KEY POINTS: The clinical significance of a number of missense variants of α-galactosidase A is often ambiguous. Defective proteostasis of some missense α-galactosidase A variants induced chronic endoplasmic reticulum stress and the unfolded protein response. Endoplasmic reticulum stress and the unfolded protein response may explain clinical manifestations of non-classic Fabry disease. BACKGROUND: Classic Fabry disease is caused by GLA mutations that result in loss of enzymatic activity of α-galactosidase A, lysosomal storage of globotriaosylceramide, and a resulting multisystemic disease. In non-classic Fabry disease, patients have some preserved α-galactosidase A activity and a milder disease course. Heterozygous female patients may also be affected. While Fabry disease pathogenesis has been mostly attributed to catalytic deficiency of mutated α-galactosidase A, lysosomal storage, and impairment of lysosomal functions, other pathogenic factors may contribute, especially in nonclassic Fabry disease. METHODS: We characterized the genetic, clinical, biochemical, molecular, cellular, and organ pathology correlates of the p.L394P α-galactosidase A variant that was identified initially in six individuals with kidney failure by the Czech national screening program for Fabry disease and by further screening in an additional 24 family members. RESULTS: Clinical findings in affected male patients revealed a milder clinical course, with approximately 15% residual α-galactosidase A activity with normal plasma lyso-globotriaosylceramide levels and abnormally low ratio of these values. None of the four available kidney biopsies showed lysosomal storage. Laboratory investigations documented intracellular retention of mutated α-galactosidase A with resulting endoplasmic reticulum stress and the unfolded protein response, which were alleviated with BRD4780, a small molecule clearing misfolded proteins from the early secretory compartment. We observed similar findings of endoplasmic reticulum stress and unfolded protein response in five kidney biopsies with several other classic and non-classic Fabry disease missense α-galactosidase A variants. CONCLUSIONS: We identified defective proteostasis of mutated α-galactosidase A resulting in chronic endoplasmic reticulum stress and unfolded protein response of α-galactosidase A expressing cells as a contributor to Fabry disease pathogenesis.

• Publication type
• Journal Article MeSH

Farber disease (FD) and spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME) are ultra-rare lysosomal storage disorders caused by deficient acid ceramidase (ACDase) activity. Although both conditions are caused by mutations in the ASAH1 gene, clinical presentations differ considerably. FD patients usually die in childhood, while SMA-PME patients can live until adulthood. There is no treatment for FD or SMA-PME. Hematopoietic stem cell transplantation (HSCT) and gene therapy strategies for the treatment of ACDase deficiency are being investigated. We have previously generated and characterized mouse models of both FD and SMA-PME that recapitulate the symptoms described in patients. Here, we show that HSCT improves lifespan, behavior, hematopoietic system anomalies, and plasma cytokine levels and significantly reduces histiocytic infiltration and ceramide accumulation throughout the tissues investigated, including the CNS, in both models of ACDase-deficient mice. HSCT was also successful in preventing lesion development and significant demyelination of the spinal cord seen in SMA-PME mice. Importantly, we note that only early and generally pre-symptomatic treatment was effective, and kidney impairment was not improved in either model.

• Keywords
• Farber disease, HSCT, Lysosomal storage disorders, central nervous system, ceramides, spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME),
• MeSH
• Ceramides metabolism   MeSH
• Farber Lipogranulomatosis * therapy   genetics   MeSH
• Acid Ceramidase * genetics   metabolism   MeSH
• Humans MeSH
• Spinal Cord metabolism   pathology   MeSH
• Disease Models, Animal MeSH
• Myoclonic Epilepsies, Progressive genetics   therapy   metabolism   MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Hematopoietic Stem Cell Transplantation * methods   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Asah1 protein, mouse MeSH Browser
• Ceramides MeSH
• Acid Ceramidase * MeSH

Acid ceramidase catalyzes the degradation of ceramide into sphingosine and a free fatty acid. Acid ceramidase deficiency results in lipid accumulation in many tissues and leads to the development of Farber disease (FD). Typical manifestations of classical FD include formation of subcutaneous nodules and joint contractures as well as the development of a hoarse voice. Healthy skin depends on a unique lipid profile to form a barrier that confers protection from pathogens, prevents excessive water loss, and mediates cell-cell communication. Ceramides comprise ~50% of total epidermis lipids and regulate cutaneous homeostasis and inflammation. Abnormal skin development including visual skin lesions has been reported in FD patients, but a detailed study of FD skin has not been performed. We conducted a pathophysiological study of the skin in our mouse model of FD. We observed altered lipid composition in FD skin dominated by accumulation of all studied ceramide species and buildup of abnormal storage structures affecting mainly the dermis. A deficiency of acid ceramidase activity also led to the activation of inflammatory IL-6/JAK/signal transducer and activator of transcription 3 and noncanonical NF-κB signaling pathways. Last, we report reduced proliferation of FD mouse fibroblasts and adipose-derived stem/stromal cells (ASC) along with impaired differentiation of ASCs into mature adipocytes.

• Keywords
• Farber disease, acid ceramidase, adipogenesis, ceramides, macrophages, skin,
• MeSH
• Adipogenesis MeSH
• Ceramides metabolism   MeSH
• Farber Lipogranulomatosis * MeSH
• Acid Ceramidase genetics   MeSH
• Disease Models, Animal MeSH
• Mice MeSH
• Inflammation MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Ceramides MeSH
• Acid Ceramidase MeSH

Mucolipidosis type IV (MLIV) is a lysosomal storage disease exhibiting progressive intellectual disability, motor impairment, and premature death. There is currently no cure or corrective treatment. The disease results from mutations in the gene encoding mucolipin-1, a transient receptor potential channel believed to play a key role in lysosomal calcium egress. Loss of mucolipin-1 and subsequent defects lead to a host of cellular aberrations, including accumulation of glycosphingolipids (GSLs) in neurons and other cell types, microgliosis and, as reported here, cerebellar Purkinje cell loss. Several studies have demonstrated that N-butyldeoxynojirimycin (NB-DNJ, also known as miglustat), an inhibitor of the enzyme glucosylceramide synthase (GCS), successfully delays the onset of motor deficits, improves longevity, and rescues some of the cerebellar abnormalities (e.g., Purkinje cell death) seen in another lysosomal disease known as Niemann-Pick type C (NPC). Given the similarities in pathology between MLIV and NPC, we examined whether miglustat would be efficacious in ameliorating disease progression in MLIV. Using a full mucolipin-1 knockout mouse (Mcoln1-/-), we found that early miglustat treatment delays the onset and progression of motor deficits, delays cerebellar Purkinje cell loss, and reduces cerebellar microgliosis characteristic of MLIV disease. Quantitative mass spectrometry analyses provided new data on the GSL profiles of murine MLIV brain tissue and showed that miglustat partially restored the wild type profile of white matter enriched lipids. Collectively, our findings indicate that early miglustat treatment delays the progression of clinically relevant pathology in an MLIV mouse model, and therefore supports consideration of miglustat as a therapeutic agent for MLIV disease in humans.

• Keywords
• Glycosphingolipids, Lysosomal storage disease, Miglustat, Mucolipidosis type IV, Mucolipin-1, Purkinje cells, Small molecule therapy,
• MeSH
• 1-Deoxynojirimycin analogs & derivatives   therapeutic use   MeSH
• Antigens, CD metabolism   MeSH
• Gliosis drug therapy   etiology   MeSH
• Enzyme Inhibitors therapeutic use   MeSH
• Transient Receptor Potential Channels genetics   metabolism   MeSH
• Lipid Metabolism drug effects   genetics   MeSH
• Disease Models, Animal MeSH
• Cerebellum pathology   MeSH
• Mucolipidoses * complications   genetics   pathology   MeSH
• Mice, Inbred C57BL MeSH
• Mice, Transgenic MeSH
• Mice MeSH
• Exploratory Behavior drug effects   MeSH
• Cell Count MeSH
• Movement Disorders drug therapy   etiology   MeSH
• Nerve Tissue Proteins metabolism   MeSH
• Psychomotor Performance drug effects   MeSH
• Purkinje Cells drug effects   pathology   MeSH
• Retina pathology   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 1-Deoxynojirimycin MeSH
• Antigens, CD MeSH
• Enzyme Inhibitors MeSH
• Transient Receptor Potential Channels MeSH
• Mcoln1 protein, mouse MeSH Browser
• miglustat MeSH Browser
• Nerve Tissue Proteins MeSH

BACKGROUND: Prediagnostic steps in suspected metachromatic leukodystrophy (MLD) rely on clinical chemical methods other than enzyme assays. We report a new diagnostic method which evaluates changes in the spectrum of molecular types of sulfatides (3-O-sulfogalactosyl ceramides) in MLD urine. METHODS: The procedure allows isolation of urinary sulfatides by solid-phase extraction on DEAE-cellulose membranes, transportation of a dry membrane followed by elution and tandem mass spectrometry (MS/MS) analysis in the clinical laboratory. Major sulfatide isoforms are normalized to the least variable component of the spectrum, which is the indigenous C18:0 isoform. This procedure does not require the use of specific internal standards and minimizes errors caused by sample preparation and measurement. RESULTS: Urinary sulfatides were analyzed in a set of 21 samples from patients affected by sulfatidosis. The combined abundance of the five most elevated isoforms, C22:0, C22:0-OH, C24:0, C24:1-OH, and C24:0-OH sulfatides, was found to give the greatest distinction between MLD-affected patients and a control group. CONCLUSIONS: The method avoids transportation of liquid urine samples and generates stable membrane-bound sulfatide samples that can be stored at ambient temperature. MS/MS sulfatide profiling targeted on the most MLD-representative isoforms is simple with robust results and is suitable for screening.

• Keywords
• ASA, CV, DEAE, DEAE-cellulose membrane, DUS, Diethylaminoethyl, Dry urinary samples, IPN, Isoforms, MLD, MS/MS, PTFE, Psap-d, S/N, SRM, Screening for metachromatic leukodystrophy, Tandem mass spectrometry, Urinary sulfatide, arylsulfatase A, coefficient of variation, dry urinary sample, isoform profile number (ratio of the sum of the major five isoforms and the C18:0 sulfatide), metachromatic leukodystrophy, polytetrafluoroethylene, prosaposin deficiency, selected reaction monitoring, signal to noise ratio, tandem mass spectrometry,
• MeSH
• DEAE-Cellulose MeSH
• Child MeSH
• Solid Phase Extraction MeSH
• Infant MeSH
• Middle Aged MeSH
• Humans MeSH
• Membranes, Artificial MeSH
• Leukodystrophy, Metachromatic diagnosis   urine   MeSH
• Adolescent MeSH
• Specimen Handling standards   MeSH
• Child, Preschool MeSH
• Reference Standards MeSH
• Case-Control Studies MeSH
• Sulfoglycosphingolipids urine   MeSH
• Tandem Mass Spectrometry MeSH
• Desiccation MeSH
• Check Tag
• Child MeSH
• Infant MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Male MeSH
• Child, Preschool MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• DEAE-Cellulose MeSH
• Membranes, Artificial MeSH
• Sulfoglycosphingolipids MeSH

The aim of this retrospective study was to determine the prevalence of lysosomal storage disorders (LSDs) in the Czech Republic. The data on cases diagnosed between 1975 and 2008 were collected and analyzed. The overall prevalence of LSDs in the Czech population (12.25 per 100,000) is comparable to that reported for the countries with well-established and advanced diagnostics of LSDs such as the Netherlands (14 per 100,000), Australia (12.9 per 100,000) and Italy (12.1 per 100,000). Relatively higher prevalence of LSDs was reported in the north of Portugal (25 per 100,000). Thirty-four different LSDs were diagnosed in a total of 478 individuals. Gaucher disease was the most frequent LSD with a birth prevalence of 1.13 per 100,000 births. The most frequent LSD groups were lipidoses, mucopolysaccharidoses, and neuronal ceroid lipofuscinoses, with combined prevalences of 5.0, 3.72, and 2.29 per 100,000 live births, respectively. Glycoproteinoses (0.57 per 100,000 live births), glycogenosis type II (0.37), and mucolipidoses (0.31) rarely occur in the Czech population, and a range of other LSDs have not been detected at all over the past three decades. Knowledge of the birth prevalence and carrier frequency of particular disorders is important in genetic counselling for calculation of the risk for the disorder in the other members of affected families. Earlier diagnosis of these disorders will permit timely intervention and may also result in lowering of the number of newborns with LSDs.

• MeSH
• Genetic Predisposition to Disease epidemiology   MeSH
• Genetic Counseling MeSH
• Heterozygote MeSH
• Humans MeSH
• Lysosomal Storage Diseases epidemiology   genetics   MeSH
• Infant, Newborn MeSH
• Prevalence MeSH
• Retrospective Studies MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Infant, Newborn MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Geographicals
• Australia epidemiology   MeSH
• Czech Republic epidemiology   MeSH
• Italy epidemiology   MeSH
• Netherlands epidemiology   MeSH
• Portugal epidemiology   MeSH