galactocerebroside and Leukodystrophy--Globoid-Cell

To establish cut-off values of lysosomal storage disease (LSD)-related enzymes by tandem mass spectrometry.. A total of 26 689 newborns and 7 clinically confirmed LSD children underwent screening for LSDs (glycogen storage disease typeⅡ, Fabry disease, mucopolysaccharidosis type Ⅰ, Krabbe disease, Niemann-Pick disease A/B and Gaucher disease). The activities of LSD-related enzymes were detected by tandem mass spectrometry. The 20% of the median enzyme activity of each batch of acid β-glucocerebrosidase, acid sphingomyelinase, β-galactocerebroside, α-. The enzyme activities of 7 clinically confirmed cases were all lower than the cut-off values. Among 26 689 newborns, 142 cases (0.53%) were suspected positive for LSDs, including 25 cases of β-galactocerebroside deficiency, 1 case of α-. The established cut-off values of LSD-related enzyme activities detected by tandem mass spectrometry can be used for screening LSDs in neonates, and the enzyme activity would be affected by temperature and humidity.

Topics: alpha-Galactosidase; alpha-Glucosidases; Child; Fabry Disease; Galactosylceramides; Glucosylceramidase; Glycogen Storage Disease Type II; Humans; Iduronidase; Infant; Infant, Newborn; Leukodystrophy, Globoid Cell; Lysosomal Storage Diseases; Neonatal Screening; Sphingomyelin Phosphodiesterase; Tandem Mass Spectrometry

Krabbe's disease (KD) is a degenerative lysosomal storage disease resulting from deficiency of β-galactocerebrosidase activity. Over 100 mutations are known to cause the disease, and these usually occur in compound heterozygote patterns. In affected patients, nonsense mutations leading to a nonfunctional enzyme are often found associated with other mutations. The twitcher mouse is a naturally occurring model of KD, containing in β-galactocerebrosidase a premature stop codon, W339X. Recent studies have shown that selected compounds may induce the ribosomal bypass of premature stop codons without affecting the normal termination codons. The rescue of β-galactocerebrosidase activity induced by treatment with premature termination codon (PTC) 124, a well-characterized compound known to induce ribosomal read-through, was investigated on oligodendrocytes prepared from twitcher mice and on human fibroblasts from patients bearing nonsense mutations. The effectiveness of the nonsense-mediated mRNA decay (NMD) inhibitor 1 (NMDI1), a newly identified inhibitor of NMD, was also tested. Incubation of these cell lines with PTC124 and NMDI1 increased the levels of mRNA and rescued galactocerebrosidase enzymatic activity in a dose-dependent manner. The low but sustained expression of β-galactocerebrosidase in oligodendrocytes was sufficient to improve the morphology of the differentiated cells. Our in vitro approach provides the basis for further investigation of ribosomal read-through as an alternative therapeutic strategy to ameliorate the quality of life in selected KD patients. © 2016 Wiley Periodicals, Inc.

Topics: Animals; Animals, Newborn; Cell Line, Transformed; Codon, Terminator; Disease Models, Animal; Dose-Response Relationship, Drug; Fibroblasts; Galactosylceramidase; Galactosylceramides; Humans; Leukodystrophy, Globoid Cell; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Nonsense Mediated mRNA Decay; Oligodendroglia; Oxadiazoles; RNA, Messenger

This chapter describes in detail a practical procedure for the preparation of radiolabeled galactocerebroside and its use in the assay of galactocerebrosidase (GalCase), the enzyme deficient in globoid cell leukodystrophy (Krabbe disease). The reference range for leukocytes and fibroblasts is 0.9-4.4 and 8-36 nmoles substrate hydrolyzed per hour per milligram of protein, respectively. Because of its low abundance this enzyme is difficult to assay in certain situations, such as prenatal diagnosis by chorionic villus sampling. To obviate this a modified assay is used where only the radiolabeled substrate is included in the incubation. This provides a clear separation between affected samples and unaffected controls. The methods detailed here should be reproducible in any laboratory.

Topics: Cells, Cultured; Female; Fibroblasts; Galactose Oxidase; Galactosylceramidase; Galactosylceramides; Humans; Leukodystrophy, Globoid Cell; Oxidation-Reduction; Pregnancy; Prenatal Diagnosis; Tritium

The pigmentary type of orthochromatic leukodystrophy (van Bogaert-Nyssen disease) is a hardly known neurological disorder usually with late onset that is very difficult to diagnose in vivo. Neuropathologically, the disorder features noninflammatory demyelination and the presence of pigmented macrophages and astrocytes that may contain iron. Clinically, van Bogaert-Nyssen disease can lead to death within a few years and is characterized by dementia, psychiatric abnormalities, epileptic seizures, spastic pareses, and occasionally extrapyramidal motor symptoms. This report presents a typical case and an overview of the literature. Furthermore, galactocerebroside could be documented in remaining macrophages and astrocytes by immunohistochemistry. This possibly indicates a dysfunction in sphingolipid breakdown and could relate the pigmented form of orthochromatic leukodystrophy to the genetically defined globoid cell leukodystrophy (Krabbe's disease). Thus, the rather heterogeneous pool of orthochromatic leukodystrophies could be further narrowed.

Topics: Adult; Astrocytes; Brain; Brain Diseases, Metabolic, Inborn; Demyelinating Diseases; Diagnosis, Differential; Fatal Outcome; Female; Galactosylceramides; Humans; Inclusion Bodies; Iron; Leukodystrophy, Globoid Cell; Lipofuscin; Macrophages; Microscopy, Electron; Myelin Sheath; Peripheral Nerves; Sphingolipidoses

Globoid-cell leukodystrophy (GLD) is an autosomal recessive inherited disorder caused by the deficiency of galactocerebrosidase, the lysosomal enzyme responsible for the degradation of the myelin glycolipid galactocerebroside. Although the most common form of the disease is the classical infantile form (Krabbe disease), later-onset forms also have been described. We have analyzed the galactocerebrosidase gene in 17 patients (nine families) with late-onset GLD and in 1 patient with classical Krabbe disease. Half of the patients were heterozygous for the large gene deletion associated with the 502C-->T polymorphism, the most common mutation in infantile patients. Several novel mutations that result in deficient galactocerebrosidase activity were also identified in these patients. They include the missense mutations R63H, G95S, M101L, G268S, Y298C, and I234T; the nonsense mutation S7X; a one-base deletion (805delG); a mutation that interferes with the splicing of intron 1; and a 34-nt insertion in the RNA, caused by the aberrant splicing of intron 6. All of these genetic defects are clustered in the first 10 exons of the galactocerebrosidase gene and therefore affect the 50-kD subunit of the mature enzyme. Studies on the distribution and enzymatic activity of the polymorphic alleles 1637T/C (I546/T546) provided support for previous data that had indicated the existence of two galactocerebrosidase forms with different catalytic activities in the general population. Our data also indicate that the mutations occur preferentially in the "low activity" 1637C allele.

Topics: Adolescent; Age of Onset; Animals; Child; Child, Preschool; COS Cells; Female; Galactosylceramides; Gene Deletion; Heterozygote; Humans; Infant; Leukodystrophy, Globoid Cell; Male; Oligonucleotide Probes; Pedigree; Point Mutation; Polymorphism, Genetic; RNA, Messenger

6-Hexadecanoylamino-4-methylumbelliferyl-beta-D-galactopyranoside (HMGal) has been shown to be a specific fluorogenic substrate of galactocerebrosidase and to facilitate the simple enzymatic diagnosis of Krabbe disease in human patients and in twitcher mice. HMGal hydrolysis at pH 4.5 is optimally stimulated by sodium taurocholate (0.25%) and oleic acid (0.05%) with a Km of 0.150, 0.04 and 0.03 mM, respectively for control mouse kidney, human fibroblasts and leukocytes. In control samples, the specific activity (nmol/mg prot./h) for HMGal is higher than for the natural substrate, galactocerebroside, and is severely deficient in the twitcher mouse and in patients with Krabbe disease. Comparative investigation of galactocerebrosidase activity in fibroblasts, leukocytes and brain with radioactive and fluorogenic substrates reveals a good agreement between the results of the two methods. Galactocerebroside (Gal-Cer) is a competitive inhibitor of HMGal hydrolysis in mouse kidney homogenates while GM1-ganglioside has no inhibitory effect in the same assay system. The sensitivity and specificity of this fluorogenic substrate for galactocerebrosidase provides a simple and rapid method for the diagnosis of Krabbe disease, and for the purification of this enzyme from normal tissues.

Topics: Animals; Brain; Carbohydrate Sequence; Cells, Cultured; Clinical Enzyme Tests; Dose-Response Relationship, Drug; Female; Fibroblasts; Fluorescent Dyes; Galactosides; Galactosylceramidase; Galactosylceramides; Humans; Hydrolysis; Hymecromone; Leukocytes; Leukodystrophy, Globoid Cell; Male; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Oleic Acid; Oleic Acids; Substrate Specificity; Taurocholic Acid

The effects of psychosine on the metabolism of myelin associated glycolipids such as galactocerebroside and sulfatide in mouse brain cell cultures were investigated in order to clarify the mechanism of demyelination in globoid cell leukodystrophy (Krabbe's disease). The incorporation of 3H-galactose into cerebroside and sulfatide was studied in the presence of psychosine (1-3 micrograms/ml medium). These data indicated that psychosine inhibited the incorporation of 3H-galactose into cerebroside and sulfatide not in astroglial cell culture but in oligodendroglial cell culture. Oligodendrocytes produce myelin in the central nervous system, and cerebroside and sulfatide are major components of myelin. These results suggest that psychosine influences the lipid metabolisms of myelin and subsequently leads to the demyelination in Krabbe's disease.

Topics: Animals; Cells, Cultured; Demyelinating Diseases; Galactosylceramides; Leukodystrophy, Globoid Cell; Mice; Oligodendroglia; Psychosine; Sphingosine; Sulfoglycosphingolipids

Enzyme replacement with liposomes containing beta-galactosidase obtained from charonia lumpas was carried out in murine globoid cell leukodystrophy (GLD). Charonia lumpas beta-galactosidase was able to hydrolyze galactocerebroside trapped into liposomes prepared from lecithin, cholesterol and sulfatide (molar ratio; 7:2:1). Liposomes containing charonia lumpas beta-galactosidase were successfully incorporated into the mouse tissues. 3H-galactocerebroside labeled liposomes were also incorporated into mouse liver, spleen and other tissues. The accumulation rate of 3H-galactocerebroside into twithcer mice liver and spleen was almost 40 to 100 times higher than those of controls and degraded to 70 to 80% of accumulated radioactivity of 3H-galactocerebroside by single injection of liposomes containing charonia lumpas beta-galactosidase. Results suggest that exogeneous enzyme trapped in liposomes can be useful for the correction of accumulated compound.

Topics: Animals; Aspergillus oryzae; beta-Galactosidase; Brain; Cattle; Cerebrosides; Disease Models, Animal; Fabaceae; Galactosidases; Galactosylceramides; Hydrolysis; Kidney; Leukodystrophy, Globoid Cell; Liposomes; Liver; Mice; Mice, Neurologic Mutants; Mollusca; Plants, Medicinal; Spleen