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

Prosaposin deficiency (pSap-d) and saposin B deficiency (SapB-d) are both lipid storage disorders caused by mutations in the PSAP gene that codes for the 65-70 kDa prosaposin protein, which is the precursor for four sphingolipid activator proteins, saposins A-D. We report on two new patients with PSAP gene defects; one, with pSap-d, who had a severe neurovisceral dystrophy and died as a neonate, and the other with SapB-d, who presented with a metachromatic leukodystrophy-like disorder but had normal arylsulfatase activity. Screening for urinary sphingolipids was crucial to the diagnosis of both patients, with electrospray ionization tandem mass spectrometry also providing quantification. The pSap-d patient is the first case with this condition where urinary sphingolipids have been investigated. Multiple sphingolipids were elevated, with globotriaosylceramide showing the greatest increase. Both patients had novel mutations in the PSAP gene. The pSap-d patient was homozygous for a splice-acceptor site mutation two bases upstream of exon 10. This mutation led to a premature stop codon and yielded low levels of transcript. The SapB-d patient was a compound heterozygote with a splice-acceptor site variant exclusively affecting the SapB domain on one allele, and a 2 bp deletion leading to a null, that is, pSap-d mutation, on the other allele. Phenotypically, pSap-d is a relatively uniform disease of the neonate, whereas SapB-d is heterogeneous with a spectrum similar to that in metachromatic leukodystrophy. The possible existence of genotypes and phenotypes intermediate between those of pSap-d and the single saposin deficiencies is speculated.

• MeSH
• Child MeSH
• Heterozygote MeSH
• Homozygote MeSH
• Infant MeSH
• Skin pathology   MeSH
• Humans MeSH
• Magnetic Resonance Imaging MeSH
• Leukodystrophy, Metachromatic genetics   metabolism   pathology   MeSH
• RNA Splice Sites genetics   MeSH
• Brain abnormalities   pathology   MeSH
• Mutation * MeSH
• DNA Mutational Analysis MeSH
• Codon, Nonsense MeSH
• Infant, Newborn MeSH
• Child, Preschool MeSH
• Saposins deficiency   genetics   MeSH
• Sequence Deletion MeSH
• Sphingolipids urine   MeSH
• Check Tag
• Child MeSH
• Infant MeSH
• Humans MeSH
• Male MeSH
• Infant, Newborn MeSH
• Child, Preschool MeSH
• Publication type
• Journal Article MeSH
• Case Reports MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA Splice Sites MeSH
• Codon, Nonsense MeSH
• PSAP protein, human MeSH Browser
• Saposins MeSH
• Sphingolipids MeSH

A neuropathologic study of three cases of prosaposin (pSap) deficiency (ages at death 27, 89 and 119 days), carried out in the standard autopsy tissues, revealed a neurolysosomal pathology different from that in the non-neuronal cells. Non-neuronal storage is represented by massive lysosomal accumulation of glycosphingolipids (glucosyl-, galactosyl-, lactosyl-, globotriaosylceramides, sulphatide, and ceramide). The lysosomes in the central and peripheral neurons were distended by pleomorphic non-lipid aggregates lacking specific staining and autofluorescence. Lipid storage was borderline in case 1, and at a low level in the other cases. Neurolysosomal storage was associated with massive ubiquitination, which was absent in the non-neuronal cells and which did not display any immunohistochemical aggresomal properties. Confocal microscopy and cross-correlation function analyses revealed a positive correlation between the ubiquitin signal and the late endosomal/lysosomal markers. We suppose that the neuropathology most probably reflects excessive influx of non-lipid material (either in bulk or as individual molecules) into the neurolysosomes. The cortical neurons appeared to be uniquely vulnerable to pSap deficiency. Whereas in case 1 they populated the cortex, in cases 2 and 3 they had been replaced by dense populations of both phagocytic microglia and astrocytes. We suggest that this massive neuronal loss reflects a cortical neuronal survival crisis precipitated by the lack of pSap. The results of our study may extend the knowledge of the neurotrophic function of pSap, which should be considered essential for the survival and maintenance of human cortical neurons.

• MeSH
• Antigens, Differentiation, Myelomonocytic metabolism   MeSH
• Antigens, CD metabolism   MeSH
• Child MeSH
• Glial Fibrillary Acidic Protein metabolism   MeSH
• Glycosphingolipids metabolism   MeSH
• GPI-Linked Proteins MeSH
• Humans MeSH
• Lysosomal Storage Diseases metabolism   pathology   MeSH
• Lysosomes metabolism   pathology   MeSH
• Cell Adhesion Molecules, Neuronal metabolism   MeSH
• Cerebral Cortex pathology   MeSH
• Neurons pathology   ultrastructure   MeSH
• Infant, Newborn MeSH
• Autopsy MeSH
• Saposins deficiency   physiology   MeSH
• Ubiquitin metabolism   MeSH
• Check Tag
• Child MeSH
• Humans MeSH
• Male MeSH
• Infant, Newborn MeSH
• Publication type
• Journal Article MeSH
• Case Reports MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antigens, Differentiation, Myelomonocytic MeSH
• Antigens, CD MeSH
• CD68 antigen, human MeSH Browser
• Glial Fibrillary Acidic Protein MeSH
• Glycosphingolipids MeSH
• GPI-Linked Proteins MeSH
• limbic system-associated membrane protein MeSH Browser
• Cell Adhesion Molecules, Neuronal MeSH
• PSAP protein, human MeSH Browser
• Saposins MeSH
• Ubiquitin MeSH

Gaucher disease (GD), deficiency of acid glucosylceramidase (GlcCer-ase) is characterized by deficient degradation of beta-glucosylceramide (GlcCer). It is well known that, in GD, the lysosomal accumulation of uncleaved GlcCer is limited to macrophages, which are gradually converted to storage cells with well known cytology--Gaucher cells (GCs). On the basis of previous studies of the disorder and of a comparison with other lysosomal enzymopathies affecting degradation of the GlcCer-based glycosphingolipid series, it is hypothesized that in other cell types (i.e. non-macrophage cells) the uncleaved GlcCer, in GlcCer-ase deficiency, is transferred to other cell compartments, where it may be processed and even accumulated to various degrees. The consequence of the abnormal extralysosomal load may differ according to the cell type and compartment targeted and may be influenced by genetically determined factors, by a number of acquired conditions, including the current metabolic situation. The sequelae of the uncleaved GlcCer extralysosomal transfer may range from probably innocent or positive stimulatory, to the much more serious, in which it interferes with a variety of cell functions, and in extreme cases, can lead to cell death. This alternative processing of uncleaved GlcCer may help to explain tissue alterations seen in GD that have, so far, resisted explanation based simply on the presence of GCs. Paralysosomal alternative processing may thus go a long way towards filling a long-standing gap in the understanding of the molecular pathology of the disorder. The impact of this alternative process will most likely be inversely proportional to the level of residual GlcCer-ase activity. Lysosomal sequestration of GlcCer in these cells is either absent or in those exceptional cases where it does occur, it is exceptional and rudimentary. It is suggested that paralysosomal alternative processing of uncleaved GlcCer is the main target for enzyme replacement therapy. The mechanism responsible for GlcCer transfer remains to be elucidated. It may also help in explaining the so far unclear origin of glucosylsphingosine (GlcSph) and define the mutual relation between these two processes.

• MeSH
• Models, Biological MeSH
• Biological Transport MeSH
• Glucosylceramidase deficiency   therapeutic use   MeSH
• Glucosylceramides metabolism   MeSH
• Humans MeSH
• Lysosomes chemistry   metabolism   MeSH
• Macrophages metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Glucosylceramidase MeSH
• Glucosylceramides MeSH

Immunohistochemical studies of the presence of lactosylceramide (LacCer) in lysosomal storage disorders (LSDs) were done using anti-LacCer monoclonal antibody of the CDw 17 type (clone MG-2). No sign of an association between LacCer and the lysosomal system in normal cells was observed, except for histiocytes active in phagocytosis. A comparative study of a group of LSDs showed a general tendency for LacCer to increase in storage cells in Niemann-Pick disease type C (NPC), and types A and B, GM1 gangliosidosis, acid lipase deficiency, glycogen storage disease type II and mucopolysaccharidoses. LacCer accumulated in storage cells despite normal activity of relevant lysosomal degrading enzymes. The accumulation of LacCer displayed variability within storage cell populations, and was mostly expressed in neurons in NPC. An absence of the increase in LacCer in storage cells above control levels was seen in neuronal ceroid lipofuscinoses (neurons and cardiocytes) and in Fabry disease. Gaucher and Krabbe cells showed significantly lower levels, or even the absence, of LacCer compared with control macrophages. Results of immunohistochemistry were corroborated by semiquantitative lipid thin-layer chromatography (TLC). It is suggested that different associations of LacCer with the lysosomal storage process may reflect differences in glycosphingolipid turnover induced by the storage-compromised lysosomal/endosomal system.

• MeSH
• Biomarkers analysis   MeSH
• Antigens, CD analysis   metabolism   MeSH
• Chromatography, Thin Layer methods   MeSH
• Child MeSH
• Adult MeSH
• Histiocytes chemistry   metabolism   pathology   MeSH
• Immunohistochemistry methods   MeSH
• Liver chemistry   metabolism   pathology   MeSH
• Lactosylceramides analysis   metabolism   MeSH
• Humans MeSH
• Lysosomal Storage Diseases classification   metabolism   pathology   MeSH
• Macrophages chemistry   metabolism   pathology   MeSH
• Cerebral Cortex chemistry   metabolism   pathology   MeSH
• Neurons chemistry   metabolism   pathology   MeSH
• Spleen chemistry   metabolism   pathology   MeSH
• Check Tag
• Child MeSH
• Adult MeSH
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• Biomarkers MeSH
• Antigens, CD MeSH
• CDw17 antigen MeSH Browser
• Lactosylceramides MeSH