Establishing Treatment Effectiveness in Fabry Disease: Observation-Based Recommendations for Improvement.

Fabry disease (FD, OMIM #301500) is caused by pathogenic GLA gene (OMIM #300644) variants, resulting in a deficiency of the α-galactosidase A enzyme with accumulation of its substrate globotriaosylceramide and its derivatives. The phenotype of FD is highly variable, with distinctive disease features and course in classical male patients but more diverse and often nonspecific features in non-classical and female patients. FD-specific therapies have been available for approximately two decades, yet establishing robust evidence for long-term effectiveness remains challenging. This review aims to identify the factors contributing to this lack of robust evidence for the treatment of FD with enzyme replacement therapy (ERT) (agalsidase-alfa and -beta and pegunigalsidase alfa) and chaperone therapy (migalastat). Major factors that have been identified are study population heterogeneity (concerning sex, age, phenotype, disease stage) and differences in study design (control groups, outcomes assessed), as well as the short duration of studies. To address these challenges, we advocate for patient matching to improve control group compatibility in future FD therapy studies. We recommend international collaboration and harmonization, facilitated by an independent FD registry. We propose a stepwise approach for evaluating the effectiveness of novel treatments, including recommendations for surrogate outcomes and required study duration.

Application of an α-galactosidase from Bacteroides fragilis on structural analysis of raffinose family oligosaccharides.

Raffinose family oligosaccharides (RFOs) have diverse structures and exhibit various biological activities. When using RFOs as prebiotics, their structures need to be identified. If we first knew whether an RFO was classical or non-classical, structural identification would become much easier. Here, we cloned and expressed an α-galactosidase (BF0224) from Bacteroides fragilis which showed strict specificity for hydrolyzing α-Gal-(1 â†’ 6)-Gal linkages in RFOs. BF0224 efficiently distinguished classical from non-classical RFOs by identifying the resulting hydrolyzed oligo- and mono-saccharides with HPAEC-PAD-MS. Using this strategy, we identified a non-classical RFO from Pseudostellaria heterophylla (Miquel) Pax with DP6 (termed PHO-6), as well as a classical RFO from Lycopus lucidus Turcz. with DP7 (termed LTO-7). To characterize these RFO structures, we employed four other commercial or reported α-galactosidases in combination with NMR and methylation analysis. Using this approach, we elucidated the accurate chemical structure of PHO-6 and LTO-7. Our study provides an efficient analytical approach to structurally analyze RFOs. This enzyme-based strategy also can be applied to structural analysis of other glycans.

Inflammatory cytokine expression in Fabry disease: impact of disease phenotype and alterations under enzyme replacement therapy.

Objectives: The aim of this study is to explore the expression of inflammatory cytokines (ICs) in Fabry disease (FD), the correlation between ICs and FD phenotypes, and the impact of enzyme replacement therapy (ERT) on IC expression. Methods: We recruited 67 FD patients and 44 healthy controls (HCs) and detected concentrations of the following ICs: interferon-γ, interleukin (IL)-1ß, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12P70, IL-17A, IL-17F, IL-22, tumor necrosis factor (TNF)-α, and TNF-ß. We also analyzed the impact of ERT on IC expression in FD patients and the relationship between IC expression and sex, genotype, phenotype, disease burden, and biomarkers. Results: Most ICs were significantly higher in FD patients than in HCs. A number of ICs were positively correlated with clinical aspects, including disease burden (Mainz Severity Score Index [MSSI]) and cardiac and renal markers. IL-8 was higher in the high MSSI (P-adj=0.026*) than in the low MSSI. Conclusions: ICs were upregulated in FD patients, indicating the role of the innate immune process in FD etiology. ERT ameliorated FD-related inflammatory activation, at least to some extent. IC expression was positively correlated with disease burden and clinical markers in FD. Our findings indicated that the inflammatory pathway may be a promising therapeutic target for FD.

CRISPR/Cas9-mediated suppression of A4GALT rescues endothelial cell dysfunction in a fabry disease vasculopathy model derived from human induced pluripotent stem cells.

BACKGROUND AND AIMS: The objective of this study was to investigate the efficacy of CRISPR/Cas9-mediated A4GALT suppression in rescuing endothelial dysfunction in Fabry disease (FD) endothelial cells (FD-ECs) derived from human induced pluripotent stem cells (hiPSCs). METHODS: We differentiated hiPSCs (WT (wild-type), WTC-11), GLA-mutant hiPSCs (GLA-KO, CMC-Fb-002), and CRISPR/Cas9-mediated A4GALT-KO hiPSCs (GLA/A4GALT-KO, Fb-002-A4GALT-KO) into ECs and compared FD phenotypes and endothelial dysfunction. We also analyzed the effect of A4GALT suppression on reactive oxygen species (ROS) formation and transcriptome profiles through RNA sequencing. RESULTS: GLA-mutant hiPSC-ECs (GLA-KO and CMC-Fb-002) showed downregulated expression of EC markers and significantly reduced α-GalA expression with increased Gb-3 deposition and intra-lysosomal inclusion bodies. However, CRISPR/Cas9-mediated A4GALT suppression in GLA/A4GALT-KO and Fb-002-A4GALT-KO hiPSC-ECs increased expression levels of EC markers and rescued these FD phenotypes. GLA-mutant hiPSC-ECs failed to form tube-like structure in tube formation assays, showing significantly decreased migration of cells into the scratched wound area. In contrast, A4GALT suppression improved tube formation and cell migration capacity. Western blot analysis revealed that MAPK and AKT phosphorylation levels were downregulated while SOD and catalase were upregulated in GLA-KO hiPSC-ECs. However, suppression of A4GALT restored these protein alterations. RNA sequencing analysis demonstrated significant transcriptome changes in GLA-mutant EC, especially in angiogenesis, cell death, and cellular response to oxidative stress. However, these were effectively restored in GLA/A4GALT-KO hiPSC-ECs. CONCLUSIONS: CRISPR/Cas9-mediated A4GALT suppression rescued FD phenotype and endothelial dysfunction in GLA-mutant hiPSC-ECs, presenting a potential therapeutic approach for FD-vasculopathy.

Relationship between Capillaroscopic Architectural Patterns and Different Variant Subgroups in Fabry Disease: Analysis of Cases from a Multidisciplinary Center.

Anderson-Fabry disease (AFD) is a genetic lysosomal storage disorder caused by mutations in the α-galactosidase A gene, leading to impaired lysosomal function and resulting in both macrovascular and microvascular alterations. AFD patients often exhibit increased intima-media thickness (IMT) and reduced flow-mediated dilation (FMD), indicating non-atherosclerotic arterial thickening and the potential for cardiovascular events. Nailfold capillaroscopy, a non-invasive diagnostic tool, has shown potential in diagnosing and monitoring microcirculatory disorders in AFD, despite limited research. This study evaluates nailfold capillaroscopy findings in AFD patients, exploring correlations with GLA gene variant subgroups (associated with classical or late-onset phenotypes and variants of uncertain significance (VUSs)), and assessing morpho-functional differences between sexes. It aims to determine whether capillaroscopy can assist in the early identification of individuals with multiorgan vascular involvement. A retrospective observational study was conducted with 25 AFD patients from AOUP "G. Rodolico-San Marco" in Catania (2020-2023). Patients underwent genetic testing, enzyme activity evaluation, and nailfold capillaroscopy using Horus basic HS 200 videodermatoscopy. Parameters like angiotectonic disorder, vascular areas, capillary density, and intimal thickening were assessed. The study identified significant differences in capillaroscopy findings among patients with different GLA gene variant subgroups. Classic AFD variant patients showed reduced capillary length and signs of erythrocyte aggregation and dilated subpapillary plexus. No correlation was found between enzymatic activity and capillaroscopy parameters. However, Lyso-Gb3 levels were positively correlated with average capillary length (ῤ = 0.453; p = 0.059). Sex-specific differences in capillaroscopy findings were observed in neoangiogenesis and average capillary length, with distinct implications for men and women. This study highlights the potential of nailfold capillaroscopy in the diagnostic process and clinical management of AFD, particularly in relation to specific GLA gene mutations, as a valuable tool for the early diagnosis and monitoring of AFD.

Evaluation of the Impact of an Enzymatic Preparation Catalyzing the Decomposition of Raffinose from Poor-Quality Beets during the White Sugar Production Process.

The study investigates the efficacy of an enzymatic preparation primarily with α-galactosidase activity for improving the quality of white sugar from poor-quality sugar beets. Focused on overcoming raffinose accumulation challenges in sugar beets, especially those harvested prematurely or stored for extended periods, an innovative exploration of enzymatic application in an industrial setting for the first time was conducted. By integrating theoretical calculations and experimental data, the findings reveal that α-galactosidase preparation notably diminishes raffinose content in beet juice, thus enhancing the sucrose yield and overall sugar quality. A reliable method to process lower-quality beets, promising enhanced efficiency in sugar production, was presented. The study also highlights the economic benefits of incorporating enzyme preparation into the production process, demonstrating a notable return on investment and underscoring the potential of enzymatic treatments to address industry challenges.

Inflammation, Oxidative Stress, and Endothelial Dysfunction in the Pathogenesis of Vascular Damage: Unraveling Novel Cardiovascular Risk Factors in Fabry Disease.

Anderson-Fabry disease (AFD), a genetic disorder caused by mutations in the α-galactosidase-A (GLA) gene, disrupts lysosomal function, leading to vascular complications. The accumulation of globotriaosylceramide (Gb3) in arterial walls triggers upregulation of adhesion molecules, decreases endothelial nitric oxide synthesis, and induces reactive oxygen species production. This cascade results in fibrotic thickening, endothelial dysfunction, hypercontractility, vasospasm, and a pro-thrombotic phenotype. AFD patients display increased intima-media thickness (IMT) and reduced flow-mediated dilation (FMD), indicating heightened cardiovascular risk. Nailfold capillaroscopy (NFC) shows promise in diagnosing and monitoring microcirculatory disorders in AFD, though it remains underexplored. Morphological evidence of AFD as a storage disorder can be demonstrated through electron microscopy and immunodetection of Gb3. Secondary pathophysiological disturbances at cellular, tissue, and organ levels contribute to the clinical manifestations, with prominent lysosomal inclusions observed in vascular, cardiac, renal, and neuronal cells. Chronic accumulation of Gb3 represents a state of ongoing toxicity, leading to increased cell turnover, particularly in vascular endothelial cells. AFD-related vascular pathology includes increased renin-angiotensin system activation, endothelial dysfunction, and smooth muscle cell proliferation, resulting in IMT increase. Furthermore, microvascular alterations, such as atypical capillaries observed through NFC, suggest early microvascular involvement. This review aims to unravel the complex interplay between inflammation, oxidative stress, and endothelial dysfunction in AFD, highlighting the potential connections between metabolic disturbances, oxidative stress, inflammation, and fibrosis in vascular and cardiac complications. By exploring novel cardiovascular risk factors and potential diagnostic tools, we can advance our understanding of these mechanisms, which extend beyond sphingolipid accumulation to include other significant contributors to disease pathogenesis. This comprehensive approach can pave the way for innovative therapeutic strategies and improved patient outcomes.

Fabry Disease Rat Model Develops Age- and Sex-Dependent Anterior Segment Ocular Abnormalities.

Purpose: Fabry disease is an X-linked lysosomal storage disorder that results in multi-systemic renal, cardiovascular, and neuropathological damage, including in the eyes. We evaluated anterior segment ocular abnormalities based on age, sex (male and female), and genotype (wild-type, knockout [KO] male, heterozygous [HET] female, and KO female) in a rat model of Fabry disease. Methods: The α-Gal A KO and WT rats were divided into young (6-24 weeks), adult (25-60 weeks), and aged (61+ weeks) groups. Intraocular pressure (IOP) was measured. Eyes were clinically scored for corneal and lens opacity as well as evaluated for corneal epithelial integrity and tear break-up time (TBUT). Anterior chamber depth (ACD) and central corneal thickness (CCT) using anterior segment-optical coherence tomography (AS-OCT). Results: The Fabry rats showed an age-dependent increase in IOP, predominantly in the male genotype. TBUT was decreased in both male and female groups with aging. Epithelial integrity was defective in KO males and HET females with age. However, it was highly compromised in KO females irrespective of age. Corneal and lens opacities were severely affected irrespective of sex or genotype in the aging Fabry rats. AS-OCT quantification of CCT and ACD also demonstrated age-dependent increases but were more pronounced in Fabry versus WT genotypes. Conclusions: Epithelial integrity, corneal, and lens opacities worsened in Fabry rats, whereas IOP and TBUT changes were age-dependent. Similarly, CCT and ACD were age-related but more pronounced in Fabry rats, providing newer insights into the anterior segment ocular abnormalities with age, sex, and genotype in a rat model of Fabry disease.

Fabry disease Enzyme Enhancement on migalastat Study: FEES.

BACKGROUND: There is limited information on the α-galactosidase A (α-Gal-A) in vivo response in Fabry patients receiving migalastat. In this single centre study, we evaluated changes from baseline in α-Gal A activity, lyso-Gb3 and other assessments in patients on migalastat. RESULTS: 79 patients were recruited (48 M:31F; median duration receiving migalastat 3.8 years [range = 0.4-14.9 years]). N215S was the commonest genotype in males (67 %) and females (29 %). Leukocyte α-Gal-A showed a positive change from baseline in males (n = 4; median = 20.05); females (n = 8; median = 26). Of these, 3 males and 1 female had N215S (median = 16.7), while 7 females and 1 male had other genotypes (median = 26). No significant changes observed in plasma α-Gal-A. Cross-sectional analysis of post-baseline data confirmed leukocyte α-Gal-A enhancement in males (n = 47; median = 20); females (n = 30; median = 72); N215S (n = 41; median = 29) and other genotypes (n = 36; median = 36.5). Plasma and dried blood spot (DBS) lyso-Gb3 correlated at baseline and post-baseline (r = 0.77 and r = 0.96; p=<0.0001). CONCLUSIONS: In the 12 patients with paired data, there was a median enzyme enhancement of 17.4 (relative change = 2.54) and 33 (relative change = 0.87) in males and in females, respectively. The cross-sectional post-baseline data in 47 patients corroborated leukocyte α-Gal-A enhancement on migalastat. Plasma and DBS lyso-Gb3 correlated well supporting DBS utility for disease monitoring.

The Missense Variant in the Signal Peptide of α-GLA Gene, c.13 A/G, Promotes Endoplasmic Reticular Stress and the Related Pathway's Activation.

Anderson-Fabry disease (AFD) is an X-linked multisystemic disorder with a heterogeneous phenotype, resulting from deficiency of the lysosomal enzyme α-galactosidase A (α-Gal A) and leading to globotriaosylceramide systemic accumulation. Lysosomal storage is not the unique player in organ failure and different mechanisms could drive tissue damage, including endoplasmic reticulum (ER) stress and its related signaling pathway's activation. We identified a new missense variant in the signal peptide of α-GLA gene, c.13 A/G, in a 55-year-old woman affected by chronic kidney disease, acroparesthesia, hypohidrosis, and deafness and exhibiting normal values of lysoGb3 and αGLA activity. The functional study of the new variant performed by its overexpression in HEK293T cells showed an increased protein expression of a key ER stress marker, GRP78, the pro-apoptotic BAX, the negative regulator of cell cycle p21, the pro-inflammatory cytokine, IL1ß, together with pNFkB, and the pro-fibrotic marker, N-cadherin. Transmission electron microscopy showed signs of ER injury and intra-lysosomal inclusions. The proband's PBMC exhibited higher expression of TGFß 1 and pNFkB compared to control. Our findings suggest that the new variant, although it did not affect enzymatic activity, could cause cellular damage by affecting ER homeostasis and promoting apoptosis, inflammation, and fibrosis. Further studies are needed to demonstrate the variant's contribution to cellular and tissue damage.

Developing Gene Therapy for Mitigating Multisystemic Pathology in Fabry Disease: Proof of Concept in an Aggravated Mouse Model.

Fabry disease (FD) is a multisystemic lysosomal storage disorder caused by the loss of α-galactosidase A (α-Gal) function. The current standard of care, enzyme replacement therapies, while effective in reducing kidney pathology when treated early, do not fully ameliorate cardiac issues, neuropathic manifestations, and risk of cerebrovascular events. Adeno-associated virus (AAV)-based gene therapies (AAV-GT) can provide superior efficacy across multiple tissues owing to continuous, endogenous production of the therapeutic enzyme and lower treatment burden. We set out to develop a robust AAV-GT to achieve optimal efficacy with the lowest feasible dose to minimize any safety risks that are associated with high-dose AAV-GTs. In this proof-of-concept study, we evaluated the effectiveness of an rAAV9 vector expressing human GLA transgene under a strong ubiquitous promoter, combined with woodchuck hepatitis virus posttranscriptional regulatory element (rAAV9-hGLA). We tested our GT at three different doses, 5e10 vg/kg, 2.5e11 vg/kg, and 6.25e12 vg/kg in the G3Stg/GLAko Fabry mouse model that has tissue Gb3 substrate levels comparable with patients with FD and develops several early FD pathologies. After intravenous injections of rAAV9-hGLA at 11 weeks of age, we observed dose-dependent increases in α-Gal activity in the key target tissues, reaching as high as 393-fold of WT in the kidneys and 6156-fold in the heart at the highest dose. Complete or near-complete substrate clearance was observed in animals treated with the two higher dose levels tested in all tissues except for the brain. We also found dose-dependent improvements in several pathological biomarkers, as well as prevention of structural and functional organ pathology. Taken together, these results indicate that an AAV-GT under a strong ubiquitous promoter has the potential to address the unmet therapeutic needs in patients with FD at relatively low doses.

8th Update on Fabry Disease: Biomarkers, Progression and Treatment Opportunities in 2024.

Fabry Update.

No evidence of Fabry disease in a patient with the new p.Met70Val GLA gene variant.

BACKGROUND: Fabry disease (FD) is a rare X-linked lysosomal storage disorder caused by variants in GLA gene leading to deficient α-galactosidase A enzyme activity. This deficiency leads to the accumulation of glycosphingolipids, particularly globotriaosylceramide (Gb3), in various tissues and organs, which can result in life-threatening complications. The clinical presentation of the disease can vary from the "classic" phenotype with pediatric onset and multi-organ involvement to the "later-onset" phenotype, which presents with predominantly cardiac symptoms. In recent years, advances in screening studies have led to the identification of an increasing number of variants of unknown significance that have not yet been described, and whose pathogenic role remains undetermined. METHODS: In this clinical report, we describe the case of an asymptomatic adult female who was found to have a new variant of unknown significance, p.Met70Val. Given the unknown pathogenic role of this variant, a thorough analysis of the potential organ involvement was conducted. The clinical data were analyzed retrospectively. RESULTS: The analysis revealed that there were no signs of significant organ involvement, and the benignity of the variant was confirmed. CONCLUSION: This case underscores the importance of a comprehensive evaluation of new variants of unknown significance to establish their pathogenicity accurately.

CRISPR/Cas9-based GLA knockout to generate the female Fabry disease human induced pluripotent stem cell line MHHi001-A-15.

The X-linked lysosomal storage disorder Fabry disease originates from GLA gene mutations causing α-galactosidase A enzyme deficiency. Here we generated the GLA knockout hiPSC line MHHi001-A-15 (GLA-KOhiPSC) as an in vitro Fabry disease model by targeting exon 2 of the GLA gene by CRISPR/Cas9 in the established control hiPSC line MHHi001-A. GLA-KOhiPSCs retained the expression of pluripotency markers, trilineage differentiation potential, as well as normal karyotype and stem cell morphology but lacked α-galactosidase A enzyme activity. The GLA-KOhiPSCs represent a potent resource to not only study the Fabry disease manifestation but also screen for novel treatment options.

Agalsidase alfa long-term effect on left ventricular hypertrophy in Fabry disease.

INTRODUCTION: Fabry disease (FD) is an X-linked lysosomal storage disorder affecting glycosphingolipid metabolism. Most FD patients have cardiac involvement, mainly manifested as left ventricular hypertrophy (LVH), leading to early death due to complications (arrhythmias, valvular disease, vascular involvement). Early initiation of enzyme replacement therapy (ERT) before fibrosis development has been associated with better cardiac outcomes in terms of left ventricular mass index (LVMI) and functional parameters. METHODS: A retrospective observational study was conducted in patients with FD treated with agalsidase alfa for at least 2 years. The primary objectives were: [a] to assess the annual rate of change in LVMI; [b] to define the overall incidence of stability, regression or progression of LVMI. RESULTS: Forty-nine patients were included in the final analysis, with a median follow-up of 7 years. The overall change in LVMI was 0.38 g/m2.73/year, without significant influence of baseline LVH, gender, age at ERT initiation, LV ejection fraction, body mass index, renal disease, and classical cardiovascular risk factors. Long-term ERT with agalsidase alfa was associated with stabilization of LVMI in 98% of patients with FD and was independent of the same covariables. CONCLUSION: Our results are in line with previous literature of comparable FD populations and probably represent the first study of its kind in Argentina. We here highlight the importance of cardiac morphometric stability as a positive outcome of ERT.

Introducción: La enfermedad de Fabry (EF) es una enfermedad de almacenamiento lisosomal ligada al cromosoma X que afecta el metabolismo de glicoesfingolípidos. La mayoría de pacientes EF tienen afectación cardíaca, manifestada principalmente como hipertrofia ventricular izquierda (HVI), que conduce a muerte prematura secundaria a complicaciones (arritmias, valvulopatías, afectación vascular). El tratamiento de reemplazo enzimático (TRE) precoz, iniciado antes del desarrollo de la fibrosis, se relaciona con mejores resultados cardíacos en términos del índice de masa ventricular izquierda (IMVI) y parámetros funcionales. Métodos: Se realizó un estudio retrospectivo observacional en que se incluyeron pacientes con EF tratados con agalsidasa alfa por al menos 2 años. Los objetivos primarios fueron: [a] evaluar el cambio anual del IMVI; [b] definir la incidencia global de estabilidad, regresión o progresión del IMVI. Resultados: Se incluyeron 49 pacientes, con seguimiento (mediana) de 7 años. El cambio global en el IMVI fue 0.38 g/m2.73/año, sin influencia significativa de HVI basal, sexo, edad de inicio de TRE, fracción de eyección del VI, índice de masa corporal, insuficiencia renal y factores de riesgo cardiovascular clásicos. La TRE a largo plazo con agalsidasa alfa se relacionó con la estabilización del IMVI en el 98% de los pacientes con EF, independientemente de las mismas covariables. Conclusión: Nuestros resultados están en línea con la bibliografía previa de poblaciones comparables y, probablemente, representan el primer estudio de este tipo en Argentina. Se destaca la importancia de la estabilidad morfométrica cardíaca como resultado positivo de la TRE.