Deficiency of the frontotemporal dementia gene GRN results in gangliosidosis.

Haploinsufficiency of GRN causes frontotemporal dementia (FTD). The GRN locus produces progranulin (PGRN), which is cleaved to lysosomal granulin polypeptides. The function of lysosomal granulins and why their absence causes neurodegeneration are unclear. Here we discover that PGRN-deficient human cells and murine brains, as well as human frontal lobes from GRN-mutation FTD patients have increased levels of gangliosides, glycosphingolipids that contain sialic acid. In these cells and tissues, levels of lysosomal enzymes that catabolize gangliosides were normal, but levels of bis(monoacylglycero)phosphates (BMP), lipids required for ganglioside catabolism, were reduced with PGRN deficiency. Our findings indicate that granulins are required to maintain BMP levels to support ganglioside catabolism, and that PGRN deficiency in lysosomes leads to gangliosidosis. Lysosomal ganglioside accumulation may contribute to neuroinflammation and neurodegeneration susceptibility observed in FTD due to PGRN deficiency and other neurodegenerative diseases.

Phenotypical changes of satellite glial cells in a murine model of GM1 -gangliosidosis.

Satellite glial cells (SGCs) of dorsal root ganglia (DRG) react in response to various injuries in the nervous system. This study investigates reactive changes within SGCs in a murine model for GM1 -gangliosidosis (GM1 ). DRG of homozygous ß-galactosidase-knockout mice and homozygous C57BL/6 wild-type mice were investigated performing immunostaining on formalin-fixed, paraffin-embedded tissue. A marked upregulation of glial fibrillary acidic protein (GFAP), the progenitor marker nestin and Ki67 within SGCs of diseased mice, starting after 4 months at the earliest GFAP, along with intracytoplasmic accumulation of ganglioside within neurons and deterioration of clinical signs was identified. Interestingly, nestin-positive SGCs were detected after 8 months only. No changes regarding inwardly rectifying potassium channel 4.1, 2, 3-cyclic nucleotide 3-phosphodiesterase, Sox2, doublecortin, periaxin and caspase3 were observed in SGCs. Iba1 was only detected in close vicinity of SGCs indicating infiltrating or tissue-resident macrophages. These results indicate that SGCs of DRG show phenotypical changes during the course of GM1 , characterized by GFAP upregulation, proliferation and expression of a neural progenitor marker at a late time point. This points towards an important role of SGCs during neurodegenerative disorders and supports that SGCs represent a multipotent glial precursor cell line with high plasticity and functionality.

Biomarkers of central nervous system inflammation in infantile and juvenile gangliosidoses.

BACKGROUND: The gangliosidoses (Tay-Sachs disease, Sandhoff disease, and GM1-gangliosidosis) are progressive neurodegenerative diseases caused by lysosomal enzyme activity deficiencies and consequent accumulation of gangliosides in the central nervous system (CNS). The infantile forms are distinguished from the juvenile forms by age of onset, rate of disease progression, and age of death. There are no approved treatments for the gangliosidoses. In search of potential biomarkers of disease, we quantified 188 analytes in CSF and serum from living human patients with longitudinal (serial) measurements. Notably, several associated with inflammation were elevated in the CSF of infantile gangliosidosis patients, and less so in more slowly progressing forms of juvenile gangliosidosis, but not in MPS disease. Thirteen CSF and two serum biomarker candidates were identified. Five candidate biomarkers were distinguished by persistent elevation in the CSF of patients with the severe infantile phenotype: ENA-78, MCP-1, MIP-1α, MIP-1ß, and TNFR2. Correspondence of abnormal elevation with other variables of disease-i.e., severity of clinical phenotype, differentiation from changes in serum, and lack of abnormality in other neurodegenerative lysosomal diseases-identifies these analytes as biomarkers of neuropathology specific to the gangliosidosis diseases.

Gangliosides and gangliosidoses: principles of molecular and metabolic pathogenesis.

Gangliosides are the main glycolipids of neuronal plasma membranes. Their surface patterns are generated by coordinated processes, involving biosynthetic pathways of the secretory compartments, catabolic steps of the endolysosomal system, and intracellular trafficking. Inherited defects in ganglioside biosynthesis causing fatal neurodegenerative diseases have been described so far almost exclusively in mouse models, whereas inherited defects in ganglioside catabolism causing various clinical forms of GM1- and GM2-gangliosidoses have long been known. For digestion, gangliosides are endocytosed and reach intra-endosomal vesicles. At the level of late endosomes, they are depleted of membrane-stabilizing lipids like cholesterol and enriched with bis(monoacylglycero)phosphate (BMP). Lysosomal catabolism is catalyzed at acidic pH values by cationic sphingolipid activator proteins (SAPs), presenting lipids to their respective hydrolases, electrostatically attracted to the negatively charged surface of the luminal BMP-rich vesicles. Various inherited defects of ganglioside hydrolases, e.g., of ß-galactosidase and ß-hexosaminidases, and of GM2-activator protein, cause infantile (with tetraparesis, dementia, blindness) and different protracted clinical forms of GM1- and GM2-gangliosidoses. Mutations yielding proteins with small residual catabolic activities in the lysosome give rise to juvenile and adult clinical forms with a wide range of clinical symptomatology. Apart from patients' differences in their genetic background, clinical heterogeneity may be caused by rather diverse substrate specificities and functions of lysosomal hydrolases, multifunctional properties of SAPs, and the strong regulation of ganglioside catabolism by membrane lipids. Currently, there is no treatment available for neuronal ganglioside storage diseases. Therapeutic approaches in mouse models and patients with juvenile forms of gangliosidoses are discussed.

Gangliosidoses.

The gangliosidoses comprise a family of lysosomal storage diseases characterized by the accumulation of complex glycosphingolipids in the nervous system and other tissues, secondary to the deficient activity of lysosomal hydrolases or their associated activator proteins. GM1 and GM2 gangliosidosis are associated with deficiency of ß-galactosidase and ß-hexosaminidase respectively. All gangliosidoses are characterized by progressive neurodegeneration, the severity of which is proportional to the residual enzyme activity. The GM1 gangliosidoses are characterized by dysostosis, organomegaly and coarsening in their most severe forms, whereas children with classic infantile GM2 gangliosidosis (Tay-Sachs disease) are usually spared systemic involvement, except in the case of the Sandhoff variant, in which organomegaly may occur. Cherry-red macular spots occur in the early onset forms of the gangliosidoses, but are less frequently seen in the less severe, later onset phenotypes. Macrocephaly, an exaggerated startle response, cognitive decline, seizures, ataxia, and progressive muscular atrophy may occur in different forms of gangliosidosis. The diagnosis is made by assay of enzyme activity, and can be confirmed by mutation analysis. Carrier screening for Tay-Sachs disease has been remarkably successful in reducing the incidence of this disease in the at-risk Ashkenazi population. There are no proven disease-modifying therapies for the gangliosidoses.

Resveratrol prevents global cerebral ischemia-induced decrease in lipid content.

OBJECTIVE: The present study was undertaken to evaluate whether resveratrol (RSV) modulates membrane lipid composition, as well as on ganglioside profile in ischemia/reperfusion injury. METHODS: Global cerebral ischemia was induced by four-vessel occlusion for 10 minutes. RSV (30 mg/kg) or vehicle was intraperitoneally administered to rats 7 days prior to ischemia. Brain structures were homogenized with chloroform/methanol for ganglioside, phospholipids, and cholesterol levels. RESULTS: RSV significantly prevented the reduction in the total content of gangliosides, phospholipids, and cholesterol in hippocampi and cerebral cortex induced by global cerebral ischemia. Although ischemia/reperfusion decreased ganglioside content, the ganglioside profiles were apparently not modified. CONCLUSIONS: Our experiments suggest that lipid metabolism is important for development of ischemic damage and indicate that RSV treatment 7 days prior to ischemia may prevent membrane lipid loss.

Sodium-calcium exchanger and lipid rafts in pig coronary artery smooth muscle.

Pig coronary artery smooth muscle expresses, among many other proteins, Na+-Ca²+-exchanger NCX1 and sarcoplasmic reticulum Ca²+ pump SERCA2. NCX1 has been proposed to play a role in refilling the sarcoplasmic reticulum Ca²+ pool suggesting a functional linkage between the two proteins. We hypothesized that this functional linkage may require close apposition of SERCA2 and NCX1 involving regions of plasma membrane like lipid rafts. Lipid rafts are specialized membrane microdomains that appear as platforms to co-localize proteins. To determine the distribution of NCX1, SERCA2 and lipid rafts, we isolated microsomes from the smooth muscle tissue, treated them with non-ionic detergent and obtained fractions of different densities by sucrose density gradient centrifugal flotation. We examined the distribution of NCX1; SERCA2; non-lipid raft plasma membrane marker transferrin receptor protein; lipid raft markers caveolin-1, flotillin-2, prion protein, GM1-gangliosides and cholesterol; and cytoskeletal markers clathrin, actin and myosin. Distribution of markers identified two subsets of lipid rafts that differ in their components. One subset is rich in caveolin-1 and flotillin-2 and the other in GM1-gangliosides, prion protein and cholesterol. NCX1 distribution correlated strongly with SERCA2, caveolin-1 and flotillin-2, less strongly with the other membrane markers and negatively with the cytoskeletal markers. These experiments were repeated with a non-detergent method of treating microsomes with sonication at high pH and similar results were obtained. These observations are consistent with the observed functional linkage between NCX1 and SERCA2 and suggest a role for NCX1 in supplying Ca²+ for refilling the sarcoplasmic reticulum.

Lysosomal storage of oligosaccharide and glycosphingolipid in imino sugar treated cells.

Sandhoff and Tay-Sachs disease are autosomal recessive GM2 gangliosidoses where a deficiency of lysosomal beta-hexosaminidase results in storage of glycoconjugates. Imino sugar (2-acetamido-1,4-imino-1,2,4-trideoxy-L-arabinitol) inhibition of beta-hexosaminidase in murine RAW264.7 macrophage-like cells led to lysosomal storage of glycoconjugates that were characterised structurally using fluorescence labelling of the free or glycolipid-derived oligosaccharides followed by HPLC and mass spectrometry. Stored glycoconjugates were confirmed as containing non-reducing GlcNAc or GalNAc residues resulting from the incomplete degradation of N-linked glycoprotein oligosaccharide and glycolipids, respectively. When substrate reduction therapeutics N-butyl-deoxynojirimycin (NB-DNJ) or N-butyldeoxygalactonojirimycin (NB-DGJ) were applied to the storage phenotype cells, an increase in glucosylated and galactosylated oligosaccharide species was observed due to endoplasmic reticulum alpha-glucosidases and lysosomal beta-galactosidase inhibition, respectively. Hexosaminidase inhibition triggered a tightly regulated cytokine-mediated inflammatory response that was normalised using imino sugars NB-DNJ and NB-DGJ, which restored the GM2 ganglioside storage burden but failed to reduce the levels of GA2 glycolipid or glycoprotein-derived N-linked oligosaccharides. Using a chemically induced gangliosidosis phenotype that can be modulated with substrate lowering drugs, the critical role of GM2 ganglioside in the progression of inflammatory disease is also demonstrated.

Neurochemical, morphological, and neurophysiological abnormalities in retinas of Sandhoff and GM1 gangliosidosis mice.

Retinal abnormalities are well documented in patients with ganglioside storage diseases. The total content and distribution of retinal glycosphingolipids was studied for the first time in control mice and in Sandhoff disease (SD) and GM1 gangliosidosis mice. Light and electron microscopy of the SD and the GM1 retinas revealed storage in ganglion cells. Similar to previous findings in rat retina, GD3 was the major ganglioside in mouse retina, while GM2 and GM1 were minor species. Total ganglioside content was 44% and 40% higher in the SD and the GM1 retinas, respectively, than in the control retinas. Furthermore, GM2 and GM1 content were 11-fold and 51-fold higher in the SD and the GM1 retinas than in the control retinas, respectively. High concentrations of asialo-GM2 and asialo-GM1 were found in the SD and the GM1 retinas, respectively, but were undetectable in the control retinas. The GSL abnormalities in the SD and the GM1 retinas reflect significant reductions in beta-hexosaminidase and beta-galactosidase enzyme activities, respectively. Although electroretinograms appeared normal in the SD and the GM1 mice, visual evoked potentials were subnormal in both mutants, indicating visual impairments. Our findings present a model system for assessing retinal pathobiology and therapies for the gangliosidoses.

Elastogenesis in cultured dermal fibroblasts from patients with lysosomal beta-galactosidase, protective protein/cathepsin A and neuraminidase-1 deficiencies.

The human GLB1 gene encodes a lysosomal beta-galactosidase (beta-Gal) and an elastin-binding protein (EBP). Defect of the EBP as a chaperon for tropoelastin and a component of receptor complex among neuraminidase-1 (NEU1) and protective protein/cathepsin A (PPCA) is suggested responsible for impaired elastogenesis in autosomal recessive beta-Gal, PPCA and NEU1 deficiencies. The purpose of this study is to determine effects of GLB1, PPCA and NEU1 gene mutations on elastogenesis in skin fibroblasts. Elastic fiber formation and the EBP mRNA expression were examined by immunofluorescence with an anti-tropoelastin antibody and RT-PCR selective for EBP in skin fibroblasts with these lysosomal enzyme deficiencies. Apparently normal elastogenesis and EBP mRNA expression were observed for fibroblasts from Morquio B disease cases with the GLB1 gene alleles (W273L/W273L, W273L/R482H and W273L/W509C substitutions, respectively), a galactosialidosis case with the PPCA allele (IVS7+3A/IVS7+3A) and a sialidosis case with the NEU1 allele (V217M/G243R) as well as normal subject. In this study, the W273L substitution in the EBP could impossibly cause the proposed defect of elastogenesis, and the typical PPCA splicing mutation and the V217M/G243R substitutions in the NEU1 might hardly have effects on elastic fiber formation in the dermal fibroblasts.

Cross-seeding of wild-type and hereditary variant-type amyloid beta-proteins in the presence of gangliosides.

We investigated the molecular mechanism underlying the ganglioside-induced initiation of the assembly of wild and hereditary variant-type amyloid beta-proteins, including Arctic-, Dutch-, and Flemish-type amyloid beta-proteins. We monitored the assembly of amyloid beta-protein by thioflavin-T assay, western blotting and electron microscopy. We also examined how externally added amyloid beta-protein assembles in a cell culture. The assembly of wild-, Arctic-, Dutch-, and Flemish-type amyloid beta-proteins were accelerated in the presence of GM1, GM1, GM3 and GD3 gangliosides. Notably, all of these amyloid beta-proteins accelerated the assembly of different type of amyloid beta-protein, following prior binding to a specific ganglioside. A specific-ganglioside-bound form of variant-type amyloid beta-protein was recognized by the antibody (4396C) specific to the GM1-ganglioside-induced altered conformation of wild-type amyloid beta-protein. Moreover, the assembly of these amyloid beta-proteins in the presence of a specific ganglioside was markedly suppressed by coincubation with 4396C. This study suggests that cross-seeding can occur between wild and hereditary variant-type amyloid beta-proteins despite differences in their amino acid sequences.

Neurobiology and cellular pathogenesis of glycolipid storage diseases.

Disorders of lysosomal metabolism often involve the accumulation of specific types of glycolipid, particularly gangliosides, because of either degradative failure or other currently unknown mechanisms. Although the precise role of gangliosides in cells remains enigmatic, the presence of specific abnormalities secondary to ganglioside accumulation in lysosomal diseases has suggested important biological functions. Chief among these is the growth of new dendrites on particular classes of mature neurons secondary to an increase in GM2 ganglioside. That GM2 has also been shown to be elevated in normal immature neurons coincident with dendritic sprouting provides a compelling argument that this ganglioside plays a role in dendritic initiation. This discovery has led to the search for other regulators of dendritic differentiation that may in some way be linked to the expression and/or function of GM2 ganglioside. Principal candidates that have emerged include tyrosine kinase receptors, small GTPases and calcium/calmodulin-dependent protein kinase II. Understanding the mechanism underlying ectopic dendritogenesis in lysosomal diseases can be expected to generate significant insight into the control of dendritic plasticity in normal brain. The detrimental aspects of ganglioside accumulation in storage diseases as well as the potential link between gangliosides and dendritogenesis also provide a strong rationale for developing pharmacological means to manipulate ganglioside expression in neurons.

Clathrin-dependent and -independent internalization of plasma membrane sphingolipids initiates two Golgi targeting pathways.

Sphingolipids (SLs) are plasma membrane constituents in eukaryotic cells which play important roles in a wide variety of cellular functions. However, little is known about the mechanisms of their internalization from the plasma membrane or subsequent intracellular targeting. We have begun to study these issues in human skin fibroblasts using fluorescent SL analogues. Using selective endocytic inhibitors and dominant negative constructs of dynamin and epidermal growth factor receptor pathway substrate clone 15, we found that analogues of lactosylceramide and globoside were internalized almost exclusively by a clathrin-independent ("caveolar-like") mechanism, whereas an analogue of sphingomyelin was taken up approximately equally by clathrin-dependent and -independent pathways. We also showed that the Golgi targeting of SL analogues internalized via the caveolar-like pathway was selectively perturbed by elevated intracellular cholesterol, demonstrating the existence of two discrete Golgi targeting pathways. Studies using SL-binding toxins internalized via clathrin-dependent or -independent mechanisms confirmed that endogenous SLs follow the same two pathways. These findings (a) provide a direct demonstration of differential SLs sorting into early endosomes in living cells, (b) provide a "vital marker" for endosomes derived from caveolar-like endocytosis, and (c) identify two independent pathways for lipid transport from the plasma membrane to the Golgi apparatus in human skin fibroblasts.

GM2 gangliosidosis AB variant: clinical and biochemical studies of a Japanese patient.

OBJECTIVE: To determine the clinical features and biochemical basis of the first Japanese patient with the GM2 gangliosidosis AB variant. METHODS: The clinical manifestations and laboratory findings in the patient were investigated. Cultured fibroblasts from the patient were analyzed by means of immunofluorescence staining with an anti-GM2 ganglioside monoclonal antibody and thin-layer chromatography and immunostaining. GM1 ganglioside catabolism in cultured cells was analyzed by pulse labeling, and the amount of GM2 activator in cells was determined by Western blot analysis. Gene analysis was performed according to standard protocols. RESULTS: The patient showed progressive neurologic manifestations of quite early onset. Muscular weakness and hypotonia became evident by 1 month of age, and the patient then developed a startle reaction, severe psychomotor retardation, and myoclonic seizures. Immunocytochemical analysis clearly revealed the accumulation of GM2 ganglioside in cultured fibroblasts from the patient, and thin-layer chromatography confirmed it. Western blot and metabolic studies showed a complete deficiency of GM2 activator. Gene analysis did not reveal any mutations in the protein coding region of the GM2 activator gene. CONCLUSION: The clinical features and biochemical basis of this Japanese patient with GM2 gangliosidosis AB variant were determined. Immunocytochemical analysis using cultured fibroblasts as samples is available for the diagnosis of this disease.

The GM2 activator protein, its roles as a co-factor in GM2 hydrolysis and as a general glycolipid transport protein.

Although there is only one documented function carried out by the GM2 activator protein in the lysosome, new information suggests that other less obvious roles may also be played by this protein in vivo. This information includes data demonstrating that the GM2 activator is a secretory, as well as a lysosomal protein, and that cells possess a carbohydrate-independent mechanism to re-capture the activator, with or without bound lipid, from the extracellular fluid. Additionally the GM2 activator has been shown to bind, solubilize and transport a broad spectrum of lipid molecules, such as glycolipids, gangliosides and at least one phosphoacylglycerol, between liposomes. At pH 7 the GM2 activator's rate of lipid transport is reduced by only 50% from its maximum rate which is achieved at approx. pH 5, suggesting that the GM2 activator may serve as a general intra- and/or inter-cellular lipid transport protein in vivo. Since the late 1970s the lysosomal form of the GM2 activator has been known to act as a substrate-specific co-factor for the hydrolysis of GM2 ganglioside by beta-hexosaminidase A. Gangliosides are a class of negatively charged glycolipids particularly abundant in neuronal cells which have been linked to numerous in vivo functions, such as memory formation and signal transduction events. Deficiency of the GM2 activator protein results in the storage of GM2 ganglioside and severe neurological disease, the AB-variant form of GM2 gangliosidosis, usually culminating in death before the age of 4 years. The exact mode-of-action of the GM2 activator in its role as a co-factor, and its specificity for various glycolipids are currently matters of debate in the literature.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis of glycosphingolipids including gangliosides.

Long chain base compositions of gangliosides containing mainly stearic acid could be determined without any chemical modification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry with delayed ion extraction (DE MALDI-TOF MS). The analytical results for the long chain base compositions of various samples of GM1 from the brain tissues of patients with different diseases at different ages confirmed that the proportion of d20:1 (icosasphingosine) and d20 (icosa-sphinganine) of the total sphingosine bases increased quickly until adolescent or adult age and then remained constant slightly exceeding 50%; this value was evidently higher than the proportion of d20:1 and d20 of GM1 in various adult mammalian brains. A long chain base composition of GM1 from the brain tissue of a patient with infantile type of GM1-gangliosidosis at 4y2m was abnormal and so was in two sibling patients with Spielmeyer-Vogt type of juvenile amaurotic idiocy at 19y and 21y in spite of that in the latter there was no accumulation of GM1 in the brain tissue. On the other hand, a patient with adult type of GM1 gangliosidosis at 66y showed a local accumulation of GM1 in the putamen and caudate nucleus, but its long chain base composition was found to be normal. It was of interest that the white matter of Eker rat with hereditary renal carcinoma contained a large amount of plasmalocerebroside as compared with the amount of cerebroside and sphingomyelin. The individual molecular species of plasmalocerebroside were identified by DE MALDI-TOF MS.

Unusual gangliosidosis in emu (Dromaius novaehollandiae).

A previous study has demonstrated an unusual gangliosidosis in emu that is characterized by the accumulation of gangliosides in the brain tissues with GM3 and GM1 predominating. To provide insight into this unique disorder of emu gangliosidosis, the current study focused on analysis of neutral glycosphingolipids and gangliosides from brain and liver tissues of affected birds and healthy controls. We found not only that the total lipid-bound sialic acid content was increased three- and fourfold in the affected brain and liver, respectively, but also that the ganglioside pattern was rather complex as compared with the control. The absolute ganglioside sialic acid content was significantly increased in the diseased tissues, with the highest elevation levels of GM3 (14-fold) and GM1 (ninefold) in the affected brain. Relative increases in content of these monosialogangliosides were also significant. GM2 was only detected in the affected brain, but not in normal controls. The neutral glycosphingolipid fraction showed accumulation of many oligosylceramides, with six- and 5.5-fold increases in lactosylceramide levels for brain and liver, respectively. The level of myelin-associated galactosylceramide (GalCer) in the brain was decreased to only 41% of that in the healthy control, whereas no difference was found in liver tissues from both groups. Besides GalCer, the brain content of sulfatide (cerebroside-sulfate esters), another myelin-associated glycolipid, decreased to only 16% of the control. The loss of myelin-associated GalCer and sulfatide strongly suggests demyelination in the affected emu brain. Our overall data are consistent with the presence of a unique form of sphingolipidosis in the affected emus, perhaps with secondary demyelination, and suggest a metabolic disorder related to total sphingolipid activator deficiency.

Intracellular degradation of sulforhodamine-GM1: use for a fluorescence-based characterization of GM2-gangliosidosis variants in fibroblasts and white blood cells.

A novel fluorescent ganglioside, sulforhodamine-GM1 was administered into cells derived from carriers and patients with different subtypes of GM2 gangliosidosis, resulting from various mutations in the gene encoding the lysosomal enzyme hexosaminidase (Hex) A. The cells used were skin fibroblasts and white blood cells, i.e. lymphocytes, monocytes and macrophages. In the severe infantile form of the GM2 gangliosidosis, Tay-Sachs disease, the sulforhodamine-GM1 was hydrolyzed within the lysosomes to the corresponding sulforhodamine-GM2 which, because of lack of Hex A activity, was not further degraded. In comparison, in the cells derived from GM2 gangliosidoses carriers, as well as pseudodeficient and adult forms of GM2 gangliosidosis, the sulforhodamine-GM2 was further processed and sequentially degraded by the lysosomal glycosidases to sulforhodamine-ceramide. The latter was converted to sulforhodamine-sphingomyelin, which was secreted into the culture medium. The fluorescence of the sulforhodamine ceramide in cell extracts and/or sulforhodamine-sphingomyelin in the culture medium was quantified and related to parallel data obtained using cells of normal individuals. This permitted distinguishing between the various GM2 gangliosidoses subtypes and relating the intracellular hydrolysis of sulforhodamine-GM1 to the genotypes of the respective GM2 gangliosidoses variants.