Parsing Fabry Disease Metabolic Plasticity Using Metabolomics

Background: 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.

Biallelic and de novo variants in ATP6V0A1 cause progressive myoclonus epilepsy and developmental and epileptic encephalopathy

The vacuolar H+-ATPase is a large multi-subunit proton pump, composed of an integral membrane V0 domain, involved in proton translocation, and a peripheral V1 domain, catalysing ATP hydrolysis. This complex is widely distributed on the membrane of various subcellular organelles, such as endosomes and lysosomes, and plays a critical role in cellular processes ranging from autophagy to protein trafficking and endocytosis. Here we identified 17 individuals from 14 unrelated families with variants in ATP6V0A1, the brain-enriched isoform in the V0 domain. Five affected subjects carried biallelic variants in this gene and presented with a phenotype of early-onset progressive myoclonus epilepsy with ataxia, while 12 individuals were found as de novo cases (missense variants) and showed severe developmental and epileptic encephalopathy. We describe that the disease-associated variants lead to failure of lysosomal hydrolysis by directly impairing acidification of the endolysosomal compartment. The R740Q mutation, which alone accounts for almost 50% of the variants identified in this cohort, causes autophagic dysfunction and a severe developmental defect in C. elegans. Altogether, our findings establish a novel cause of lysosomal disease and provide a direct link with endolysosomal acidification in the pathophysiology of these conditions.

Plasma Proteomic Analysis in Morquio A Disease

Mucopolysaccharidosis type IVA (MPS IVA) is a lysosomal disease caused by mutations in the gene encoding the enzymeN-acetylgalactosamine-6-sulfate sulfatase (GALNS), and is characterized by systemic skeletal dysplasia due to excessive storage of keratan sulfate (KS) and chondroitin-6-sulfate in chondrocytes. Although improvements in the activity of daily living and endurance tests have been achieved with enzyme replacement therapy (ERT) with recombinant human GALNS, recovery of bone lesions and bone growth in MPS IVA has not been demonstrated to date. Moreover, no correlation has been described between therapeutic efficacy and urine levels of KS, which accumulates in MPS IVA patients. The objective of this study was to assess the validity of potential biomarkers proposed by other authors and to identify new biomarkers. To identify candidate biomarkers of this disease, we analyzed plasma samples from healthy controls (n=6) and from untreated (n=8) and ERT-treated (n=5, sampled before and after treatment) MPS IVA patients using both qualitative and quantitative proteomics analyses. The qualitative proteomics approach analyzed the proteomic profile of the different study groups. In the quantitative analysis, we identified/quantified 215 proteins after comparing healthy control untreated, ERT-treated MPSIVA patients. We selected a group of proteins that were dysregulated in MPS IVA patients. We identified four potential protein biomarkers, all of which may influence bone and cartilage metabolism: fetuin-A, vitronectin, alpha-1antitrypsin, and clusterin. Further studies of cartilage and bone samples from MPS IVA patients will be required to verify the validity of these proteins as potential biomarkers of MPS IVA.

No differences in native T1 of the renal cortex between Fabry patients and healthy volunteers in clinically acquired native T1 maps by cardiovascular magnetic resonance

Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Karolinska Institutet Swedish Heart and Lung foundation Introduction Fabry disease (FD) is a lysosomal disease that causes accumulation of sphingolipids, which untreated may leadto hypertrophic cardiomyopathyand renal failure. Cardiovascular magnetic resonance imaging (CMR) can detect sphingolipid accumulationin the heart, using native T1 mapping. The kidneys are often visible in clinically acquired native T1 maps, however it is currently unknown if clinically acquired native T1 maps of the heart also can be used to detect sphingolipid accumulation in the kidneysin FD patients. Purpose To evaluate if clinically acquired native T1 maps using CMR can be used to detect sphingolipid accumulation in the kidneysin FD patients. Methods FD patients (n = 18, 41 ± 10 years, 44 % male) and healthy volunteers (n = 41, 26 ± 5 years, 49 % male) were retrospectively enrolled. Native T1 maps were acquired with a 1.5 T scanner (Magnetom Aera, Siemens Healthineers, Erlangen, Germany) usinga modified look locker inversion recovery (MOLLI) sequence with a 5s(3s)3s sampling scheme (Siemens WIP 1041). The native T1 maps were analysed using Segment (Medviso AB, Lund, Sweden). Native T1 values were measured by manually delineating regions of interest (ROI), conservatively placed with a minimum gap of 1 pixel between adjacent structures, in the renal cortex, renal medulla, myocardium, spleen, blood, and liver. Renal cortex ROIs were delineated in all slices where the renal cortex was visible and averaged across all slices. Renal medulla, spleen, and liver ROIs were drawn in the slice where most parenchyma was visible. Endo- and epicardial borders were delineated in all slices of the myocardium and averaged across all slices. Blood ROIs were placed in the midventricular slice, Figure 1. Results There were no differences in native T1 values between the patients and the healthy volunteers in the renal cortex (1034 ± 88 vs 1038 ± 51 ms, p = 0.89), blood (1632 ± 123 vs 1600 ± 104 ms, p = 0.94), spleen (1143 ± 45 vs 1134 ± 77 ms, p = 0.64) or liver (569 ± 49 vs 576 ± 45 ms, p = 0.57), and did not change when analysed with regards to sex, Figure 2. Native T1-values were lower in the myocardium of the patients compared to the healthy volunteers (937 ± 53 vs 1019 ± 35 ms, p = 0.01), and higher in the renal medulla (1635 ± 144 vs 1523 ± 70 ms, p = 0.01). Conclusions Compared to healthy volunteers, patients with FD and myocardial involvement have no differences in native T1 of the renal cortex. FD patients have higher native T1 in the renal medulla, which cannot be explained by differences in blood native T1. The findings suggest that clinically acquired native T1-maps cannot be used to detect sphingolipid accumulation in the renal cortex in FD patients.

The role of Niemann-Pick type C2 in zebrafish embryonic development

Niemann-Pick disease type C (NPC) is a rare, fatal, neurodegenerative lysosomal disease caused by mutations of either NPC1 or NPC2. NPC2 is a soluble lysosomal protein which functions in coordination with NPC1 to efflux cholesterol from the lysosomal compartment. Mutations of either gene result in the accumulation of unesterified cholesterol and other lipids in the late endosome/lysosome, while reducing cellular cholesterol bioavailability. Zygotic null npc2m/m zebrafish showed significant unesterified cholesterol accumulation at larval stages, a reduction in body size, and motor and balance defects in adulthood. However, the phenotype at embryonic stages was milder than expected, suggesting a possible role of maternal Npc2 in embryonic development. Maternal-zygotic npc2m/m zebrafish exhibited significant developmental defects including defective otic vesicle development/absent otoliths, abnormal head/brain development, curved/twisted body axes, no circulating blood cells, and died by 72 hpf. RNA-seq analysis conducted on 30 hpf npc2+/m and MZnpc2m/m embryos revealed a significant reduction in the expression of notch3 and other downstream genes in the Notch signaling pathway, suggesting that impaired Notch3 signaling underlies aspects of the developmental defects observed in MZnpc2m/m zebrafish.

Mucopolysaccharidosis Type I: Current Treatments, Limitations, and Prospects for Improvement

Mucopolysaccharidosis type I (MPS I) is a lysosomal disease, caused by a deficiency of the enzyme alpha-L-iduronidase (IDUA). IDUA catalyzes the degradation of the glycosaminoglycans dermatan and heparan sulfate (DS and HS, respectively). Lack of the enzyme leads to pathologic accumulation of undegraded HS and DS with subsequent disease manifestations in multiple organs. The disease can be divided into severe (Hurler syndrome) and attenuated (Hurler-Scheie, Scheie) forms. Currently approved treatments consist of enzyme replacement therapy (ERT) and/or hematopoietic stem cell transplantation (HSCT). Patients with attenuated disease are often treated with ERT alone, while the recommended therapy for patients with Hurler syndrome consists of HSCT. While these treatments significantly improve disease manifestations and prolong life, a considerable burden of disease remains. Notably, treatment can partially prevent, but not significantly improve, clinical manifestations, necessitating early diagnosis of disease and commencement of treatment. This review discusses these standard therapies and their impact on common disease manifestations in patients with MPS I. Where relevant, results of animal models of MPS I will be included. Finally, we highlight alternative and emerging treatments for the most common disease manifestations.

Decrease in Myelin-Associated Lipids Precedes Neuronal Loss and Glial Activation in the CNS of the Sandhoff Mouse as Determined by Metabolomics

Sandhoff disease (SD) is a lysosomal disease caused by mutations in the gene coding for the β subunit of β-hexosaminidase, leading to deficiency in the enzymes β-hexosaminidase (HEX) A and B. SD is characterised by an accumulation of gangliosides and related glycolipids, mainly in the central nervous system, and progressive neurodegeneration. The underlying cellular mechanisms leading to neurodegeneration and the contribution of inflammation in SD remain undefined. The aim of the present study was to measure global changes in metabolism over time that might reveal novel molecular pathways of disease. We used liquid chromatography-mass spectrometry and 1H Nuclear Magnetic Resonance spectroscopy to profile intact lipids and aqueous metabolites, respectively. We examined spinal cord and cerebrum from healthy and Hexb−/− mice, a mouse model of SD, at ages one, two, three and four months. We report decreased concentrations in lipids typical of the myelin sheath, galactosylceramides and plasmalogen-phosphatidylethanolamines, suggesting that reduced synthesis of myelin lipids is an early event in the development of disease pathology. Reduction in neuronal density is progressive, as demonstrated by decreased concentrations of N-acetylaspartate and amino acid neurotransmitters. Finally, microglial activation, indicated by increased amounts of myo-inositol correlates closely with the late symptomatic phases of the disease.

Clinical experience of replacing enzyme replacement therapy in a patient with mucopolysaccharidosis type II

Mucopolysaccharidosis type II (MPS II, Hunter syndrome) is an inherited chronic progressive lysosomal disease associated with recessive X-linked inheritance. MPS II is classified as an orphan disease and occurs at a rate of 1.3 per 100,000 white boys. Hunter syndrome is the most common type of mucopolysaccharidosis, accounting for about 50% of MPS types. The diseases pathogenesis is based on a violation of the stepwise cleavage of glycosaminoglycans (GAG) heparansulfate and dermatansulfate caused by a deficiency of the iduronate-2-sulfatase enzyme encoded by theIDSgene. The existing deficiency or complete absence of iduronate-2-sulfatase leads to a violation of the final stage of glycosaminoglycan catabolism and the accumulation heparansulfate and dermatansulfate in all organs and tissues. Currently, there are two drugs registered in the Russian Federation for pathogenetic enzyme replacement therapy of MPS: idursulfase and idursulfase beta. This refers to the expansion of the therapeutic options for Hunter syndrome patients in the event of severe adverse events. It allows choosing the treatment regimen that will be optimal for the patient and will significantly improve the quality of life. In this article, the authors share their own experience of changing enzyme replacement therapy in an MPS II child patient.

Misfolding of Lysosomal α-Galactosidase a in a Fly Model and Its Alleviation by the Pharmacological Chaperone Migalastat

Fabry disease, an X-linked recessive lysosomal disease, results from mutations in the GLA gene encoding lysosomal α-galactosidase A (α-Gal A). Due to these mutations, there is accumulation of globotriaosylceramide (GL-3) in plasma and in a wide range of cells throughout the body. Like other lysosomal enzymes, α-Gal A is synthesized on endoplasmic reticulum (ER) bound polyribosomes, and upon entry into the ER it undergoes glycosylation and folding. It was previously suggested that α-Gal A variants are recognized as misfolded in the ER and undergo ER-associated degradation (ERAD). In the present study, we used Drosophila melanogaster to model misfolding of α-Gal A mutants. We did so by creating transgenic flies expressing mutant α-Gal A variants and assessing development of ER stress, activation of the ER stress response and their relief with a known α-Gal A chaperone, migalastat. Our results showed that the A156V and the A285D α-Gal A mutants underwent ER retention, which led to activation of unfolded protein response (UPR) and ERAD. UPR could be alleviated by migalastat. When expressed in the fly’s dopaminergic cells, misfolding of α-Gal A and UPR activation led to death of these cells and to a shorter life span, which could be improved, in a mutation-dependent manner, by migalastat.