Inflammatory mechanisms involved in the pathophysiology of Fabry disease and the clinical response to enzyme replacement therapy

La maladie de Fabry (MF) est une maladie lysosomale liée à l’X, due à des mutations du gène GLA codant pour l’alpha-galactosidase A, caractérisée par une accumulation en glycosphingolipides. L’enzymothérapie substitutive (ES) par agalsidase alfa ou beta, a une efficacité inégale selon les patients. Ce travail a exploré les mécanismes inflammatoires impliqués dans la réponse à l’ES et dans la physiopathologie propre de la MF. Après élaboration d’un test ELISA permettant le dépistage concomitant des anticorps (Ac) anti-agalsidase alfa et beta, on observait un risque accru de développer ces Ac chez les patients de phénotype classique, sans retentissement clinique évident mais associé à des taux de lysoGb3 plasmatiques plus élevés suggérant un pronostic péjoratif. Ces Ac, notamment les IgG4, avaient un pouvoir inhibiteur enzymatique dose-dépendant. Alors que la réactivité croisée entre les deux ES était totale et que le risque de développer des Ac n’était pas lié à l’ES reçue, l’analyse des leucocytes circulants a mis en évidence une élévation du pourcentage de cellules NK chez les patients traités par agalsidase beta suggérant son immunogénicité accrue. Indépendamment du traitement, on observait des perturbations des lymphocytes T CD4 et CD8 CCR7- ayant un profil atypique immature CD27+ et CD57-. L’expression du CCR7 était inversement corrélée aux concentrations de lysoGb3 et de sphingosine-1-phosphate (S1P) dans les lymphocytes T CD4+. Enfin, on observait une augmentation du S1P chez les patients non-classiques. Notre travail fournit des outils indispensables au développement de thérapies géniques et ouvre des pistes de recherche sur la maturation lymphocytaire T et la S1P.Fabry disease (FD) is an X-linked disorder due to mutations in the GLA gene that lead to defects in alpha-galactosidase A enzyme activity and subsequent accumulation of glycosphingolipids. A pro-inflammatory condition has been described in FD, but very few studies have investigated the peripheral blood mononuclear cells (PBMCs). Enzyme replacement therapy (ERT) has been the historical treatment of FD and has shown inconstant benefits. In the present work, we investigated the inflammatory mechanisms involved in the response to ERT and in the pathophysiology of the disease. We first developed an ELISA that allows the detection of anti-agalsidase antibodies (Abs) developed against both available ERT. We showed that the prevalence of Abs depends on the clinical phenotype of the patients. The cross-reactivity towards both ERT was complete. Abs, notably the IgG4, had a dose-dependent inhibitory effect on ERT. Abs were not associated with clinical events, but higher lysoGb3 levels suggesting a poorer associated prognosis. In PBMCs, we observed higher percentages of NK cells in Fabry patients under agalsidase beta, suggesting its higher immunogenicity. In Fabry patients, we observed disturbances in T cell maturation with higher percentages of CD27+ and lower percentages of CD57+ cells among the CCR7- populations. The expression of CCR7 was inversely correlated with the concentrations of lysoGb3 and sphingosine-1-phosphate (S1P) in CD4 T cells. We showed that S1P was significantly higher in non-classic patients. Hence we developed essential tools in the perspective of future gene therapies and suggest new topics of research such as the role of S1P or T-cell maturation in FD

Prevalence of Cancer in Acid Sphingomyelinase Deficiency

Acid sphingomyelinase deficiency (ASMD) is an inherited lysosomal disease characterised by a diffuse accumulation of sphingomyelin that cannot be catabolised into ceramide and phosphocholine. We studied the incidence of cancer in ASMD patients. We retrospectively reviewed the medical records of the adult chronic visceral ASMD patients in our cohort. Thirty-one patients (12 females, 19 males) were included with a median age of 48.7 y. (IQ: 30.3–55.1). Five cancers were observed in 1 female (breast cancer) and 4 males (two lung cancers, one thyroid cancer and one bladder cancer), resulting in a prevalence of 16.1%. The existence of cancer was associated with a more severe ASMD characterised by a larger spleen (25 cm (22.5–25) vs. 18 cm (17–20); p = 0.042); lower diffusing capacity of the lung for carbon monoxide (DLCO; 29.5 % (17.8–43.0) vs. 58.5 % (49.8–69.5%); p = 0.01) and tobacco use (100% vs. 45%; p = 0.04). Three patients died, all from cancer (p = 0.002). The prevalence of cancer appeared to be strikingly elevated in our cohort of patients, without any specificity in the type of cancer. Systematic screening for cancer should be performed, and carcinogenic substances such as tobacco should be avoided in patients with ASMD

Exploration des mécanismes inflammatoires impliqués dans la physiopathologie de la maladie de Fabry et la réponse à l’enzymothérapie substitutive

Fabry disease (FD) is an X-linked disorder due to mutations in the GLA gene that lead to defects in alpha-galactosidase A enzyme activity and subsequent accumulation of glycosphingolipids. A pro-inflammatory condition has been described in FD, but very few studies have investigated the peripheral blood mononuclear cells (PBMCs). Enzyme replacement therapy (ERT) has been the historical treatment of FD and has shown inconstant benefits. In the present work, we investigated the inflammatory mechanisms involved in the response to ERT and in the pathophysiology of the disease. We first developed an ELISA that allows the detection of anti-agalsidase antibodies (Abs) developed against both available ERT. We showed that the prevalence of Abs depends on the clinical phenotype of the patients. The cross-reactivity towards both ERT was complete. Abs, notably the IgG4, had a dose-dependent inhibitory effect on ERT. Abs were not associated with clinical events, but higher lysoGb3 levels suggesting a poorer associated prognosis. In PBMCs, we observed higher percentages of NK cells in Fabry patients under agalsidase beta, suggesting its higher immunogenicity. In Fabry patients, we observed disturbances in T cell maturation with higher percentages of CD27+ and lower percentages of CD57+ cells among the CCR7- populations. The expression of CCR7 was inversely correlated with the concentrations of lysoGb3 and sphingosine-1-phosphate (S1P) in CD4 T cells. We showed that S1P was significantly higher in non-classic patients. Hence we developed essential tools in the perspective of future gene therapies and suggest new topics of research such as the role of S1P or T-cell maturation in FD.La maladie de Fabry (MF) est une maladie lysosomale liée à l’X, due à des mutations du gène GLA codant pour l’alpha-galactosidase A, caractérisée par une accumulation en glycosphingolipides. L’enzymothérapie substitutive (ES) par agalsidase alfa ou beta, a une efficacité inégale selon les patients. Ce travail a exploré les mécanismes inflammatoires impliqués dans la réponse à l’ES et dans la physiopathologie propre de la MF. Après élaboration d’un test ELISA permettant le dépistage concomitant des anticorps (Ac) anti-agalsidase alfa et beta, on observait un risque accru de développer ces Ac chez les patients de phénotype classique, sans retentissement clinique évident mais associé à des taux de lysoGb3 plasmatiques plus élevés suggérant un pronostic péjoratif. Ces Ac, notamment les IgG4, avaient un pouvoir inhibiteur enzymatique dose-dépendant. Alors que la réactivité croisée entre les deux ES était totale et que le risque de développer des Ac n’était pas lié à l’ES reçue, l’analyse des leucocytes circulants a mis en évidence une élévation du pourcentage de cellules NK chez les patients traités par agalsidase beta suggérant son immunogénicité accrue. Indépendamment du traitement, on observait des perturbations des lymphocytes T CD4 et CD8 CCR7- ayant un profil atypique immature CD27+ et CD57-. L’expression du CCR7 était inversement corrélée aux concentrations de lysoGb3 et de sphingosine-1-phosphate (S1P) dans les lymphocytes T CD4+. Enfin, on observait une augmentation du S1P chez les patients non-classiques. Notre travail fournit des outils indispensables au développement de thérapies géniques et ouvre des pistes de recherche sur la maturation lymphocytaire T et la S1P

Of the importance of the clinical phenotypes in the interpretation of the studies dealing with Fabry disease

Abstract Fabry disease (OMIM #301500) is an X-linked disorder caused by alpha-galactosidase A deficiency with two major clinical phenotypes: classic and non-classic of different prognosis. From 2001, enzyme replacement therapies with agalsidase alfa and beta have been available. In this letter we underline the different clinical and technical considerations the readers have to be aware of to interpret the results of studies dealing with Fabry disease and anti-agalsidase antibodies. We reaffirm that antibodies preferentially develop in the severe classic Fabry phenotype, which can mislead into interpreting that antibodies are associated with much severe clinical events

Anti-mitochondrial antibodies are not a hallmark of severity in idiopathic inflammatory myopathies

International audienceAnti‐mitochondrial antibodies type 2 (AMA2) are the hallmarks of primary biliary cholangitis (PBC) [1]. AMA2 have also been described in 11.3% of idiopathic inflammatory myopathies (IIM) then associatedwith cardiac involvement, muscular atrophy and granuloma (table 1) [2]

Strong increase of leukocyte apha‐galactosidase A activity in two male patients with Fabry disease following oral chaperone therapy

International audienceBACKGROUND: Fabry disease (OMIM 301500) is an X-linked disorder caused by alpha-galactosidase A (α-Gal A) deficiency. The administration of a pharmacologic chaperone (migalastat) in Fabry patients with amenable mutations has been reported to improve or stabilize organ damages and reduce lyso-Gb3 plasma level. An increase of α-Gal A activity has been observed in vitro in cells expressing amenable GLA mutations when incubated with migalastat. The impact of the drug on α-Gal A in vivo activity has been poorly studied.METHODS: We conducted a retrospective analysis of two unrelated male Fabry patients with p.Asn215Ser (p.N215S) variant.RESULTS: We report the important increase of α-Gal A activity in blood leukocytes reaching normal ranges of activity after about 1 year of treatment with migalastat. Cardiac parameters improved or stabilized with the treatment.CONCLUSION: We confirm in vivo the effects of migalastat that have been observed in N215S carriers in vitro. The increase of α-Gal A activity may be the strongest marker for biochemical efficacy. The normalization of enzyme activity could become the new therapeutic target to achieve

Predictive biological patterns in Fabry disease revealed by integrative omics machine learning analysis

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CD8+T-bet+ cells as a predominant biomarker for inclusion body myositis

International audienceBackground: Myositis is a heterogeneous group of muscular auto-immune diseases with clinical and pathological criteria that allow the classification of patients into different sub-groups. Inclusion body myositis is the most frequent myositis above fifty years of age. Diagnosing inclusion body myositis requires expertise and is challenging. Little is known concerning the pathogenic mechanisms of this disease in which conventional suppressive-immune therapies are inefficacious.Objectives: Our aim was to deepen our understanding of the immune mechanisms involved in inclusion body myositis and identify specific biomarkers.Methods: Using a panel of thirty-six markers and mass cytometry, we performed deep immune profiling of peripheral blood cells from inclusion body myositis patients and healthy donors, divided into two cohorts: test and validation cohorts. Potential biomarkers were compared to myositis controls (anti-Jo1-, anti-3-hydroxyl-3-methylglutaryl CoA reductase-, and anti-signal recognition particle-positive patients).Results: Unsupervised analyses revealed substantial changes only within CD8+ cells. We observed an increase in the frequency of CD8+ cells that expressed high levels of T-bet, and containing mainly both effector and terminally differentiated memory cells. The senescent marker CD57 was overexpressed in CD8+T-bet+ cells of inclusion body myositis patients. As expected, senescent CD8+T-bet+ CD57+ cells of both patients and healthy donors were CD28nullCD27nullCD127null. Surprisingly, non-senescent CD8+T-bet+ CD57- cells in inclusion body myositis patients expressed lower levels of CD28, CD27, and CD127, and expressed higher levels of CD38 and HLA-DR compared to healthy donors. Using classification and regression trees alongside receiver operating characteristics curves, we identified and validated a frequency of CD8+T-bet+ cells >51.5% as a diagnostic biomarker specific to inclusion body myositis, compared to myositis control patients, with a sensitivity of 94.4%, a specificity of 88.5%, and an area under the curve of 0.97.Conclusion: Using a panel of thirty-six markers by mass cytometry, we identify an activated cell population (CD8+T-bet+ CD57- CD28lowCD27lowCD127low CD38+ HLA-DR+) which could play a role in the physiopathology of inclusion body myositis, and identify CD8+T-bet+ cells as a predominant biomarker of this disease

Dissecting Fabry disease biological plasticity using network-based metabolic phenotyping

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Parsing Fabry Disease Metabolic Plasticity Using Metabolomics

International audienceBackground: Fabry disease (FD) is an X-linked lysosomal disease due to a deficiency in the activity of the lysosomal α-galactosidase A (GalA), a key enzyme in the glycosphingolipid degradation pathway. FD is a complex disease with a poor genotype–phenotype correlation. FD could involve kidney, heart or central nervous system impairment that significantly decreases life expectancy. The advent of omics technologies offers the possibility of a global, integrated and systemic approach well-suited for the exploration of this complex disease. Materials and Methods: Sixty-six plasmas of FD patients from the French Fabry cohort (FFABRY) and 60 control plasmas were analyzed using liquid chromatography and mass spectrometry-based targeted metabolomics (188 metabolites) along with the determination of LysoGb3 concentration and GalA enzymatic activity. Conventional univariate analyses as well as systems biology and machine learning methods were used. Results: The analysis allowed for the identification of discriminating metabolic profiles that unambiguously separate FD patients from control subjects. The analysis identified 86 metabolites that are differentially expressed, including 62 Glycerophospholipids, 8 Acylcarnitines, 6 Sphingomyelins, 5 Aminoacids and 5 Biogenic Amines. Thirteen consensus metabolites were identified through network-based analysis, including 1 biogenic amine, 2 lysophosphatidylcholines and 10 glycerophospholipids. A predictive model using these metabolites showed an AUC-ROC of 0.992 (CI: 0.965–1.000). Conclusion: These results highlight deep metabolic remodeling in FD and confirm the potential of omics-based approaches in lysosomal diseases to reveal clinical and biological associations to generate pathophysiological hypotheses