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Objectives: Gangliosides (GGs) are considered as diagnostic biomarkers and therapeutic targets and agents. The goal of this study was to develop a tandem mass spectrometry (MS/MS) method for the simultaneous measurement of both GM1 and GM2 gangliosides in human cerebrospinal fluid (CSF) samples in order to be able to determine their concentrations in patients with Tay-Sachs and Sandhoff disease and assess whether drugs or transplantation affect their concentrations., Design and Methods: An API-4000 tandem mass spectrometer equipped with TurboIonSpray source and Shimadzu HPLC system was employed to perform the analysis using isotope dilution with deuterium labeled internal standards. To a 1.5 mL conical plastic Eppendorf centrifuge tube, 40 microL of human CSF sample was added and mixed with 400 microL of internal standard solution for deproteinization. After centrifugation, 100 microL of supernatant was injected onto a C-18 column. After a 2.5 min wash, the switching valve was activated and the analytes were eluted from the column with a water/methanol gradient into the MS/MS system. Quantification by multiple reaction-monitoring (MRM) analysis was performed in the negative mode., Results: The within-day coefficients of variation were <3% for GM1 and <2% for GM2 and the between-day coefficients of variation were <5% for both GM1 and GM2 at all concentrations tested. Accuracy ranged between 98% and 102% for both analytes. Good linearity was also obtained within the concentration range of 10-200 ng/mL (6.5-129.3 nmol/L) for GM1 and 5-100 ng/mL (3.6-72.3 nmol/L) for GM2 (r> or =0.995)., Conclusions: A new simple, accurate, and fast isotope dilution tandem mass spectrometry method was developed for the simultaneous quantification of GM1 and GM2 gangliosides in a small amount of human CSF. Concentrations were measured in "normal" CSF and in CSF from patients with Tay-Sachs disease.

This case report documents clinical and molecular findings in two littermate kittens of the Japanese domestic cat with GM2 gangliosidosis variant 0. Analysis included detailed physical, magnetic resonance imaging, biochemical, pathological and genetic examinations. At first, these littermate kittens showed typical cerebellar signs at approximately 2 months of age. About 2 months later, they progressively showed other neurological signs and subsequently died at about 7 months of age. Magnetic resonance imaging just before the death showed an enlarged ventricular system, T1 hyperintensity in the internal capsule, and T2 hyperintensity in the white matter of the whole brain. Histological findings suggested a type of lysosomal storage disease. Biochemical studies demonstrated that the kittens were affected with GM2 gangliosidosis variant 0, and a DNA assay finally demonstrated that these animals were homozygous for the mutation, which the authors had identified in a different family of the Japanese domestic cat. The findings in the present cases provide useful information about GM2 gangliosidosis variant 0 in Japanese domestic cats.

A five-month-old, female Japanese domestic shorthair cat with proportionate dwarfism developed neurological disorders, including ataxia, decreased postural responses and generalised body and head tremors, at between two and five months of age. Leucocytosis due to lymphocytosis with abnormal cytoplasmic vacuolations was observed. The concentration of G(M2)-ganglioside in its cerebrospinal fluid was markedly higher than in normal cats, and the activities of beta-hexosaminidases A and B in its leucocytes were markedly reduced. On the basis of these biochemical data, the cat was diagnosed antemortem with G(M2)-gangliosidosis variant 0 (Sandhoff-like disease). The neurological signs became more severe and the cat died at 10 months of age. Histopathologically, neurons throughout the central nervous system were distended, and an ultrastructural study revealed membranous cytoplasmic bodies in these distended neurons. The compound which accumulated in the brain was identified as G(M2)-ganglioside, confirming G(M2)-gangliosidosis. A family study revealed that there were probable heterozygous carriers in which the activities of leucocyte beta-hexosaminidases A and B were less than half the normal value. The Sandhoff-like disease observed in this family of Japanese domestic cats is the first occurrence reported in Japan.

In the present study, laboratory techniques were used to diagnose canine GM2-gangliosidosis using blood and cerebrospinal fluid (CSF) that can be collected noninvasively from living individuals. Lysosomal acid beta-hexosaminidase (Hex) was measured spectrofluorometrically using 4-methylumbelliferyl N-acetyl-beta-D-glucosaminide and 4-methylumbelliferyl 7-(6-sulfo-2-acetamido-2-deoxy-beta-D-glucopyranoside) as substrates. Main isoenzymes A and B of Hex in leukocytes were also analyzed using cellulose acetate membrane electrophoresis. GM2-ganglioside in CSF was detected and determined quantitatively by using thin-layer chromatography/enzyme-immunostaining method with anti-GM2-ganglioside antibody. In normal dogs, Hex activities could be determined in leukocytes, serum, and CSF and the total activities were markedly reduced in all the enzyme sources in a dog with Sandhoff disease. Electrophoresis of a leukocyte lysate from a normal dog showed that the Hex A and Hex B were not separated distinctively with formation of a broad band, whereas there were no bands in electrophoresis of a lysate from a dog with Sandhoff disease, showing a deficiency in the total enzyme activity. GM2-ganglioside could be detected and determined quantitatively in as little as 100 microl of canine CSE GM2-ganglioside in CSF in a dog with Sandhoff disease increased to 46 times the normal level. In conclusion, the methods in the present study are useful for diagnosis of canine GM2-gangliosidosis. These techniques enable definitive and early diagnosis of canine GM2-gangliosidosis even if tissues and organs cannot be obtained.

A 35-year-old Japanese man with juvenile Sandhoff disease is described. He showed progressive neurogenic muscular atrophy, cerebellar ataxia and mental deterioration, beginning at age 10 years. The accumulation of GM2 ganglioside in the submucosal nerve cell was confirmed by positive immunostaining using anti-GM2 ganglioside antibody. Biochemical evaluation revealed nearly absent beta-hexosaminidase A and B activities in leukocytes and cultured fibroblasts. Hydrolysis of [3H]globoside I in the intact fibroblasts was apparently disturbed but the rate of hydrolysis was higher than those seen in cells from patients with infantile Sandhoff disease. Analysis of the beta-hexosaminidase beta-subunit gene of the patient disclosed a point mutation (a G-to-A transition) within intron 12. The mutation generates a new splice junction resulting in a 24-base insertion between exons 12 and 13 in the processed mRNA and consequently an 8-amino acid insertion in the translation product. This mutation is identical to that originally found in a Canadian patient with juvenile Sandhoff disease. A possible relationship with the clinical phenotype and the gene abnormality is discussed.

Brain biopsy has been used for the diagnosis of the variant AB of infantile GM2 gangliosidosis. Accumulation of ganglioside GM2 (300 ng of neuraminic acid per milliliter) was observed in the CSF of a patient with this disorder. GM2 was found also in the CSF of a patient with classic Tay-Sachs disease. Normal CSF did not contain any measurable amounts of GM2. In addition, a glycolipid with a mobility, by thin-layer chromatography, similar to that of paragloboside was observed in the CSF of the patient with the variant AB of GM2 gangliosidosis. These findings indicate that the variant AB can be diagnosed by demonstrating accumulation of GM2 in the CSF of patients with normal hexosaminidase activity.

A child with AB variant GM2 gangliosidosis who had progressive intellectual deterioration and seizures commencing at the age of 12 months is described. Neuronal loss, and neuronal and astrocytic inclusions characteristic of the gangliosidoses, were seen on cortical biopsy. GM2 ganglioside was detected in the CSF. As CNS ganglioside accumulation in this condition occurs in the presence of normal leukocyte hexosaminidase A and B levels, spinal fluid assay for GM2 ganglioside may serve as a valuable aid in diagnosis.