Hematopoietic stem cell gene therapy improves outcomes in a clinically relevant mouse model of multiple sulfatase deficiency.

Multiple sulfatase deficiency (MSD) is a severe, lysosomal storage disorder caused by pathogenic variants in the gene SUMF1, encoding the sulfatase modifying factor formylglycine-generating enzyme. Patients with MSD exhibit functional deficiencies in all cellular sulfatases. The inability of sulfatases to break down their substrates leads to progressive and multi-systemic complications in patients, similar to those seen in single-sulfatase disorders such as metachromatic leukodystrophy and mucopolysaccharidoses IIIA. Here, we aimed to determine if hematopoietic stem cell transplantation with ex vivo SUMF1 lentiviral gene therapy could improve outcomes in a clinically relevant mouse model of MSD. We first tested our approach in MSD patient-derived cells and found that our SUMF1 lentiviral vector improved protein expression, sulfatase activities, and glycosaminoglycan accumulation. In vivo, we found that our gene therapy approach rescued biochemical deficits, including sulfatase activity and glycosaminoglycan accumulation, in affected organs of MSD mice treated post-symptom onset. In addition, treated mice demonstrated improved neuroinflammation and neurocognitive function. Together, these findings suggest that SUMF1 HSCT-GT can improve both biochemical and functional disease markers in the MSD mouse.

A novel iPSC model reveals selective vulnerability of neurons in multiple sulfatase deficiency.

Multiple sulfatase deficiency (MSD) is an ultra-rare, inherited lysosomal storage disease caused by mutations in the gene sulfatase modifying factor 1 (SUMF1). MSD is characterized by the functional deficiency of all sulfatase enzymes, leading to the storage of sulfated substrates including glycosaminoglycans (GAGs), sulfolipids, and steroid sulfates. Patients with MSD experience severe neurological impairment, hearing loss, organomegaly, corneal clouding, cardiac valve disease, dysostosis multiplex, contractures, and ichthyosis. Here, we generated a novel human model of MSD by reprogramming patient peripheral blood mononuclear cells to establish an MSD induced pluripotent stem cell (iPSC) line (SUMF1 p.A279V). We also generated an isogenic control iPSC line by correcting the pathogenic variant with CRISPR/Cas9 gene editing. We successfully differentiated these iPSC lines into neural progenitor cells (NPCs) and NGN2-induced neurons (NGN2-iN) to model the neuropathology of MSD. Mature neuronal cells exhibited decreased SUMF1 gene expression, increased lysosomal stress, impaired neurite outgrowth and maturation, reduced sulfatase activities, and GAG accumulation. Interestingly, MSD iPSCs and NPCs did not exhibit as severe of phenotypes, suggesting that as neurons differentiate and mature, they become more vulnerable to loss of SUMF1. In summary, we demonstrate that this human iPSC-derived neuronal model recapitulates the cellular and biochemical features of MSD. These cell models can be used as tools to further elucidate the mechanisms of MSD pathology and for the development of therapeutics.

Biochemical signatures of disease severity in multiple sulfatase deficiency.

Sulfatases catalyze essential cellular reactions, including degradation of glycosaminoglycans (GAGs). All sulfatases are post-translationally activated by the formylglycine generating enzyme (FGE) which is deficient in multiple sulfatase deficiency (MSD), a neurodegenerative lysosomal storage disease. Historically, patients were presumed to be deficient of all sulfatase activities; however, a more nuanced relationship is emerging. Each sulfatase may differ in their degree of post-translational modification by FGE, which may influence the phenotypic spectrum of MSD. Here, we evaluate if residual sulfatase activity and accumulating GAG patterns distinguish cases from controls and stratify clinical severity groups in MSD. We quantify sulfatase activities and GAG accumulation using three complementary methods in MSD participants. Sulfatases differed greatly in their tolerance of reduction in FGE-mediated activation. Enzymes that degrade heparan sulfate (HS) demonstrated lower residual activities than those that act on other GAGs. Similarly, HS-derived urinary GAG subspecies preferentially accumulated, distinguished cases from controls, and correlated with disease severity. Accumulation patterns of specific sulfatase substrates in MSD provide fundamental insights into sulfatase regulation and will serve as much-needed biomakers for upcoming clinical trials. This work highlights that biomarker investigation of an ultra-rare disease can simultaneously inform our understanding of fundamental biology and advance clinical trial readiness efforts.

New mouse models with hypomorphic SUMF1 variants mimic attenuated forms of multiple sulfatase deficiency.

Multiple sulfatase deficiency (MSD) is an ultrarare lysosomal storage disorder due to deficiency of all known sulfatases. MSD is caused by mutations in the Sulfatase Modifying Factor 1 (SUMF1) gene encoding the enzyme responsible for the post-translational modification and activation of all sulfatases. Most MSD patients carry hypomorph SUMF1 variants resulting in variable degrees of residual sulfatase activities. In contrast, Sumf1 null mice with complete deficiency in all sulfatase enzyme activities, have very short lifespan with significant pre-wean lethality, owing to a challenging preclinical model. To overcome this limitation, we genetically engineered and characterized in mice two commonly identified patient-based SUMF1 pathogenic variants, namely p.Ser153Pro and p.Ala277Val. These pathogenic missense variants correspond to variants detected in patients with attenuated MSD presenting with partial-enzyme deficiency and relatively less severe disease. These novel MSD mouse models have a longer lifespan and show biochemical and pathological abnormalities observed in humans. In conclusion, mice harboring the p.Ser153Pro or the p.Ala277Val variant mimic the attenuated MSD and are attractive preclinical models for investigation of pathogenesis and treatments for MSD.

A systematic review and meta-analysis of published cases reveals the natural disease history in multiple sulfatase deficiency.

Multiple Sulfatase Deficiency (MSD, MIM#272200) is an ultra-rare lysosomal storage disorder arising from mutations in the SUMF1 gene, which encodes the formylglycine-generating enzyme (FGE). FGE is necessary for the activation of sulfatases, a family of enzymes that are involved in the degradation of sulfated substrates such as glycosaminoglycans and sulfolipids. SUMF1 mutations lead to functionally impaired FGE and individuals with MSD demonstrate clinical signs of single sulfatase deficiencies, including metachromatic leukodystrophy (MLD) and several mucopolysaccharidosis (MPS) subtypes. Comprehensive information related to the natural history of MSD is missing. We completed a systematic literature review and a meta-analysis on data from published cases reporting on MSD. As available from these reports, we extracted clinical, genetic, biochemical, and brain imaging information. We identified 75 publications with data on 143 MSD patients with a total of 53 unique SUMF1 mutations. The mean survival was 13 years (95% CI 9.8-16.2 years). Seventy-five clinical signs and 11 key clusters of signs were identified. The most frequently affected organs systems were the nervous, skeletal, and integumentary systems. The most frequent MRI features were abnormal myelination and cerebral atrophy. Individuals with later onset MSD signs and survived longer than those with signs at birth. Less severe mutations, low disease burden and achievement of independent walking positively correlated with longer survival. Despite the limitations of our approach, we were able to define clinical characteristics and disease outcomes in MSD. This work will provide the foundation of natural disease history data needed for future clinical trial design.

Natural history of multiple sulfatase deficiency: Retrospective phenotyping and functional variant analysis to characterize an ultra-rare disease.

Multiple sulfatase deficiency (MSD) is an ultra-rare neurodegenerative disorder caused by pathogenic variants in SUMF1. This gene encodes formylglycine-generating enzyme (FGE), a protein required for sulfatase activation. The clinical course of MSD results from additive effect of each sulfatase deficiency, including metachromatic leukodystrophy (MLD), several mucopolysaccharidoses (MPS II, IIIA, IIID, IIIE, IVA, VI), chondrodysplasia punctata, and X-linked ichthyosis. While it is known that affected individuals demonstrate a complex and severe phenotype, the genotype-phenotype relationship and detailed clinical course is unknown. We report on 35 cases enrolled in our retrospective natural history study, n = 32 with detailed histories. Neurologic function was longitudinally assessed with retrospective scales. Biochemical and computational modeling of novel SUMF1 variants was performed. Genotypes were classified based on predicted functional change, and each individual was assigned a genotype severity score. The median age at symptom onset was 0.25 years; median age at diagnosis was 2.7 years; and median age at death was 13 years. All individuals demonstrated developmental delay, and only a subset of individuals attained ambulation and verbal communication. All subjects experienced an accumulating systemic symptom burden. Earlier age at symptom onset and severe variant pathogenicity correlated with poor neurologic outcomes. Using retrospective deep phenotyping and detailed variant analysis, we defined the natural history of MSD. We found that attenuated cases can be distinguished from severe cases by age of onset, attainment of ambulation, and genotype. Results from this study can help inform prognosis and facilitate future study design.

Multiple Sulfatase Deficiency: A Disease Comprising Mucopolysaccharidosis, Sphingolipidosis, and More Caused by a Defect in Posttranslational Modification.

Multiple sulfatase deficiency (MSD, MIM #272200) is an ultra-rare disease comprising pathophysiology and clinical features of mucopolysaccharidosis, sphingolipidosis and other sulfatase deficiencies. MSD is caused by impaired posttranslational activation of sulfatases through the formylglycine generating enzyme (FGE) encoded by the sulfatase modifying factor 1 (SUMF1) gene, which is mutated in MSD. FGE is a highly conserved, non-redundant ER protein that activates all cellular sulfatases by oxidizing a conserved cysteine in the active site of sulfatases that is necessary for full catalytic activity. SUMF1 mutations result in unstable, degradation-prone FGE that demonstrates reduced or absent catalytic activity, leading to decreased activity of all sulfatases. As the majority of sulfatases are localized to the lysosome, loss of sulfatase activity induces lysosomal storage of glycosaminoglycans and sulfatides and subsequent cellular pathology. MSD patients combine clinical features of all single sulfatase deficiencies in a systemic disease. Disease severity classifications distinguish cases based on age of onset and disease progression. A genotype- phenotype correlation has been proposed, biomarkers like excreted storage material and residual sulfatase activities do not correlate well with disease severity. The diagnosis of MSD is based on reduced sulfatase activities and detection of mutations in SUMF1. No therapy exists for MSD yet. This review summarizes the unique FGE/ sulfatase physiology, pathophysiology and clinical aspects in patients and their care and outlines future perspectives in MSD.

Complex care of individuals with multiple sulfatase deficiency: Clinical cases and consensus statement.

Multiple sulfatase deficiency (MSD) is an ultra-rare neurodegenerative disorder that results in defective sulfatase post-translational modification. Sulfatases in the body are activated by a unique protein, formylglycine-generating enzyme (FGE) that is encoded by SUMF1. When FGE is absent or insufficient, all 17 known human sulfatases are affected, including the enzymes associated with metachromatic leukodystrophy (MLD), several mucopolysaccharidoses (MPS II, IIIA, IIID, IVA, VI), chondrodysplasia punctata, and X-linked ichthyosis. As such, individuals demonstrate a complex and severe clinical phenotype that has not been fully characterized to date. In this report, we describe two individuals with distinct clinical presentations of MSD. Also, we detail a comprehensive systems-based approach to the management of individuals with MSD, from the initial diagnostic evaluation to unique multisystem issues and potential management options. As there have been no natural history studies to date, the recommendations within this report are based on published studies and consensus opinion and underscore the need for future research on evidence-based outcomes to improve management of children with MSD.

A152T tau allele causes neurodegeneration that can be ameliorated in a zebrafish model by autophagy induction.

Mutations in the gene encoding tau (MAPT) cause frontotemporal dementia spectrum disorders. A rare tau variant p.A152T was reported as a risk factor for frontotemporal dementia spectrum and Alzheimer's disease in an initial case-control study. Such findings need replication in an independent cohort. We analysed an independent multinational cohort comprising 3100 patients with neurodegenerative disease and 4351 healthy control subjects and found p.A152T associated with significantly higher risk for clinically defined frontotemporal dementia and progressive supranuclear palsy syndrome. To assess the functional and biochemical consequences of this variant, we generated transgenic zebrafish models expressing wild-type or A152T-tau, where A152T caused neurodegeneration and proteasome compromise. Impaired proteasome activity may also enhance accumulation of other proteins associated with this variant. We increased A152T clearance kinetics by both pharmacological and genetic upregulation of autophagy and ameliorated the disease pathology observed in A152T-tau fish. Thus, autophagy-upregulating therapies may be a strategy for the treatment for tauopathies.

Expanding the genetic cause of multiple sulfatase deficiency: A novel SUMF1 variant in a patient displaying a severe late infantile form of the disease.

Multiple sulfatase deficiency (MSD) is a rare inherited metabolic disease caused by defective cellular sulfatases. Activity of sulfatases depends on post-translational modification catalyzed by formylglycine-generating enzyme (FGE), encoded by the SUMF1 gene. SUMF1 pathologic variants cause MSD, a syndrome presenting with a complex phenotype. We describe the first Polish patient with MSD caused by a yet undescribed pathologic variant c.337G>A [p.Glu113Lys] (i.e. p.E113K) in heterozygous combination with the known deletion allele c.519+5_519+8del [p.Ala149_Ala173del]. The clinical picture of the patient initially suggested late infantile metachromatic leukodystrophy, with developmental delay followed by regression of visual, hearing and motor abilities as the most apparent clinical symptoms. Transient signs of ichthyosis and minor dysmorphic features guided the laboratory workup towards MSD. Since MSD is a rare disease and there is a variable clinical spectrum, we thoroughly describe the clinical outcome of our patient. The FGE-E113K variant, expressed in cell culture, correctly localized to the endoplasmic reticulum but was retained intracellularly in contrast to the wild type FGE. Analysis of FGE-mediated activation of steroid sulfatase in immortalized MSD cells revealed that FGE-E113K exhibited only approx. 15% of the activity of wild type FGE. Based on the crystal structure we predict that the exchange of glutamate-113 against lysine should induce a strong destabilization of the secondary structure, possibly affecting the folding for correct disulfide bridging between C235-C346 as well as distortion of the active site groove that could affect both the intracellular stability as well as the activity of FGE. Thus, the novel variant of the SUMF1 gene obviously results in functionally impaired FGE protein leading to a severe late infantile type of MSD.

Bone marrow transplantation increases sulfatase activity in somatic tissues in a multiple sulfatase deficiency mouse model.

BACKGROUND: Multiple Sulfatase Deficiency (MSD) is an ultra-rare autosomal recessive disorder characterized by deficient enzymatic activity of all known sulfatases. MSD patients frequently carry two loss of function mutations in the SUMF1 gene, encoding a formylglycine-generating enzyme (FGE) that activates 17 different sulfatases. MSD patients show common features of other lysosomal diseases like mucopolysaccharidosis and metachromatic leukodystrophy, including neurologic impairments, developmental delay, and visceromegaly. There are currently no approved therapies for MSD patients. Hematopoietic stem cell transplant (HSCT) has been applied with success in the treatment of certain lysosomal diseases. In HSCT, donor-derived myeloid cells are a continuous source of active sulfatase enzymes that can be taken up by sulfatase-deficient host cells. Thus, HSCT could be a potential approach for the treatment of MSD. METHODS: To test this hypothesis, we used a clinically relevant mouse model for MSD, B6-Sumf1(S153P/S153P) mice, engrafted with bone marrow cells, Sumf1+/+, from B6-PtprcK302E mice (CD45.1 immunoreactive). RESULTS: After 10 months post-transplant, flow cytometric analysis shows an average of 90% of circulating leukocytes of donor origin (Sumf1(+/+)). Enzymatic activity for ARSA, ARSB, and SGSH is significantly increased in spleen of B6-Sumf1(S153P/S153P) recipient mice. In non-lymphoid organs, only liver and heart show a significant correction of sulfatase activity and GAG accumulation. Frequency of inflammatory cells and lysosomal pathology is significantly reduced in liver and heart, while no significant improvement is detected in brain. CONCLUSIONS: Our results indicate that HSCT could be a suitable approach to treat MSD-pathology affecting peripheral organs, however that benefit to CNS pathology might be limited.

Multiple Sulfatase Deficiency (MSD) is a rare genetic disorder caused by loss-of-function variations in the SUMF1 gene. This deficiency results in the accumulation of toxic compounds, leading to developmental delays and neurological impairments. In a bone marrow transplant (BMT), donor cells are infused into the patient and secrete active proteins that can help remove those toxic compounds. We carried out BMT in a mouse model for MSD and saw beneficial effects on peripheral organs, such as the liver and heart, but less change in neurological symptoms. Our results will be useful for the design of potential cell therapy approaches that could be used clinically to treat MSD.

Effective Gene Therapy for Metachromatic Leukodystrophy Achieved with Minimal Lentiviral Genomic Integrations.

Metachromatic leukodystrophy (MLD) is a fatal lysosomal storage disease (LSD) characterized by the deficient enzymatic activity of arylsulfatase A (ARSA). Combined autologous hematopoietic stem cell transplant (HSCT) with lentiviral (LV) based gene therapy has great potential to treat MLD. However, if enzyme production is inadequate, this could result in continued loss of motor function, implying a high vector copy number (VCN) requirement for optimal enzymatic output. This may place children at increased risk for genomic toxicity due to higher VCN. We increased the expression of ARSA cDNA at single integration by generating novel LVs, optimizing ARSA expression, and enhancing safety. In addition, our vectors achieved optimal transduction in mouse and human HSC with minimal multiplicity of infection (MOI). Our top-performing vector (EA1) showed at least 4X more ARSA activity than the currently EU-approved vector and a superior ability to secrete vesicle-associated ARSA, a critical modality to transfer functional enzymes from microglia to oligodendrocytes. Three-month-old Arsa -KO MLD mice transplanted with Arsa -KO BM cells transduced with 0.6 VCN of EA1 demonstrated behavior and CNS histology matching WT mice. Our novel vector boosts efficacy while improving safety as a robust approach for treating early symptomatic MLD patients.

An inducible expression system for the manipulation of autophagic flux in vivo.

Much of our understanding of the intracellular regulation of macroautophagy/autophagy comes from in vitro studies. However, there remains a paucity of knowledge about how this process is regulated within different tissues during development, aging and disease in vivo. Because upregulation of autophagy is considered a promising therapeutic strategy for the treatment of diverse disorders, it is vital that we understand how this pathway functions in different tissues and this is best done by in vivo analysis. Similarly, to understand the role of autophagy in the pathogenesis of disease, it is important to study this process in the whole animal to investigate how tissue-specific changes in flux and cell-autonomous versus non-cell-autonomous effects alter disease progression. To this end, we have developed an inducible expression system to up- or downregulate autophagy in vivo, in zebrafish. We have used a modified version of the Gal4-UAS expression system to allow inducible expression of autophagy up- or downregulating transgenes by addition of tamoxifen. Using this inducible expression system, we have tested which transgenes robustly up- or downregulate autophagy and have validated these tools using Lc3-II blots and puncta analysis and disease rescue in a zebrafish model of neurodegeneration. These tools allow the temporal control of autophagy via the administration of tamoxifen and spatial control via tissue or cell-specific ERT2-Gal4 driver lines and will enable the investigation of how cell- or tissue-specific changes in autophagic flux affect processes such as aging, inflammation and neurodegeneration in vivo.Abbreviations: ANOVA: analysis of variance; Atg: autophagy related; Bcl2l11/Bim: BCL2 like 11; d.p.f.: days post-fertilization; Cryaa: crystallin, alpha a: DMSO: dimethyl sulfoxide; Elavl3: ELAV like neuron-specific RNA binding protein 3; ER: estrogen receptor; ERT2: modified ligand-binding domain of human ESR1/estrogen receptor α; Gal4: galactose-responsive transcription factor 4; GFP: green fluorescent protein; h.p.f.: hours post-fertilization; HSP: heat-shock protein; Map1lc3/Lc3: microtubule-associated protein 1 light chain 3; RFP: red fluorescent protein; SD: standard deviation; SEM: standard error of the mean; UAS: upstream activating sequence; Ubb: ubiquitin b.

Multiple Sulfatase Deficiency from an Ophthalmologist's Perspective-Case Report and Literature Review.

Multiple sulfatase deficiency (MSD) is an extremely rare autosomal recessively inherited disease with a prevalence of 1:500.000 caused by mutations on the sulfatase-modifying-Factor 1 gene (SUMF1). MSD is most specifically characterised by a combination of developmentally retarded psychomotoric functions, neurodegeneration that entails the loss of many already acquired abilities, and by ichthyosis. Other symptoms include those associated with mucopolysaccharidosis, i.e., facial dysmorphy, dwarfism, and hepatosplenomegaly. In 50-75% of all MSD-affected patients, functional or structural ocular damage is likely. MSD seldom affects the anterior segment of the eye. The main pathology these patients present is a highly conspicuous tapetoretinal degeneration, similar to severe Retinitis pigmentosa, that leads to blindness at an early age. An initially five-year-old boy with MSD, genetically verified at his first examination in our opthalmology department (SUMF1 mutations c.776A>T, p.Asn259Ile; c.797A>T, p.Pro266Leu; c.836A>T, p.Ala279Val), and a 4, 5 year regular follow-up are described. The patient had some visual potential ("tunnel view"), which deteriorated dramatically after his fifth birthday. We observed no evidence of worsening retinal involvement in this patient in spite of his progressively worsening clinical symptoms, extending to total blindness/no light perception. OCT revealed that the outer retinal layers containing photoreceptors were diseased; the ellipsoid zone was only partially discernible and the outer nuclear layer appeared to be thinned out. The inner nuclear layer, ganglion cell layer, and retinal nerve fibre layer were indistinguishable. These anomalies are indicative of a severe pathology within the retina's inner layers. Characteristic anomalies in the fundus should stimulate clinicians to suspect a case of MSD in their differential diagnosis, and thus to order thorough genetic and paediatric diagnostics.

Drug screening identifies tazarotene and bexarotene as therapeutic agents in multiple sulfatase deficiency.

Multiple sulfatase deficiency (MSD, MIM #272200) results from pathogenic variants in the SUMF1 gene that impair proper function of the formylglycine-generating enzyme (FGE). FGE is essential for the posttranslational activation of cellular sulfatases. MSD patients display reduced or absent sulfatase activities and, as a result, clinical signs of single sulfatase disorders in a unique combination. Up to date therapeutic options for MSD are limited and mostly palliative. We performed a screen of FDA-approved drugs using immortalized MSD patient fibroblasts. Recovery of arylsulfatase A activity served as the primary readout. Subsequent analysis confirmed that treatment of primary MSD fibroblasts with tazarotene and bexarotene, two retinoids, led to a correction of MSD pathophysiology. Upon treatment, sulfatase activities increased in a dose- and time-dependent manner, reduced glycosaminoglycan content decreased and lysosomal position and size normalized. Treatment of MSD patient derived induced pluripotent stem cells (iPSC) differentiated into neuronal progenitor cells (NPC) resulted in a positive treatment response. Tazarotene and bexarotene act to ultimately increase the stability of FGE variants. The results lay the basis for future research on the development of a first therapeutic option for MSD patients.

Unexpected Phenotype Reversion and Survival in a Zebrafish Model of Multiple Sulfatase Deficiency.

Multiple sulfatase deficiency (MSD) is a rare recessively inherited Mendelian disorder that manifests with developmental delay, neurodegeneration, skeletal deformities, facial dysmorphism, congenital growth retardation, and other clinical signs. The disorder is caused by mutations in the SUMF1 gene, which encodes the formylglycine-generating enzyme (FGE), and responsible for the activation of sulfatases. Mutations in SUMF1 result in reduced or absent FGE function with consequent compromised activities of its client sulfatases. This leads to an accumulation of enzyme substrates, such as glycosaminoglycans and sulfolipids, within lysosomes and subsequently impaired lysosome function and cellular pathology. Currently, there are no disease modifying therapeutic options for MSD patients, hence the need for more suitable animal models to investigate the disorder. Here, we describe the characterisation of a sumf1 null zebrafish model, which has negligible sulfatase activity. Our sumf1 -/- zebrafish model successfully recapitulates the pathology of MSD such as cranial malformation, altered bone development, an enlarged population of microglia, and growth retardation during early development but lacks early lethality of mouse Sumf1 -/- models. Notably, we provide evidence of recovery in MSD pathology during later developmental stages, resulting in homozygous mutants that are viable. Hence, our data suggest the possibility of a unique compensatory mechanism that allows the sumf1 -/- null zebrafish to survive better than human MSD patients and mouse Sumf1 -/- models.

Bi-allelic VPS16 variants limit HOPS/CORVET levels and cause a mucopolysaccharidosis-like disease.

Lysosomal storage diseases, including mucopolysaccharidoses, result from genetic defects that impair lysosomal catabolism. Here, we describe two patients from two independent families presenting with progressive psychomotor regression, delayed myelination, brain atrophy, neutropenia, skeletal abnormalities, and mucopolysaccharidosis-like dysmorphic features. Both patients were homozygous for the same intronic variant in VPS16, a gene encoding a subunit of the HOPS and CORVET complexes. The variant impaired normal mRNA splicing and led to an ~85% reduction in VPS16 protein levels in patient-derived fibroblasts. Levels of other HOPS/CORVET subunits, including VPS33A, were similarly reduced, but restored upon re-expression of VPS16. Patient-derived fibroblasts showed defects in the uptake and endosomal trafficking of transferrin as well as accumulation of autophagosomes and lysosomal compartments. Re-expression of VPS16 rescued the cellular phenotypes. Zebrafish with disrupted vps16 expression showed impaired development, reduced myelination, and a similar accumulation of lysosomes and autophagosomes in the brain, particularly in glia cells. This disorder resembles previously reported patients with mutations in VPS33A, thus expanding the family of mucopolysaccharidosis-like diseases that result from mutations in HOPS/CORVET subunits.

Molecular basis of multiple sulfatase deficiency, mucolipidosis II/III and Niemann-Pick C1 disease - Lysosomal storage disorders caused by defects of non-lysosomal proteins.

Multiple sulfatase deficiency (MSD), mucolipidosis (ML) II/III and Niemann-Pick type C1 (NPC1) disease are rare but fatal lysosomal storage disorders caused by the genetic defect of non-lysosomal proteins. The NPC1 protein mainly localizes to late endosomes and is essential for cholesterol redistribution from endocytosed LDL to cellular membranes. NPC1 deficiency leads to lysosomal accumulation of a broad range of lipids. The precise functional mechanism of this membrane protein, however, remains puzzling. ML II, also termed I cell disease, and the less severe ML III result from deficiencies of the Golgi enzyme N-acetylglucosamine 1-phosphotransferase leading to a global defect of lysosome biogenesis. In patient cells, newly synthesized lysosomal proteins are not equipped with the critical lysosomal trafficking marker mannose 6-phosphate, thus escaping from lysosomal sorting at the trans Golgi network. MSD affects the entire sulfatase family, at least seven members of which are lysosomal enzymes that are specifically involved in the degradation of sulfated glycosaminoglycans, sulfolipids or other sulfated molecules. The combined deficiencies of all sulfatases result from a defective post-translational modification by the ER-localized formylglycine-generating enzyme (FGE), which oxidizes a specific cysteine residue to formylglycine, the catalytic residue enabling a unique mechanism of sulfate ester hydrolysis. This review gives an update on the molecular bases of these enigmatic diseases, which have been challenging researchers since many decades and so far led to a number of surprising findings that give deeper insight into both the cell biology and the pathobiochemistry underlying these complex disorders. In case of MSD, considerable progress has been made in recent years towards an understanding of disease-causing FGE mutations. First approaches to link molecular parameters with clinical manifestation have been described and even therapeutical options have been addressed. Further, the discovery of FGE as an essential sulfatase activating enzyme has considerable impact on enzyme replacement or gene therapy of lysosomal storage disorders caused by single sulfatase deficiencies.

Mannose phosphate isomerase deficiency-congenital disorder of glycosylation (MPI-CDG) with cerebral venous sinus thrombosis as first and only presenting symptom: A rare but treatable cause of thrombophilia.

Mannose phosphate isomerase deficiency-congenital disorder of glycosylation (MPI-CDG; formerly named CDG type 1b) is characterized by the clinical triad of hepatopathy, protein-losing enteropathy, and hyperinsulinemic hypoglycemia in combination with coagulation disorder (thrombophilia, depletion of antithrombin, proteins C and S, factor XI). In the majority of patients, MPI-CDG manifests during early infancy or childhood. Here, we present a 15-year-old female patient with unremarkable medical history suffering from acute cerebral venous sinus thrombosis necessitating interventional thrombectomy and neurosurgical decompression. Diagnostic work-up of thrombophilia revealed deficiency of antithrombin (AT), proteins C and S, and factor XI. Detailed evaluation identified MPI-CDG as the underlying cause of disease. After initiation of mannose therapy, coagulation parameters normalized. The girl fully recovered without any neurologic sequelae, and remains free of further thrombotic events or any other clinical and laboratory abnormalities on follow-up 1 year after start of mannose treatment. In conclusion, we here present the significant case of MPI-CDG with a severe cerebral venous sinus thrombosis as the first and only symptom of the disease. In light of the high frequency of AT deficiency on one hand, and the excellent treatability of MPI-CDG on the other hand, CDG screening should be included as a routine analysis in all patients presenting with unexplained coagulation disorder, especially when comprising AT deficiency.

Zebrafish disease model of human RNASET2-deficient cystic leukoencephalopathy displays abnormalities in early microglia.

Human infantile-onset RNASET2-deficient cystic leukoencephalopathy is a Mendelian mimic of in utero cytomegalovirus brain infection with prenatally developing inflammatory brain lesions. We used an RNASET2-deficient zebrafish model to elucidate the underlying disease mechanisms. Mutant and wild-type zebrafish larvae brain development between 2 and 5 days post fertilization (dpf) was examined by confocal live imaging in fluorescent reporter lines of the major types of brain cells. In contrast to wild-type brains, RNASET2-deficient larvae displayed increased numbers of microglia with altered morphology, often containing inclusions of neurons. Furthermore, lysosomes within distinct populations of the myeloid cell lineage including microglia showed increased lysosomal staining. Neurons and oligodendrocyte precursor cells remained unaffected. This study provides a first look into the prenatal onset pathomechanisms of human RNASET2-deficient leukoencephalopathy, linking this inborn lysosomal disease to the innate immune system and other immune-related childhood encephalopathies like Aicardi-Goutières syndrome (AGS).