RESUMEN
BACKGROUND: Fabry disease is a progressive, X chromosome-linked lysosomal storage disorder with multiple organ dysfunction. Due to the absence or reduced activity of alpha-galactosidase A (AGAL), glycosphingolipids, primarily globotriaosyl-ceramide (Gb3), concentrate in cells. In heterozygous women, symptomatology is heterogenous and currently routinely used fluorometry-based assays measuring mean activity mostly fail to uncover AGAL dysfunction. The aim was the development of a flow cytometry assay to measure AGAL activity in individual cells. METHODS: Conventional and multispectral imaging flow cytometry was used to detect AGAL activity. Specificity was validated using the GLA knockout (KO) Jurkat cell line and AGAL inhibitor 1-deoxygalactonojirimycin. The GLA KO cell line was generated via CRISPR-Cas9-based transfection, validated with exome sequencing, gene expression and substrate accumulation. RESULTS: Flow cytometric detection of specific AGAL activity is feasible with fluorescently labelled Gb3. In the case of Jurkat cells, a substrate concentration of 2.83 nmol/mL and 6 h of incubation are required. Quenching of the aspecific exofacial binding of Gb3 with 20% trypan blue solution is necessary for the specific detection of lysosomal substrate accumulation. CONCLUSION: A flow cytometry-based assay was developed for the quantitative detection of AGAL activity at the single-cell level, which may contribute to the diagnosis of Fabry patients.
Asunto(s)
Citometría de Flujo , alfa-Galactosidasa , Humanos , Citometría de Flujo/métodos , Células Jurkat , alfa-Galactosidasa/metabolismo , alfa-Galactosidasa/genética , Enfermedad de Fabry/metabolismo , Enfermedad de Fabry/enzimología , Enfermedad de Fabry/diagnóstico , 1-Desoxinojirimicina/farmacología , 1-Desoxinojirimicina/análogos & derivadosRESUMEN
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.
Asunto(s)
Enfermedad de Fabry , Fenotipo , alfa-Galactosidasa , Humanos , Enfermedad de Fabry/genética , Enfermedad de Fabry/patología , Enfermedad de Fabry/enzimología , alfa-Galactosidasa/genética , alfa-Galactosidasa/metabolismo , Masculino , Adulto , Variación Genética , Trihexosilceramidas/metabolismo , Persona de Mediana Edad , Piel/patología , Células Endoteliales/patología , Células Endoteliales/metabolismo , Mutación , Glucolípidos/metabolismo , EsfingolípidosRESUMEN
Fabry disease (FD) is an X-linked lysosomal storage disorder caused by mutations of the GLA gene that result in a deficiency of the enzymatic activity of α-galactosidase A and consequent accumulation of glycosphingolipids in body fluids and lysosomes of the cells throughout the body. GB3 accumulation occurs in virtually all cardiac cells (cardiomyocytes, conduction system cells, fibroblasts, and endothelial and smooth muscle vascular cells), ultimately leading to ventricular hypertrophy and fibrosis, heart failure, valve disease, angina, dysrhythmias, cardiac conduction abnormalities, and sudden death. Despite available therapies and supportive treatment, cardiac involvement carries a major prognostic impact, representing the main cause of death in FD. In the last years, knowledge has substantially evolved on the pathophysiological mechanisms leading to cardiac damage, the natural history of cardiac manifestations, the late-onset phenotypes with predominant cardiac involvement, the early markers of cardiac damage, the role of multimodality cardiac imaging on the diagnosis, management and follow-up of Fabry patients, and the cardiac efficacy of available therapies. Herein, we provide a comprehensive and integrated review on the cardiac involvement of FD, at the pathophysiological, anatomopathological, laboratory, imaging, and clinical levels, as well as on the diagnosis and management of cardiac manifestations, their supportive treatment, and the cardiac efficacy of specific therapies, such as enzyme replacement therapy and migalastat.
Asunto(s)
Arritmias Cardíacas/terapia , Terapia de Reemplazo Enzimático , Enfermedad de Fabry/terapia , alfa-Galactosidasa/administración & dosificación , alfa-Galactosidasa/metabolismo , Animales , Arritmias Cardíacas/enzimología , Arritmias Cardíacas/etiología , Enfermedad de Fabry/complicaciones , Enfermedad de Fabry/enzimología , HumanosRESUMEN
In the present study the use of extracellular vesicles (EVs) as vehicles for therapeutic enzymes in lysosomal storage disorders was explored. EVs were isolated from mammalian cells overexpressing alpha-galactosidase A (GLA) or N-sulfoglucosamine sulfohydrolase (SGSH) enzymes, defective in Fabry and Sanfilippo A diseases, respectively. Direct purification of EVs from cell supernatants was found to be a simple and efficient method to obtain highly active GLA and SGSH proteins, even after EV lyophilization. Likewise, EVs carrying GLA (EV-GLA) were rapidly uptaken and reached the lysosomes in cellular models of Fabry disease, restoring lysosomal functionality much more efficiently than the recombinant enzyme in clinical use. In vivo, EVs were well tolerated and distributed among all main organs, including the brain. DiR-labelled EVs were localized in brain parenchyma 1 h after intra-arterial (internal carotid artery) or intravenous (tail vein) administrations. Moreover, a single intravenous administration of EV-GLA was able to reduce globotriaosylceramide (Gb3) substrate levels in clinically relevant tissues, such kidneys and brain. Overall, our results demonstrate that EVs from cells overexpressing lysosomal enzymes act as natural protein delivery systems, improving the activity and the efficacy of the recombinant proteins and facilitating their access to organs neglected by conventional enzyme replacement therapies.
Asunto(s)
Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/trasplante , Enfermedades por Almacenamiento Lisosomal/terapia , Vehículos Farmacéuticos , Animales , Encéfalo/metabolismo , Células CHO , Clonación Molecular , Cricetulus , Enfermedad de Fabry/enzimología , Enfermedad de Fabry/terapia , Células HEK293 , Humanos , Hidrolasas/metabolismo , Enfermedades por Almacenamiento Lisosomal/enzimología , Lisosomas , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Vehículos Farmacéuticos/metabolismo , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapéutico , Trihexosilceramidas/metabolismo , alfa-Galactosidasa/metabolismoRESUMEN
The late-onset type of Fabry disease (FD) with GLA IVS4 + 919G > A mutation has been shown to lead to cardiovascular dysfunctions. In order to eliminate variations in other aspects of the genetic background, we established the isogenic control of induced pluripotent stem cells (iPSCs) for the identification of the pathogenetic factors for FD phenotypes through CRISPR/Cas9 genomic editing. We adopted droplet digital PCR (ddPCR) to efficiently capture mutational events, thus enabling isolation of the corrected FD from FD-iPSCs. Both of these exhibited the characteristics of pluripotency and phenotypic plasticity, and they can be differentiated into endothelial cells (ECs). We demonstrated the phenotypic abnormalities in FD iPSC-derived ECs (FD-ECs), including intracellular Gb3 accumulation, autophagic flux impairment, and reactive oxygen species (ROS) production, and these abnormalities were rescued in isogenic control iPSC-derived ECs (corrected FD-ECs). Microarray profiling revealed that corrected FD-derived endothelial cells reversed the enrichment of genes in the pro-inflammatory pathway and validated the downregulation of NF-κB and the MAPK signaling pathway. Our findings highlighted the critical role of ECs in FD-associated vascular dysfunctions by establishing a reliable isogenic control and providing information on potential cellular targets to reduce the morbidity and mortality of FD patients with vascular complications.
Asunto(s)
Células Endoteliales , Enfermedad de Fabry/terapia , Edición Génica , Células Madre Pluripotentes Inducidas , Mutación , alfa-Galactosidasa/genética , Proteína 9 Asociada a CRISPR , Enfermedad de Fabry/enzimología , Enfermedad de Fabry/genética , Enfermedad de Fabry/patología , Humanos , Inflamación , FenotipoRESUMEN
Fabry disease is a rare lysosomal storage disorder characterized by a deficiency of α-galactosidase A (GLA), a lysosomal hydrolase. The enzyme replacement therapy administering naked GLA shows several drawbacks including poor biodistribution, limited efficacy, and relatively high immunogenicity in Fabry patients. An attractive strategy to overcome these problems is the use of nanocarriers for encapsulating the enzyme. Nanoliposomes functionalized with RGD peptide have already emerged as a good platform to protect and deliver GLA to endothelial cells. However, low colloidal stability and limited enzyme entrapment efficiency could hinder the further pharmaceutical development and the clinical translation of these nanoformulations. Herein, the incorporation of the cationic miristalkonium chloride (MKC) surfactant to RGD nanovesicles is explored, comparing two different nanosystems-quatsomes and hybrid liposomes. In both systems, the positive surface charge introduced by MKC promotes electrostatic interactions between the enzyme and the nanovesicles, improving the loading capacity and colloidal stability. The presence of high MKC content in quatsomes practically abolishes GLA enzymatic activity, while low concentrations of the surfactant in hybrid liposomes stabilize the enzyme without compromising its activity. Moreover, hybrid liposomes show improved efficacy in cell cultures and a good in vitro/in vivo safety profile, ensuring their future preclinical and clinical development.
Asunto(s)
Terapia de Reemplazo Enzimático , Enfermedad de Fabry/terapia , Nanoestructuras/química , alfa-Galactosidasa/metabolismo , Enfermedad de Fabry/enzimología , Humanos , Oligopéptidos/química , Tamaño de la Partícula , Propiedades de Superficie , Tensoactivos/químicaRESUMEN
Enzyme and chaperone therapies are used to treat Fabry disease. Such treatments are expensive and require intrusive biweekly infusions; they are also not particularly efficacious. In this pilot, single-arm study (NCT02800070), five adult males with Type 1 (classical) phenotype Fabry disease were infused with autologous lentivirus-transduced, CD34+-selected, hematopoietic stem/progenitor cells engineered to express alpha-galactosidase A (α-gal A). Safety and toxicity are the primary endpoints. The non-myeloablative preparative regimen consisted of intravenous melphalan. No serious adverse events (AEs) are attributable to the investigational product. All patients produced α-gal A to near normal levels within one week. Vector is detected in peripheral blood and bone marrow cells, plasma and leukocytes demonstrate α-gal A activity within or above the reference range, and reductions in plasma and urine globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3) are seen. While the study and evaluations are still ongoing, the first patient is nearly three years post-infusion. Three patients have elected to discontinue enzyme therapy.
Asunto(s)
Enfermedad de Fabry/enzimología , Enfermedad de Fabry/terapia , Terapia Genética/métodos , Lentivirus/genética , alfa-Galactosidasa/genética , alfa-Galactosidasa/uso terapéutico , Adulto , Antígenos CD34 , Células de la Médula Ósea , Enfermedad de Fabry/genética , Vectores Genéticos , Células Madre Hematopoyéticas , Humanos , Leucocitos , Masculino , Persona de Mediana Edad , Trihexosilceramidas/sangre , Trihexosilceramidas/orinaRESUMEN
Fabry disease (FD) is a rare X-linked glycosphingolipidosis caused by mutations in GLA, a gene responsible for encoding α-galactosidase A, an enzyme required for degradation of glycosphingolipids, mainly globotriaosylceramide (Gb3) in all cells of the body. FD patients present a broad spectrum of clinical phenotype and many symptoms are shared with other diseases, making diagnosis challenging. Here we describe a novel GLA variant located in the 5' splice site of the intron 3, in four members of a family with neuropsychiatric symptoms. Analysis of the RNA showed the variant promotes alteration of the wild type donor site, affecting splicing and producing two aberrant transcripts. The functional characterization showed absence of enzymatic activity in cells expressing both transcripts, confirming their pathogenicity. The family presents mild signs of FD, as angiokeratoma, cornea verticillata, acroparesthesia, tinnitus, vertigo, as well as accumulation of plasma lyso-Gb3 and urinary Gb3. Interestingly, the man and two women present psychiatric symptoms, as depression or schizophrenia. Although psychiatric illnesses, especially depression, are frequently reported in patients with FD and studies have shown that the hippocampus is an affected brain structure in these patients, it is not clear whether the Gb3 accumulation in the brain is responsible for these symptoms or they are secondary. Therefore, new studies are needed to understand whether the accumulation of Gb3 could produce neuronal alterations leading to psychiatric symptoms.
Asunto(s)
Encéfalo/metabolismo , Enfermedad de Fabry/genética , Mutación , alfa-Galactosidasa/genética , Adolescente , Enfermedad de Fabry/enzimología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Adulto Joven , alfa-Galactosidasa/metabolismoRESUMEN
Cell-penetrating peptides (CPPs) can deliver payloads into cells by forming complexes with bioactive molecules via either covalent or non-covalent bonds. Previously, we reported polyhistidine (H16 peptide: HHHHHHHHHHHHHHHH-NH2) as a new CPP. This peptide is anticipated to be a valuable new carrier for drug delivery to intracellular lysosomes; the peptide can transport macromolecules into these organelles. In the present study, we examined the application of the H16 peptide as a drug delivery system (DDS) to reverse to lysosomal storage disease (LSD) in cells in vitro. LSDs are metabolic disorders caused by the loss of specific lysosomal enzymes. The majority of lysosomal enzymes are acidic proteins and we utilized this common feature for our DDS. We synthesized a polylysine-polyhistidine fusion peptide (K10H16 peptide: KKKKKKKKKKGHHHHHHHHHHHHHHHH-NH2) and developed a simple method for transporting acidic proteins into intracellular lysosomes via formation of complexes of enzymes with the K10H16 peptide by electrostatic interaction. First, we demonstrated our strategy using maltose-binding protein-fused green fluorescent protein (MBP-GFP) to model an acidic protein. The K10H16 peptide bound to MBP-GFP and transported it into intracellular lysosomes. Further, alpha-galactosidase A (GLA), one of the lysosomal enzymes associated with LSD, was also delivered to intracellular lysosomes by the peptide. The complex between K10H16 peptide and GLA restored typical proliferation to LSD cells, which otherwise grew more slowly than normal cells. These results suggest that K10H16 peptide replenished lysosomal enzyme deficiency in LSD cells. The K10H16 peptide may be useful as a DDS for LSD therapy.
Asunto(s)
Péptidos de Penetración Celular/administración & dosificación , Péptidos de Penetración Celular/química , Sistemas de Liberación de Medicamentos , Terapia de Reemplazo Enzimático/métodos , Enfermedades por Almacenamiento Lisosomal/tratamiento farmacológico , Enfermedades por Almacenamiento Lisosomal/enzimología , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Secuencia de Aminoácidos , Células Cultivadas , Enfermedad de Fabry/tratamiento farmacológico , Enfermedad de Fabry/enzimología , Histidina/química , Humanos , Polilisina/química , Transporte de Proteínas , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/química , alfa-Galactosidasa/administración & dosificaciónRESUMEN
Fabry Disease (FD) is a systemic disorder that can result in cardiovascular, renal, and neurovascular disease leading to reduced life expectancy. FD should be considered in the differential of all patients with unexplained left ventricular hypertrophy (LVH). We therefore performed a prospective screening study in Edmonton and Hong Kong using Dried Blood Spot (DBS) testing on patients with undiagnosed LVH. Participants found to have unexplained LVH on echocardiography were invited to participate and subsequently subjected to DBS testing. DBS testing was used to measure α-galactosidase (α-GAL) enzyme activity and for mutation analysis of the α-galactosidase (GLA) gene, both of which are required to make a diagnosis of FD. DBS testing was performed as a screening tool on patients (n = 266) in Edmonton and Hong Kong, allowing for detection of five patients with FD (2% prevalence of FD) and one patient with hydroxychloroquine-induced phenocopy. Left ventricular mass index (LVMI) by GLA genotype showed a higher LVMI in patients with IVS4 + 919G > A mutations compared to those without the mutation. Two patients were initiated on ERT and hydroxychloroquine was discontinued in the patient with a phenocopy of FD. Overall, we detected FD in 2% of our screening cohort using DBS testing as an effective and easy to administer screening tool in patients with unexplained LVH. Utilizing DBS testing to screen for FD in patients with otherwise undiagnosed LVH is clinically important due to the availability of effective therapies and the value of cascade screening in extended families.
Asunto(s)
Enfermedad de Fabry/diagnóstico , Enfermedad de Fabry/enzimología , Hipertrofia Ventricular Izquierda/diagnóstico , Hipertrofia Ventricular Izquierda/enzimología , Tamizaje Masivo/métodos , alfa-Galactosidasa/genética , Adulto , Anciano , Anciano de 80 o más Años , Análisis Mutacional de ADN , Diagnóstico Diferencial , Pruebas con Sangre Seca , Ecocardiografía , Enfermedad de Fabry/epidemiología , Femenino , Genotipo , Hong Kong/epidemiología , Humanos , Hipertrofia Ventricular Izquierda/epidemiología , Masculino , Persona de Mediana Edad , Mutación , Fenotipo , Estudios ProspectivosRESUMEN
Objectives Fabry disease (FD, OMIM #301500) is a rare and progressive X-linked lysosomal storage disorder. FD is caused by mutations in the GLA gene on chromosome Xq22. Methods In this article, we aimed to present the largest sample of GLA mutation spectrum including common and novel variants in Turkish population. GLA gene sequence analysis was performed on the subjects who applied to the department of medical genetics with the preliminary diagnosis of FD between 2013 and 2018. Results We detected 22 different mutations as two novel [(p.F69S(c.206T>C), p.P205A (c.613C>G)] and 20 previously reported GLA mutations in 47 individuals from 22 unrelated families. These mutations included 14 missense mutations, four nonsense mutations, two small deletions, one small deletion/insertion and one small insertion. Major clinical findings of the female case with p.F69S(c.206T>C) mutation were cornea verticillata, acroparesthesia, angiokeratoma, psychiatric and gastrointestinal symptoms. Other novel mutation (p.P205A [c.613C>G]) was carried by a male case presenting gastrointestinal symptoms. Conclusions We described clinical findings of two cases that had novel mutations to provide more insight in genotype-phenotype correlation. We presented the largest mutation spectrum in Turkish population and reviewed previous mutations in this article.
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Biomarcadores/análisis , Enfermedad de Fabry/genética , Mutación , alfa-Galactosidasa/genética , Adulto , Niño , Enfermedad de Fabry/enzimología , Enfermedad de Fabry/epidemiología , Enfermedad de Fabry/patología , Familia , Femenino , Estudios de Asociación Genética , Genotipo , Humanos , Masculino , Fenotipo , Pronóstico , Turquía/epidemiologíaRESUMEN
BACKGROUND: Fabry disease (FD) is an X-linked lysosomal storage and multi-system disorder due to mutations in the α-galactosidase A (α-GalA) gene. We investigated the impact of individual amino acid exchanges in the α-GalA 3D-structure on the clinical phenotype of FD patients. PATIENTS AND METHODS: We enrolled 80 adult FD patients with α-GalA missense mutations and stratified them into three groups based on the amino acid exchange location in the α-GalA 3D-structure: patients with active site mutations, buried mutations and other mutations. Patient subgroups were deep phenotyped for clinical and laboratory parameters and FD-specific treatment. RESULTS: Patients with active site or buried mutations showed a severe phenotype with multi-organ involvement and early disease manifestation. Patients with other mutations had a milder phenotype with less organ impairment and later disease onset. α-GalA activity was lower in patients with active site or buried mutations than in those with other mutations (P < 0.01 in men; P < 0.05 in women) whilst lyso-Gb3 levels were higher (P < 0.01 in men; <0.05 in women). CONCLUSIONS: The type of amino acid exchange location in the α-GalA 3D-structure determines disease severity and temporal course of symptom onset. Patient stratification using this parameter may become a useful tool in the management of FD patients.
Asunto(s)
Enfermedad de Fabry/genética , alfa-Galactosidasa/genética , Enfermedad de Fabry/complicaciones , Enfermedad de Fabry/diagnóstico , Enfermedad de Fabry/enzimología , Humanos , Conformación Molecular , Mutación MissenseRESUMEN
Fabry disease is a rare X-linked lysosomal disease, in which mutations in the gene encoding α-galactosidase A result in progressive cellular accumulation of globotriaosylceramide (GL-3) in various organs including the skin, kidney, and heart, often leading to life-threatening conditions. Enzyme replacement therapy is currently the standard therapy for the disease, to which two α-galactosidase A formulations have been approved: agalsidase α (Replagal®, Shire) and agalsidase ß (Fabrazyme®, Sanofi). We have recently developed a biosimilar of agalsidase ß, JR-051, and investigated its pharmacokinetics and pharmacodynamics to assess its bioequivalence to agalsidase ß. In a randomized phase I study, healthy adult male volunteers were treated with JR-051 or agalsidase ß and the pharmacokinetics of the drugs were compared. The ratio of geometric means (90% confidence interval [CI]) of the AUC0-24 and Cmax for JR-051 over agalsidase ß were 0.91 (0.8294, 1.0082) and 0.90 (0.7992, 1.0125), respectively. In a 52-week, single-arm, phase II/III study, patients with Fabry disease switched therapy from agalsidase ß to JR-051 to evaluate its pharmacodynamics. The mean (95% CI) plasma GL-3 concentrations at weeks 26 and 52 relative to pre-JR-051 administration were 1.03 (0.91, 1.15) and 0.96 (0.86, 1.06), respectively, which were within the pre-determined bioequivalence acceptance range (0.70, 1.43). The mean (95% CI) plasma globotriaosylsphingosine (lyso-GL-3) concentrations at weeks 26 and 52 relative to pre-JR-051 administration were 1.07 (0.92, 1.23) and 1.13 (1.03, 1.22), respectively. Estimated glomerular filtration rate and left ventricular mass index, as renal and cardiac function indicators, showed no notable changes from baseline throughout the study period, and no new safety concerns were identified. In conclusion, these studies demonstrated bioequivalence of JR-051 to agalsidase ß in terms of its pharmacokinetics and pharmacodynamics. JR-051 offers a potential new treatment option for patients with Fabry disease.
Asunto(s)
Biomarcadores/sangre , Biosimilares Farmacéuticos/administración & dosificación , Terapia de Reemplazo Enzimático/métodos , Enfermedad de Fabry/terapia , Glucolípidos/sangre , Esfingolípidos/sangre , beta-Galactosidasa/administración & dosificación , Adolescente , Adulto , Anciano , Biosimilares Farmacéuticos/farmacocinética , Biosimilares Farmacéuticos/farmacología , Estudios de Casos y Controles , Niño , Método Doble Ciego , Enfermedad de Fabry/enzimología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Distribución Tisular , Adulto JovenRESUMEN
Fabry's disease (FD) is an X-linked lysosomal storage disorder caused by the deficient activity of the lysosomal enzyme α-galactosidase A (α-Gal A) leading to intracellular accumulation of globotriaosylceramide (Gb3). Patients with amenable mutations can be treated with migalastat, a recently approved oral pharmacologic chaperone to increase endogenous α-Gal A activity. We assessed safety along with cardiovascular, renal, and patient-reported outcomes and disease biomarkers in a prospective observational multicenter study after 12 months of migalastat treatment under "real-world" conditions. Fifty-nine (28 females) patients (34 (57.6%) pretreated with enzyme replacement therapy) with amenable mutations were recruited. Migalastat was generally safe and well tolerated. Females and males presented with a reduction of left ventricular mass index (primary end point) (-7.2 and -13.7 g/m2 , P = 0.0050 and P = 0.0061). FD-specific manifestations and symptoms remained stable (all P > 0.05). Both sexes presented with a reduction of estimated glomerular filtration rate (secondary end point) (-6.9 and -5.0 mL/minute/1.73 m2 ; P = 0.0020 and P = 0.0004, respectively), which was most prominent in patients with low blood pressure (P = 0.0271). α-Gal A activity increased in male patients by 15% from 29% to 44% of the normal wild-type activity (P = 0.0106) and plasma lyso-Gb3 levels were stable in females and males (P = 0.3490 and P = 0.2009). Reevaluation of mutations with poor biochemical response revealed no marked activity increase in a zero activity background. We conclude that therapy with migalastat was generally safe and resulted in an amelioration of left ventricular mass. In terms of impaired renal function, blood pressure control seems to be an unattended important goal.
Asunto(s)
1-Desoxinojirimicina/análogos & derivados , Enfermedad de Fabry/tratamiento farmacológico , alfa-Galactosidasa/metabolismo , 1-Desoxinojirimicina/efectos adversos , 1-Desoxinojirimicina/uso terapéutico , Adulto , Biomarcadores/sangre , Enfermedad de Fabry/diagnóstico , Enfermedad de Fabry/enzimología , Enfermedad de Fabry/fisiopatología , Femenino , Predisposición Genética a la Enfermedad , Alemania , Tasa de Filtración Glomerular/efectos de los fármacos , Glucolípidos/sangre , Humanos , Masculino , Persona de Mediana Edad , Mutación , Estudios Prospectivos , Esfingolípidos/sangre , Factores de Tiempo , Resultado del Tratamiento , Función Ventricular Izquierda/efectos de los fármacos , Remodelación Ventricular/efectos de los fármacos , alfa-Galactosidasa/genéticaRESUMEN
BACKGROUND: Fabry disease is a progressive X-linked lysosomal disorder. In this subgroup analysis of the global phase III ATTRACT study, the efficacy and safety of oral migalastat, a pharmacologic chaperone, were investigated in Japanese patients with Fabry disease. METHODS: Patients were randomly assigned to receive migalastat (150 mg every other day) or to continue biweekly enzyme replacement therapy infusions (ERT; agalsidase alfa 0.2 mg/kg or agalsidase beta 1.0 mg/kg) for 18 months followed by a 12-month open-label extension during which all patients received migalastat. End points included glomerular filtration rate (estimated and measured), left ventricular mass index (LVMi), composite clinical outcomes, leukocyte alpha-galactosidase A activity, plasma globotriaosylsphingosine (lyso-Gb3), and safety. RESULTS: Data from 7 Japanese patients (migalastat, 5; ERT, 2), mean age 55 years, with high disease burden, were analyzed. All patients in the migalastat group completed the open-label comparison and extension periods. At 18 months, efficacy in the Japanese patient population was similar to that in the overall ATTRACT population. Migalastat treatment increased leukocyte alpha-galactosidase A activity, stabilized renal function, and decreased LVMi. Plasma lyso-Gb3 levels remained low and stable. Additionally, the long-term extension study showed that efficacy of migalastat was maintained for up to 48 months. Migalastat was safe and well tolerated in the Japanese patients, as in the overall ATTRACT population. CONCLUSION: Migalastat can be used to treat Japanese patients with Fabry disease with GLA mutations amenable to migalastat according to the dosage and administration approved in other countries. TRIAL REGISTRATION NUMBERS: ClinicalTrials.gov, NCT01218659 and NCT02194985.
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1-Desoxinojirimicina/análogos & derivados , Enfermedad de Fabry/tratamiento farmacológico , 1-Desoxinojirimicina/administración & dosificación , 1-Desoxinojirimicina/efectos adversos , Administración Oral , Adulto , Enfermedad de Fabry/enzimología , Enfermedad de Fabry/genética , Femenino , Predisposición Genética a la Enfermedad , Humanos , Japón , Masculino , Persona de Mediana Edad , Mutación , Estudios Prospectivos , Factores de Tiempo , Resultado del Tratamiento , alfa-Galactosidasa/genética , alfa-Galactosidasa/metabolismoRESUMEN
The lysosomal storage disorder Fabry disease is characterized by a deficiency of the lysosomal enzyme α-Galactosidase A. The observation that missense variants in the encoding GLA gene often lead to structural destabilization, endoplasmic reticulum retention and proteasomal degradation of the misfolded, but otherwise catalytically functional enzyme has resulted in the exploration of alternative therapeutic approaches. In this context, we have investigated proteostasis regulators (PRs) for their potential to increase cellular enzyme activity, and to reduce the disease-specific accumulation of the biomarker globotriaosylsphingosine in patient-derived cell culture. The PRs also acted synergistically with the clinically approved 1-deoxygalactonojirimycine, demonstrating the potential of combination treatment in a therapeutic application. Extensive characterization of the effective PRs revealed inhibition of the proteasome and elevation of GLA gene expression as paramount effects. Further analysis of transcriptional patterns of the PRs exposed a variety of genes involved in proteostasis as potential modulators. We propose that addressing proteostasis is an effective approach to discover new therapeutic targets for diseases involving folding and trafficking-deficient protein mutants.
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Enfermedad de Fabry/genética , Enfermedades por Almacenamiento Lisosomal/genética , Proteostasis/genética , alfa-Galactosidasa/genética , 1-Desoxinojirimicina/análogos & derivados , 1-Desoxinojirimicina/uso terapéutico , Biomarcadores/metabolismo , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Enfermedad de Fabry/tratamiento farmacológico , Enfermedad de Fabry/enzimología , Enfermedad de Fabry/patología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Enfermedades por Almacenamiento Lisosomal/tratamiento farmacológico , Enfermedades por Almacenamiento Lisosomal/enzimología , Enfermedades por Almacenamiento Lisosomal/patología , Lisosomas/enzimología , Lisosomas/genética , Lisosomas/metabolismo , Mutación Missense/genética , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Transporte de Proteínas/efectos de los fármacos , Esfingosina/análogos & derivados , Esfingosina/metabolismoRESUMEN
The term "pharmacological chaperone" was introduced 20 years ago. Since then the approach with this type of drug has been proposed for several diseases, lysosomal storage disorders representing the most popular targets. The hallmark of a pharmacological chaperone is its ability to bind a protein specifically and stabilize it. This property can be beneficial for curing diseases that are associated with protein mutants that are intrinsically active but unstable. The total activity of the affected proteins in the cell is lower than normal because they are cleared by the quality control system. Although most pharmacological chaperones are reversible competitive inhibitors or antagonists of their target proteins, the inhibitory activity is neither required nor desirable. This issue is well documented by specific examples among which those concerning Fabry disease. Direct specific binding is not the only mechanism by which small molecules can rescue mutant proteins in the cell. These drugs and the properly defined pharmacological chaperones can work together with different and possibly synergistic modes of action to revert a disease phenotype caused by an unstable protein.
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Enfermedad de Fabry , Chaperonas Moleculares/uso terapéutico , Mutación Missense , alfa-Galactosidasa , Enfermedad de Fabry/tratamiento farmacológico , Enfermedad de Fabry/enzimología , Enfermedad de Fabry/genética , Humanos , alfa-Galactosidasa/genética , alfa-Galactosidasa/metabolismoRESUMEN
INTRODUCTION: The lifespan of patients with Fabry disease (FD) is shorter than that seen in the general population. Leukocyte telomere length (LTL) and telomerase activity (TA) are potential markers of biologic aging. The aim of the current study was to determine the LTL and TA in FD patients and to assess the correlation between LTL and TA and renal involvement. METHODS: We included 33 FD patients and 66 healthy matched controls. LTL and TA were measured using a quantitative PCR assay and gene expression assay. FD patients were stratified by renal function (estimated glomerular filtration rate [eGFR] higher or lower than 60 mL/min/1.73 m2) and proteinuria (urine protein creatinine ratio higher or lower than 0.5 g/g). RESULTS: LTL was significantly shorter (0.69 vs. 0.73, p = 0.015) and TA significantly higher (1.55 vs. 1.19, p = 0.047) in FD patients compared to controls. Males with FD had significantly shorter LTL (p = 0.020) and lower, but non-significant, TA compared to male controls (p = 0.333). Female FD patients had similar LTL (p = 0.285) but significantly higher TA compared to female controls (p = 0.005). LTL was not influenced by eGFR, but TA was significantly lower in the low eGFR group (p = 0.003). CONCLUSIONS: FD patients have significantly shorter LTL, but significantly higher TA compared to healthy controls. Increased TA activity in FD patients could be the compensation mechanism to prevent LTL decrease (and accelerated ageing), which seems to be exhausted at the advanced stage of renal disease.
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Envejecimiento/fisiología , Enfermedad de Fabry/fisiopatología , Enfermedades Renales/fisiopatología , Telomerasa/metabolismo , Telómero , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/metabolismo , Enfermedades Cardiovasculares/metabolismo , Estudios de Casos y Controles , Enfermedad de Fabry/tratamiento farmacológico , Enfermedad de Fabry/enzimología , Femenino , Humanos , Mediadores de Inflamación/metabolismo , Enfermedades Renales/enzimología , Masculino , Persona de Mediana Edad , Reacción en Cadena en Tiempo Real de la Polimerasa , Adulto Joven , alfa-Galactosidasa/uso terapéuticoRESUMEN
α-Galactosidase, (E.C. 3.2.1.22) is an exoglycosidase that target galactooligosaccharides such as raffinose, melibiose, stachyose and branched polysaccharides like galactomannans and galacto-glucomannans by catalysing the hydrolysis of α-1,6 linked terminal galactose residues. The enzyme has been isolated and characterized from microbial, plant and animal sources. This ubiquitous enzyme possesses physiological significance and immense industrial potential. Optimization of the growth conditions and efficient purification strategies can lead to a significant increase in the enzyme production. To boost commercial productivity, cloning of novel α-galactosidase genes and their heterologous expression in suitable host has gained popularity. Enzyme immobilization leads to its greater reutilization, superior thermostability, pH tolerance and increased activity. The enzyme is well explored in food industry in the removal of raffinose family oligosaccharides (RFOs) in soymilk and sugar crystallization process. It also improves animal feed quality and biomass processing. Applications of the enzyme is in the area of biomedicine includes therapeutic advances in treatment of Fabry disease, blood group conversion and removal of α-gal type immunogenic epitopes in xenotransplantation. With considerable biotechnological applications, this enzyme has been vastly commercialized and holds greater future prospects.
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Biotecnología , Enzimas Inmovilizadas/química , alfa-Galactosidasa/química , Clonación Molecular , Estabilidad de Enzimas , Enzimas Inmovilizadas/genética , Enzimas Inmovilizadas/metabolismo , Enfermedad de Fabry/tratamiento farmacológico , Enfermedad de Fabry/enzimología , Humanos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/uso terapéutico , Especificidad por Sustrato , Trasplante Heterólogo , alfa-Galactosidasa/biosíntesis , alfa-Galactosidasa/genética , alfa-Galactosidasa/uso terapéuticoRESUMEN
BACKGROUND: Sporadic Parkinson's disease (PD) patients have lower α-galactosidase A (α-GAL A) enzymatic activity and Fabry disease (FD) patients potentially carry an increased risk of PD. OBJECTIVE: Determination of PD prevalence in FD and clinical, biochemical and vascular neuroimaging description of FD pedigrees with concomitant PD. METHODS: Clinical screening for PD in 229 FD patients belonging to 31 families, harbouring GLA gene mutation p.F113L, and subsequent pedigree analysis. Gender-stratified comparison of FD+/PD+ patients with their family members with FD but without PD (FD+/PD-) regarding Mainz scores, plasma & leukocytes α-GAL A enzymatic activity, urinary Gb3 and plasma Lyso-Gb3, vascular brain neuroimaging. RESULTS: Prevalence of PD in FD was 1.3% (3/229) (3% in patients aged ≥50 years). Three FD patients, one female (73 years old) (P1) and two males (60 and 65 years old) (P2 and P3), three different pedigrees, presented akinetic-rigid PD, with weak response to levodopa (16% - 36%), and dopaminergic deficiency on 18F-DOPA PET. No pathogenic mutations were found in a PD gene panel. FD+/PD+ patients had worse clinical severity of FD (above upper 75% IQR in Mainz scores), and cortico-subcortical white matter/small vessel lesions. P3 patient was under enzyme therapy, started 1 year before PD diagnosis. P2-P3 patients had higher leucocyte α-GAL A activity (2,2-3 vs.1,0 (median)(nmol/h/mg)). CONCLUSION: We have shown a high prevalence of PD in a late-onset phenotype of FD, presenting high cerebrovascular burden and weak response to levodopa. Further studies will untangle how much of this PD phenotype is due to Gb3 deposition versus cerebrovascular lesions in the nigro-striatal network.