We monitored biomarkers, such as plasma globotriaosylsphingosine (lyso-Gb3), urinary globotriaosylceramide (Gb3), urinary mulberry bodies, renal and cardiac parameters, and disease severity during the treatment period. Although plasma lyso-Gb3 and urinary Gb3 levels quickly decreased within two months after the initiation of ERT with agalsidase-α, they gradually increased thereafter. The urinary mulberry bodies continued to appear. Both the ADA titer and serum mediated GLA inhibition rates started to increase after two months. Moreover, 3.5 years after ERT, the vacuolated podocyte area in the renal biopsy decreased slightly from 23.1 to 18.9%. However, plasma lyso-Gb3 levels increased, and urinary Gb3, mulberry body levels, and ADA titers remained high. Therefore, we switched to agalsidase-ß which reduced, but did not normalize, plasma lyso-Gb3 levels and stabilized renal and cardiac parameters. Disease severity was attenuated. However, urinary Gb3 and mulberry body levels did not decrease noticeably in the presence of high ADA titers. The kidneys take up a small amount of the administered recombinant enzyme, and the clearance of Gb3 that has accumulated in the kidney may be limited despite the switching from agalsidase-α to agalsidase-ß.

Profiles of Globotriaosylsphingosine Analogs and Globotriaosylceramide Isoforms Accumulated in Body Fluids from Various Phenotypic Fabry Patients.

Objectives Fabry disease is characterized by the systemic accumulation of globotriaosylceramide (Gb3) and globotriaosylsphingosine (Lyso-Gb3), which are widely used as biomarkers of the disease. However, few reports have described the relationship of Lyso-Gb3 analogs and Gb3 isoforms with the disease. The present study determined the profiles of Lyso-Gb3 analogs and Gb3 isoforms accumulated in body fluids from various phenotypic Fabry patients to elucidate the basis of the disease. Methods Plasma Lyso-Gb3 and related analogs were measured in 15 classic Fabry men, 6 later-onset Fabry men, 11 Fabry women, and 36 controls, while urinary Gb3 isoforms were measured in 5 classic Fabry men, 5 later-onset Fabry men, 17 Fabry women, and 11 controls, using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Furthermore, these values were monitored for a classic Fabry man, in whom neutralizing anti-drug antibodies had developed following enzyme replacement therapy (ERT). Results The levels of plasma Lyso-Gb3 analogs/urinary Gb3 isoforms were higher in Fabry patients than in controls, especially in classic Fabry men. However, minor differences in the ratio of each Lyso-Gb3 analog and Gb3 isoform with respect to the total Lyso-Gb3 analogs and Gb3 isoforms, respectively, were observed among individual classic Fabry men. Their time courses were well associated with the development and attenuation of anti-drug antibodies in a patient with classic Fabry disease during ERT. Conclusion Quantification of Lyso-Gb3 analogs and Gb3 isoforms provides us with more detailed information about the substrates that accumulated in the body fluids of Fabry patients than does quantification of Lyso-Gb3 and Gb3 alone, so this approach may be useful for elucidating the basis of Fabry disease.

Fabry disease biomarkers in patients switched from enzyme-replacement therapy to migalastat oral chaperone therapy.

Background: A biomarker profile was evaluated longitudinally in patients with Fabry disease switched from enzyme-replacement therapy (ERT) to migalastat. Methods: 16 Gb3 isoforms and eight lyso-Gb3 analogues were analyzed in plasma and urine by LC-MS/MS at baseline and at three different time points in naive participants and participants switching from either agalsidase α or ß to migalastat. Results: 29 adult participants were recruited internationally (seven centers). The Mainz Severity Score Index and mean biomarker levels remained stable (p ≥ 0.05) over a minimum of 12 months compared with baseline following the treatment switch. Conclusion: In this cohort of patients with Fabry disease with amenable mutations, in the short term, a switch from ERT to migalastat did not have a marked effect on the average biomarker profile.

Systematic gene therapy derived from an investigative study of AAV2/8 vector gene therapy for Fabry disease.

BACKGROUND: Fabry disease (FD) is a progressive multisystemic disease characterized by a lysosomal enzyme deficiency. A lack of α-galactosidase A (α-Gal A) activity results in the progressive systemic accumulation of its substrates, including globotriaosylceramide (Gb3) and globotriaosylsphingosine (Lyso-Gb3), which results in renal, cardiac, and/or cerebrovascular disease and early death. Enzyme replacement therapy (ERT) is the current standard of care for FD; however, it has important limitations, including a low half-life, limited distribution, and requirement of lifelong biweekly infusions of recombinant enzymes. METHODS: Herein, we evaluated a gene therapy approach using an episomal adeno-associated viral 2/8 (AAV2/8) vector that encodes the human GLA cDNA driven by a liver-specific expression cassette in a mouse model of FD that lacks α-Gal A activity and progressively accumulates Gb3 and Lyso-Gb3 in plasma and tissues. RESULTS: A pharmacology and toxicology study showed that administration of AAV2/8-hGLA vectors (AAV2/8-hGLA) in FD mice without immunosuppression resulted in significantly increased plasma and tissue α-Gal A activity and substantially normalized Gb3 and Lyso-Gb3 content. CONCLUSIONS: Moreover, the plasma enzymatic activity of α-Gal A continued to be stably expressed for up to 38 weeks and sometimes even longer, indicating that AAV2/8-hGLA is effective in treating FD mice, and that α-Gal A is continuously and highly expressed in the liver, secreted into plasma, and absorbed by various tissues. These findings provide a basis for the clinical development of AAV2/8-hGLA.

Consensus recommendations for the treatment and management of patients with Fabry disease on migalastat: a modified Delphi study.

Objective: Fabry disease is a progressive disorder caused by deficiency of the α-galactosidase A enzyme (α-Gal A), leading to multisystemic organ damage with heterogenous clinical presentation. The addition of the oral chaperone therapy migalastat to the available treatment options for Fabry disease is not yet universally reflected in all treatment guidelines. These consensus recommendations are intended to provide guidance for the treatment and monitoring of patients with Fabry disease receiving migalastat. Methods: A modified Delphi process was conducted to determine consensus on treatment decisions and monitoring of patients with Fabry disease receiving migalastat. The multidisciplinary panel comprised 14 expert physicians across nine specialties and two patients with Fabry disease. Two rounds of Delphi surveys were completed and recommendations on the use of biomarkers, multidisciplinary monitoring, and treatment decisions were generated based on statements that reached consensus. Results: The expert panel reached consensus agreement on 49 of 54 statements, including 16 that reached consensus in round 1. Statements that reached consensus agreement are summarized in recommendations for migalastat treatment and monitoring, including baseline and follow-up assessments and frequency. All patients with Fabry disease and an amenable mutation may initiate migalastat treatment if they have evidence of Fabry-related symptoms and/or organ involvement. Treatment decisions should include holistic assessment of the patient, considering clinical symptoms and organ involvement as well as patient-reported outcomes and patient preference. The reliability of α-Gal A and globotriaosylsphingosine as pharmacodynamic response biomarkers remains unclear. Conclusion: These recommendations build on previously published guidelines to highlight the importance of holistic, multidisciplinary monitoring for patients with Fabry disease receiving migalastat, in addition to shared decision-making regarding treatments and monitoring throughout the patient journey. GRAPHICAL ABSTRACT.

A new multiplex analysis of glucosylsphingosine and globotriaosylsphingosine in dried blood spots by tandem mass spectrometry.

Background: Gaucher's and Fabry's disease are two of the most common treatable lysosomal storage diseases, and have a wide spectrum of clinical symptoms. Early detection is important, because timely initiation of treatments can improve the disease status and prevent complications. However disease manifestations develop in childhood, diagnosis is delayed until adulthood partly due to the limitations of the currently used diagnostic pathway. The aim of this research is to develop and validate a multiplex assay and defining reference ranges, which do not exist at this moment, to improve and facilitate the entire diagnostic work up and enable treatment in an earlier stage of disease. Methods and findings: Biomarkers glucosylsphingosine (GlcSph) and globotriaosylsphingosine (Lyso-Gb3) were detected and quantified using LC-MS/MS on dried blood spots. We developed an improved and new extraction method that allowed to measure GlcSph and Lyso-Gb3 in a multiplex analytical platform. After validation of the method, samples of 1480 individuals with normal enzymatic activity were collected to determine age and gender-related reference ranges.Our combination method showed a good linearity, precision, accuracy and limit of quantification with lack of carry-over following the specific international CLSI guidelines. The suggested protocol is robust, efficient, sensitive, specific, comprehensive and relatively cheap in order to accelerate the diagnostic process for both lysosomal storage diseases. The samples, with normal enzymatic activity, defined statistical relevant and clinical correct reference ranges for each specific age group by gender. Conclusion: We report a multiplex LC-MS/MS method and relevant reference ranges that are appropriate for the targeted screening, diagnosis and follow-up of Fabry and Gaucher disease.

Brain MRI correlations with disease burden and biomarkers in Fabry disease.

OBJECTIVE: To quantitatively evaluate cerebral small vessel disease (CSVD) in brain magnetic resonance imaging (MRI) and its correlation with disease burden and markers in Fabry disease, a rare X-linked lysosomal storage disease. METHODS: We collected brain MRI data from seventy-one Chinese patients with Fabry disease. CSVD was evaluated using an age-related white matter change rating scale, Fazekas scale, enlarged perivascular spaces grading scale, lacunar infarction scale, Microbleed Anatomical Rating Scale, global cortical atrophy scale, and small-vessel disease score. Factors associated with MRI lesions, including sex, clinical subtype, disease severity, disease burden, genotype, and biomarkers, were also analyzed. RESULTS: Of 71 patients, 16 (22.5%) experienced ischemic stroke. The incidences of lacunar infarctions, white matter hyperintensities, and cerebral microbleeds were 55%, 62%, and 33%, respectively. The abnormal MRI group had later disease onset, longer disease duration, and a higher Mainz Severity Score Index (p < 0.05) than the normal MRI group. Patients with more severe clinical phenotypes also had higher CVSD-related scores. Sex and GLA mutational type were not closely associated with brain MRI lesions. Of the disease markers, the Mainz Severity Score Index and plasma globotriaosylsphingosine (Lyso-Gb3) were closely correlated with the majority of the MRI scores, whereas α-galactosidase A activity was not. CONCLUSION: Brain MRI revealed progressive lacunar infarctions, white matter hyperintensities, and decreased brain volume in patients with Fabry disease. Brain MRI lesions were closely related to onset-age; disease duration, severity, burden; and plasma Lyso-Gb3. However, they were not associated with sex, α-galactosidase A activity, or GLA mutation type.

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.

Distinctive accumulation of globotriaosylceramide and globotriaosylsphingosine in a mouse model of classic Fabry disease.

Fabry disease (FD) is an inherited disease caused by deficient α-galactosidase A activity that is characterized by the accumulation of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3). Although plasma lyso-Gb3 is a sensitive biomarker of FD, the correlation between its concentration and clinical symptoms remains unclear. To clarify the influence of plasma Gb3 and lyso-Gb3 in a symptomatic Gla tm Tg(CAG-A4GALT) FD mouse model, the total contents of Gb3, lyso-Gb3 and their analogs in various organs and plasma were determined in mice with early- (5-week-old) and late-stage (20-week-old) renal dysfunction. A marked increase in total Gb3 content in the heart, kidneys, spleen, liver, small intestine, lungs, brain, and plasma was observed in the 20-week-old mice compared to that in 5-week-old mice. In contrast, the increase in lyso-Gb3 was relatively small, and the total content in the lungs and plasma was unchanged. Lyso-Gb3 analogs {lyso-Gb3(-2) and lyso-Gb3(+18)} and Gb3 analogs {Gb3(-2) and Gb3(+18)} were observed in all organs and plasma at both ages, and the percentages of the analogs were unique to specific organs. The pattern of 37 Gb3 analogs/isoforms of liver Gb3 corresponded well with that of plasma Gb3. Although the analog pattern of plasma lyso-Gb3 did not resemble that of any organ lyso-Gb3, the relative content {lyso-Gb3: lyso-Gb3(-2)} in the sum of all organs corresponded well to that of the plasma at both ages. These data indicate that liver Gb3 may contribute to the plasma Gb3 level, while plasma lyso-Gb3 may be released from all organs, and the capacity of the plasma lyso-Gb3 pool may reach a maximum at an early stage of renal dysfunction.

Monitoring of anti-drug antibodies and disease-specific biomarkers in three patients from a Japanese Fabry family treated with enzyme replacement therapy.

We monitored anti-drug antibodies and disease-specific biomarkers in three patients with a nonsense mutation from a Japanese Fabry family treated with enzyme replacement therapy (ERT). In two male patients from the family, neutralizing anti-drug antibodies were induced at an early stage of ERT, the antibody titer peak being found at an earlier stage of ERT in the patient treated with 1.0 mg/kg agalsidase beta than in that treated with 0.2 mg/kg agalsidase alfa. Then, the antibody titers decreased with continuation of ERT. The formation of neutralizing anti-drug antibodies adversely affected the plasma globotriaosylsphingosine (Lyso-Gb3) level and urinary globotriaosylceramide (Gb3) excretion in both patients, the impact being greater in the patient treated with 0.2 mg/kg agalsidase alfa than in that treated with 1.0 mg/kg agalsidase beta. The difference might be explained by the different doses of the infused enzymes based on supersaturation of the antibodies. In a heterozygous Fabry female from the family, no sign of antibody formation was found, and both the plasma Lyso-Gb3 level and urinary Gb3 excretion, which were moderately increased at the baseline, decreased gradually. No deterioration of the manifestations or laboratory findings was observed during ERT in either of the patients. Thus, monitoring of anti-drug antibodies and biomarkers in these Fabry patients provided us with important information on their pathological condition during ERT.

Plasma Lysosphingolipid Biomarker Measurement by Liquid Chromatography Tandem Mass Spectrometry.

Plasma lysosphingolipids are highly elevated in patients with Gaucher, Krabbe, Fabry, and Niemann-Pick diseases and tend to accumulate to a greater extent than their respective primary sphingolipids in the plasma of affected patients. In this chapter, we describe two liquid chromatography tandem mass spectrometry (LC-MS/MS) methods to measure plasma concentrations of four lysosphingolipids species. The first method described measures glucosylsphingosine (lyso-GL1) and galactosylsphingosine (psychosine), biomarkers that accumulate in Gaucher and Krabbe diseases, respectively. The second method measures globotriaosylsphingosine (lyso-Gb3) and sphingosylphosphorylcholine (lyso-SPM), biomarkers for Fabry and Niemann-Pick diseases, respectively. Each method utilizes isotope-labeled internal standards and multipoint calibration curves to quantify the analytes of interest. Briefly, plasma samples are mixed with five volumes of LC-MS grade methanol containing internal standard, and protein is removed via centrifugation. Supernatant is dried and resuspended in initial mobile phase. Samples are separated by liquid chromatography using either a BEH amide column (lyso-GL1 + psychosine) or a C18 column (lyso-Gb3 + lyso-SPM). Protonated analytes are measured by selected reaction monitoring (SRM) in positive electrospray ionization mode. Using these methods, we have observed elevations of these lyso- species in Gaucher, Fabry, and Niemann-Pick and successfully distinguished different subtypes reflecting the disease severity.

Plasma neurofilament light, glial fibrillary acidic protein and lysosphingolipid biomarkers for pharmacodynamics and disease monitoring of GM2 and GM1 gangliosidoses patients.

GM2 and GM1 gangliosidoses are genetic, neurodegenerative lysosomal sphingolipid storage disorders. The earlier the age of onset, the more severe the clinical presentation and progression, with infantile, juvenile and late-onset presentations broadly delineated into separate phenotypic subtypes. Gene and substrate reduction therapies, both of which act directly on sphingolipidosis are entering clinical trials for treatment of these disorders. Simple to use biomarkers for disease monitoring are urgently required to support and expedite these clinical trials. Here, lysosphingolipid and protein biomarkers of sphingolipidosis and neuropathology respectively, were assessed in plasma samples from 33 GM2 gangliosidosis patients, 13 GM1 gangliosidosis patients, and compared to 66 controls. LysoGM2 and lysoGM1 were detectable in 31/33 GM2 gangliosidosis and 12/13 GM1 gangliosidosis patient samples respectively, but not in any controls. Levels of the axonal damage marker Neurofilament light (NF-L) were highly elevated in both GM2 and GM1 gangliosidosis patient plasma samples, with no overlap with controls. Levels of the astrocytosis biomarker Glial fibrillary acidic protein (GFAP) were also elevated in samples from both patient populations, albeit with some overlap with controls. In GM2 gangliosidosis patient plasma NF-L, Tau, GFAP and lysoGM2 were all most highly elevated in infantile onset patients, indicating a relationship to severity and phenotype. Plasma NF-L and liver lysoGM2 were also elevated in a GM2 gangliosidosis mouse model, and were lowered by treatment with a drug that slowed disease progression. These results indicate that lysosphingolipids and NF-L/GFAP have potential to monitor pharmacodynamics and pathogenic processes respectively in GM2 and GM1 gangliosidoses patients.

Clinical features and treatment effect of enzyme replacement therapy in patients with Fabry disease / 中华肾脏病杂志

Objective:To analyze the clinical features, efficacy and adverse reactions of enzyme replacement therapy (ERT) in patients with Fabry disease (FD).Methods:The clinical data of FD patients in Shandong Provincial Hospital Affiliated to Shandong First Medical University from June 2020 to September 2021 were collected and the clinical manifestations, laboratory examinations, gene mutations, and efficacy and adverse reactions of ERT were retrospectively analyzed.Results:Sixteen patients with FD were enrolled in this study, including 12 typical cases and 4 late-onset cases, with varied clinical manifestations. Compared with late-onset patients, typical patients had younger age of onset ( P=0.001), lower activity of plasma alpha-galactosidase A ( P=0.016) and higher globotriaosylsphingosine (lyso-GL-3, P=0.030). The typical patients [(13.50±10.08) years] and late-onset patients [(10.75±7.27) years] both had long delayed time of diagnosis. In 7 patients who underwent regular 6 ERT, lyso-GL-3 was significantly lower than before ( P=0.018); after 6 treatments, the pain of 5 patients was relieved than before. Three patients with irregular ERT had aggravated symptoms, and 1 case had stroke recurrence during regular treatment. No serious adverse reaction occurred with the use of agalsidase β and α. Conclusions:ERT can effectively reduce the level of plasma lyso-GL-3 in patients with FD and relieve symptoms, and has good safety. But the efficacy of ERT is dose-dependent, and clinical benefits require long-term observation and follow-up. Patients treated with ERT should have good compliance and can receive long-term regular treatment.

Endothelial Dysfunction in Fabry Disease Is Related to Glycocalyx Degradation.

Fabry disease (FD) is an X-linked multisystemic lysosomal storage disease due to a deficiency of α-galactosidase A (GLA/AGAL). Progressive cellular accumulation of the AGAL substrate globotriaosylceramide (Gb3) leads to endothelial dysfunction. Here, we analyzed endothelial function in vivo and in vitro in an AGAL-deficient genetic background to identify the processes underlying this small vessel disease. Arterial stiffness and endothelial function was prospectively measured in five males carrying GLA variants (control) and 22 FD patients under therapy. AGAL-deficient endothelial cells (EA.hy926) and monocytes (THP1) were used to analyze endothelial glycocalyx structure, function, and underlying inflammatory signals. Glycocalyx thickness and small vessel function improved significantly over time (p<0.05) in patients treated with enzyme replacement therapy (ERT, n=16) and chaperones (n=6). AGAL-deficient endothelial cells showed reduced glycocalyx and increased monocyte adhesion (p<0.05). In addition, increased expression of angiopoietin-2, heparanase and NF-κB was detected (all p<0.05). Incubation of wild-type endothelial cells with pathological globotriaosylsphingosine concentrations resulted in comparable findings. Treatment of AGAL-deficient cells with recombinant AGAL (p<0.01), heparin (p<0.01), anti-inflammatory (p<0.001) and antioxidant drugs (p<0.05), and a specific inhibitor (razuprotafib) of angiopoietin-1 receptor (Tie2) (p<0.05) improved glycocalyx structure and endothelial function in vitro. We conclude that chronic inflammation, including the release of heparanases, appears to be responsible for the degradation of the endothelial glycocalyx and may explain the endothelial dysfunction in FD. This process is partially reversible by FD-specific and anti-inflammatory treatment, such as targeted protective Tie2 treatment.

LC-MS lipidomics of renal biopsies for the diagnosis of Fabry disease.

INTRODUCTION: Lipidomics analysis or lipid profiling is a system-based analysis of all lipids in a sample to provide a comprehensive understanding of lipids within a biological system. In the last few years, lipidomics has made it possible to better understand the metabolic processes associated with several rare disorders and proved to be a powerful tool for their clinical investigation. Fabry disease is a rare X-linked lysosomal storage disorder (LSD) caused by a deficiency in α-galactosidase A (α-GAL A). This deficiency results in the progressive accumulation of glycosphingolipids, mostly globotriaosylceramide (Gb3), globotriaosylsphingosine (lyso-Gb3), as well as galabiosylceramide (Ga2) and their isoforms/analogs in the vascular endothelium, nerves, cardiomyocytes, renal glomerular podocytes, and biological fluids. OBJECTIVES: The primary objective of this study was to evaluate lipidomic signatures in renal biopsies to help understand variations in Fabry disease markers that could be used in future diagnostic tests. METHODS: Lipidomic analysis was performed by ultra-high pressure liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) on kidney biopsies that were left over after clinical pathology analysis to diagnose Fabry disease. RESULTS: We employed UHPLC-HRMS lipidomics analysis on the renal biopsy of a patient suspicious for Fabry disease. Our result confirmed α-GAL A enzyme activity declined in this patient since a Ga2-related lipid biomarker was substantially higher in the patient's renal tissue biopsy compared with two controls. This suggests this patient has a type of LSD that could be non-classical Fabry disease. CONCLUSION: This study shows that lipidomics analysis is a valuable tool for rare disorder diagnosis, which can be conducted on leftover tissue samples without disrupting normal patient care.

First two years of reimbursed enzyme replacement therapy in the treatment of Fabry disease in Poland.

Fabry disease (FD) is an ultra-rare genetic lysosomal storage disease caused by pathologic gene variants resulting in insufficient expression of α-galactosidase A. This enzyme deficiency leads to accumulation of globotriaosylceramide and globotriaosylsphingosine in plasma and in different cells throughout the body, causing major cardiovascular, renal, and nervous system complications. Until 2018, reimbursed enzyme replacement therapy (ERT) for FD was available in all European Union countries except Poland.             We present the preliminary results of the first two years of reimbursed ERT in Poland. We obtained data from the seven largest academic centers in Katowice, Kraków, Wroclaw, Poznan, Gdansk, Warszawa, and Lódz. The questionnaire included the following data: number of patients treated, number of patients qualified for ERT, and patient characteristics.             All centers returned completed questionnaires that included data for a total of 71 patients (28 men and 43 women) as of June 2021. Thirty-five patients with the diagnosis of FD confirmed by genetic testing (22 men and 13 women) had already qualified for reimbursed ERT. Mean (SD) age at the commencement of the ERT program was 39.6 (15.5) years (range 18-79 years). Mean time from the first clinical symptoms reported by the patients to the FD diagnosis was 21.1 (8.9) years, and the mean time from the final diagnosis of FD to the beginning of ERT was 4.7 (4.6) years.             FD is still underdiagnosed in Poland. To identify undiagnosed FD patients and to ensure that patients in Poland benefit fully from ERT, implementation of an effective nationwide screening strategy and close cooperation with a network of rare disease centers is advised.

Comparative urinary globotriaosylceramide analysis by thin-layer chromatography-immunostaining and liquid chromatography-tandem mass spectrometry in patients with Fabry disease.

In Fabry disease, accumulation of glycolipids, predominantly globotriaosylceramide (Gb3), affects the kidneys, and nephropathy is one of the important disorders that influence the disease severity and prognosis of patients. Urinary Gb3 has been analyzed for diagnosis and monitoring of Fabry disease. In this study, we analyzed urinary Gb3 by thin-layer chromatography (TLC)-immunostaining and liquid chromatography (LC)-tandem mass spectrometry (MS/MS). An improved qualitative method, TLC-immunostaining, revealed excessive urinary Gb3 excretion in 100 (8/8), 88 (14/16), and 74% (45/61) of the classic Fabry males, later-onset Fabry males, and Fabry females examined, respectively. This authentic method is robust, easy, economic, and hardly affected by abundant urinary sediment, and this is useful for diagnosing individual Fabry patients. LC-MS/MS can determine the level of Gb3 in urine with high sensitivity, and it revealed that the Gb3 excretion level was higher in the order of classic Fabry males, later-onset Fabry males, Fabry females, and controls, respectively, and this is expected to be a useful quantitative method not only for diagnosis but also for predicting the progression of Fabry nephropathy. As to the relation of the urinary Gb3 level and renal events, our study revealed that the urinary Gb3 level in Fabry patients experiencing renal events tended to be higher than that in ones who did not have any renal events in each phenotypic group of the disease.