Fabry disease (FD) is a lysosomal storage disorder caused by variants in the GLA gene encoding α-galactosidase A, an enzyme required for catabolism of globotriaosylceramide (Gb3). Accumulation of Gb3 in patients\' cells, tissues, and biological fluids causes clinical manifestations including ventricular hypertrophy, renal insufficiency, and strokes. This protocol describes a methodology to analyze urinary Gb3 and creatinine. Samples are diluted with an internal standard solution containing Gb3(C17:0) and creatinine-D3, centrifuged, and directly analyzed by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) using an 8.7-min method. Eight Gb3 isoforms [C16:0, C18:0, C20:0, C22:1, C22:0, C24:1, C24:0, and (C24:0)OH] are analyzed and the total is normalized to creatinine. Confirmation ions are monitored to detect potential interferences. The Gb3 limit of quantification is 0.023 µg/ml. Its interday coefficients of variation (3 concentrations measured) are ≤15.4%. This method minimizes matrix effects (≤6.5%) and prevents adsorption or precipitation of Gb3. Urine samples are stable (bias <15%) for 2 days at 21°C, 7 days at 4°C, and 4 freeze/thaw cycles, whereas prepared samples are stable for 5 days at 21°C, and 14 days at 4°C. The Gb3/creatinine age-related upper reference limits (mean + 2 standard deviations) are 29 mg/mol creatinine (<7 years) and 14 mg/mol creatinine (≥7 years). This simple, robust protocol has been fully validated (ISO 15189) and provides a valuable tool for diagnosis and monitoring of FD patients. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Analysis of urinary globotriaosylceramide (Gb3) and creatinine by UHPLC-MS/MS Support Protocol 1: Preparation of the urinary quality controls Support Protocol 2: Preparation of the urine matrix used for the Gb3 calibration curve Support Protocol 3: Preparation of the Gb3 calibrators Support Protocol 4: Preparation of the working solution containing the internal standards Support Protocol 5: Preparation of the creatinine calibrators Support Protocol 6: Preparation of the UHPLC solutions and mobile phases.

BACKGROUND: Fabry disease (FD) is characterized by deficient activity of α-galactosidase A (GLA). Consequently, globotriaosylceramide (Gb3) accumulates in various organs, causing cardiac, renal, and cerebrovascular damage. Gene therapies for FD have been investigated in humans. Strong conditioning is required for hematopoietic stem cell-targeted gene therapy (HSC-GT). However, strong conditioning leads to various side effects and should be avoided. In this study, we tested antibody-based conditioning for HSC-GT in wild-type and FD model mice.
METHODS: After preconditioning with an antibody-drug conjugate, HSC-GT using a lentiviral vector was performed in wild-type and Fabry model mice. In the wild-type experiment, the EGFP gene was introduced into HSCs and transplanted into preconditioned mice, and donor chimerism and EGFP expression were analyzed. In the FD mouse model, the GLA gene was introduced into HSCs and transplanted into preconditioned Fabry mice. GLA activity and Gb3 accumulation in the organs were analyzed.
RESULTS: In the wild-type mouse experiment, when anti-CD45 antibody-drug conjugate was used, the percentage of donor cells at 6 months was 64.5%, and 69.6% of engrafted donor peripheral blood expressed EGFP. When anti-CD117 antibody-drug conjugate and ATG were used, the percentage of donor cells at 6 months was 80.7%, and 73.4% of engrafted donor peripheral blood expressed EGFP. Although large variations in GLA activity among mice were observed in the FD mouse experiment for both preconditioning regimens, Gb3 was significantly reduced in many organs.
CONCLUSIONS: Antibody-based preconditioning may be an alternative preconditioning strategy for HSC-GT for treating FD.

Acral burning pain triggered by fever, thermal hyposensitivity and skin denervation are hallmarks of small fibre neuropathy in Fabry disease, a life-threatening X-linked lysosomal storage disorder. Variants in the gene encoding alpha-galactosidase A may lead to impaired enzyme activity with cellular accumulation of globotriaosylceramide. To study the underlying pathomechanism of Fabry-associated small fibre neuropathy, we generated a neuronal in vitro disease model using patient-derived induced pluripotent stem cells from three Fabry patients and one healthy control. We further generated an isogenic control line via gene editing. We subjected induced pluripotent stem cells to targeted peripheral neuronal differentiation and observed intra-lysosomal globotriaosylceramide accumulations in somas and neurites of Fabry sensory neurons using super-resolution microscopy. At functional level, patch-clamp analysis revealed a hyperpolarizing shift of voltage-gated sodium channel steady-state inactivation kinetics in isogenic control neurons compared with healthy control neurons (P < 0.001). Moreover, we demonstrate a drastic increase in Fabry sensory neuron calcium levels at 39°C mimicking clinical fever (P < 0.001). This pathophysiological phenotype was accompanied by thinning of neurite calibres in sensory neurons differentiated from induced pluripotent stem cells derived from Fabry patients compared with healthy control cells (P < 0.001). Linear-nonlinear cascade models fit to spiking responses revealed that Fabry cell lines exhibit altered single neuron encoding properties relative to control. We further observed mitochondrial aggregation at sphingolipid accumulations within Fabry sensory neurites utilizing a click chemistry approach together with mitochondrial dysmorphism compared with healthy control cells. We pioneer pilot insights into the cellular mechanisms contributing to pain, thermal hyposensitivity and denervation in Fabry small fibre neuropathy and pave the way for further mechanistic in vitro studies in Fabry disease and the development of novel treatment approaches.

Fabry disease (FD) is an X-linked disease characterized by an accumulation of glycosphingolipids, notably of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lysoGb3) leading to renal failure, cardiomyopathy, and cerebral strokes. Inflammatory processes are involved in the pathophysiology. We investigated the immunological phenotype of peripheral blood mononuclear cells in Fabry patients depending on the clinical phenotype, treatment, Gb3, and lysoGb3 levels and the presence of anti-drug antibodies (ADA). Leucocytes from 41 male patients and 20 controls were analyzed with mass cytometry using both unsupervised and supervised algorithms. FD patients had an increased expression of CD27 and CD28 in memory CD45- and CD45 + CCR7-CD4 T cells (respectively p < 0.014 and p < 0.02). Percentage of CD45RA-CCR7-CD27 + CD28+ cells in CD4 T cells was correlated with plasma lysoGb3 (r = 0.60; p = 0.0036) and phenotype (p < 0.003). The correlation between Gb3 and CD27 in CD4 T cells almost reached significance (r = 0.33; p = 0.058). There was no immune profile associated with the presence of ADA. Treatment with agalsidase beta was associated with an increased proportion of Natural Killer cells. These findings provide valuable insights for understanding FD, linking Gb3 accumulation to inflammation, and proposing new prognostic biomarkers.

BACKGROUND: Fabry disease is a multisystemic disorder characterized by deposition of globotriaosylceramide (Gb3) and its deacylated form in multiple organs, sometimes localized in specific systems such as the nervous or cardiovascular system. As disease-modifying therapies are now available, early diagnosis is paramount to improving life quality and clinical outcomes. Despite the widespread use of non-invasive techniques for assessing organ damage, such as cardiac magnetic resonance imaging (MRI) for patients with cardiac disease, organ biopsy remains the gold standard to assess organ involvement.
METHODS: The cases of two patients, father and daughter with a W162C mutation, are described. The father presented with late-onset, cardiac Fabry disease, subsequently developing systolic dysfunction and heart failure. His daughter, while asymptomatic and with normal cardiac assessment (except for slightly reduced native T1 values by cardiac MRI), had already initial myocyte Gb3 deposits on the endomyocardial biopsy, allowing her to start therapy precociously and potentially modifying the course of her disease. A review of the literature concerning the W162C mutation is then provided, showing that it is usually associated to classic, multisystemic Fabry disease rather than the cardiac-restricted form as in these two cases.
CONCLUSIONS: Three main points can be concluded from this report. First, the W162C mutation can present with a more variegate phenotype than that predicted on a molecular basis. Second, endomyocardial biopsy was shown in this case to precede non-invasive investigation in determining organ involvement, justifying further studies on this potentially reliable technique, Third, difficulties can arise in the management of asymptomatic female carriers.

Fabry disease (FD) is a life-limiting disorder characterized by intracellular globotriaosylceramide (Gb3) accumulations. The underlying α-galactosidase A (α-GAL A) deficiency is caused by variants in the gene GLA. Variants of unknown significance (VUS) are frequently found in GLA and challenge clinical management. Here, we investigated a 49-year old man with cryptogenic lacunar cerebral stroke and the chance finding of the VUS S126G, who was sent to our center for diagnosis and initiation of a costly and life-long FD-specific treatment. We combined clinical examination with in vitro investigations of dermal fibroblasts (HDF), induced pluripotent stem cells (iPSC), and iPSC-derived sensory neurons. We analyzed α-GAL A activity in iPSC, Gb3 accumulation in all three cell types, and action potential firing in sensory neurons. Neurological examination and small nerve fiber assessment was normal except for reduced distal skin innervation. S126G iPSC showed normal α-GAL A activity compared to controls and no Gb3 deposits were found in all three cell types. Baseline electrophysiological characteristics of S126G neurons showed no difference compared to healthy controls as investigated by patch-clamp recordings. We pioneer multi-level cellular characterization of the VUS S126G using three cell types derived from a patient and provide further evidence for the benign nature of S126G in GLA, which is of great importance in the management of such cases in clinical practice.

Fabry disease or also called Anderson-Fabry disease (FD) is a rare disease caused by pathogenic variants in the GLA gene, located on the X chromosome. This gene is involved in the metabolism of glycosphingolipids and its pathogenic variants cause a deficit or absence of α-galactosidase A causing the deposition of globotriaosylceramide throughout the body. Females have a variable phenotypic expression and a better prognosis than males. This is due to the X chromosome inactivation phenomenon. We present a clinical case of Fabry disease in a female with predominantly renal involvement and demonstrate how the X chromosome inactivation phenomenon is tissue dependent, showing preferential inactivation of the mutated allele at the renal level.

UNASSIGNED: While cardiovascular complications are the most common cause of mortality in Fabry disease, the relationship between globotriaosylceramide (GL-3) accumulation, the hallmark of Fabry cardiomyopathy, and cardiac hypertrophy has not been fully elucidated.
UNASSIGNED: We developed unbiased stereology protocols to quantify the ultrastrcture of Fabry cardiomyopathy. Endomyocardial biopsies from 10 adult male enzyme replacement therapy naïve Fabry patients with IVS4 + 919G>A GLA mutation were studied. The findings were correlated with cardiac MRI and clinical data.
UNASSIGNED: Ultrastructural parameters showed significant relationships with key imaging and clinical and functional variables. Average cardiomyocyte volume and GL-3 volume per cardiomyocyte were progressively increased with age. Eighty-four percent of left ventricular mass index (LVMI) variability was explained by cardiomyocyte nuclear volume, age and plasma globotriaosylsphingosine with cardiomyocyte nuclear volume being the only independent predictor of LVMI. Septal thickness was directly and left ventricular ejection fraction (LVEF) was inversely correlated with cardiomyocyte GL-3 accumulation. Sixty-five percent of left ventricular ejection fraction (LVEF) variability was explained by cardiomyocyte GL3 volume, serum α-galactosidase-A activity and age with cardiomyocyte GL3 volume being the only independent predictor of LVEF. Residual α-galactosidase-A activity was directly correlated with myocardial microvasculature density.
UNASSIGNED: The unbiased stereological methods introduced in this study unraveled novel relationships between cardiomyocyte structure and important imaging and clinical parameters. These novel tools can help better understand Fabry cardiomyopathy pathophysiology.

UNASSIGNED: The diagnosis of Fabry disease (FD) has relevant implications related to the management. Thus, a clear assignment of GLA variant pathogenicity is crucial. This systematic review and meta-analysis aimed to investigate the prevalence of FD in high-risk populations and newborns and evaluate the impact of different GLA variant classifications on the estimated prevalence of FD.
UNASSIGNED: We searched the EMBASE and PubMed databases on February 21, 2023. Observational studies evaluating the prevalence of FD and reporting the identified GLA variants were included. GLA variants were re-evaluated for their pathogenicity significance using the American College of Medical Genetics and Genomics criteria and the ClinVar database. The pooled prevalence of FD among different settings was calculated. The study was registered on PROSPERO (CRD42023401663) and followed the Preferred Reporting Items for Systematic Reviews and Meta-Analysis guidelines.
UNASSIGNED: Of the 3941 studies identified, 110 met the inclusion criteria. The pooled prevalence of FD was significantly different according to the clinical setting and criteria used for the pathogenicity assessment. Using the American College of Medical Genetics and Genomics criteria, the pooled prevalence was 1.2% in patients with left ventricular hypertrophy/hypertrophic cardiomyopathy (26 studies; 10 080 patients screened), 0.3% in end-stage renal disease/chronic kidney disease (38 studies; 62 050 patients screened), 0.7% in stroke (25 studies; 15 295 patients screened), 0.7% in cardiac conduction disturbance requiring pacemaker (3 studies; 1033 patients screened), 1.0% in small-fiber neuropathy (3 studies; 904 patients screened), and 0.01% in newborns (15 studies; 11 108 793 newborns screened). The pooled prevalence was different if the GLA variants were assessed using the ClinVar database, and most patients with a discrepancy in the pathogenicity assignment carried 1 of the following variants: p.A143T, p.D313Y, and p.E66Q.
UNASSIGNED: This systematic review and meta-analysis describe the prevalence of FD among newborns and high-risk populations, highlighting the need for a periodic reassessment of the GLA variants in the context of recent clinical, biochemical, and histological data.
UNASSIGNED: URL: https://crd.york.ac.uk/PROSPERO/; Unique identifier: CRD42023401663.

OBJECTIVE: Fabry disease, an X-linked lysosomal storage disorder, causes progressive globotriaosylceramide accumulation in cells throughout the body. Characteristic multiorgan manifestations include renal dysfunction (Fabry nephropathy) and associated urinary tract complications. Enzyme replacement therapy (ERT) has been available since 2001, but contemporary real-world data are lacking regarding Fabry nephropathy risks and treatment outcomes.
METHODS: This retrospective cohort study analyzed electronic medical records data for 10,637 Fabry disease patients from the TriNetX research database. Kidney and urinary tract outcomes were evaluated over two decades, 2000-2010 and 2011-2020. Outcomes assessed included chronic kidney disease (CKD), urinary tract infections, urinary incontinence, obstruction, renal insufficiency, and end-stage renal disease (ESRD).
RESULTS: The prevalence of stage 4-5 CKD nearly doubled between 2000-2010 and 2011-2020, while ESRD prevalence rose over 4-fold. Incidence rates showed similar marked elevations across renal and urologic complications. Females and Black patients experienced disproportionate escalations in kidney and urinary tract morbidity.
CONCLUSIONS: This large cohort study revealed significantly increased Fabry nephropathy and associated urologic complications over the past two decades, contradicting expectations of reduced morbidity with ERT availability. The findings highlight needs to optimize screening, treatment strategies, monitoring practices, and address disparities to curb rising disease burden and improve patient outcomes.