Morquio B disease: From pathophysiology towards diagnosis.

Morquio B disease is an attenuated phenotype within the spectrum of beta galactosidase (GLB1) deficiencies. It is characterised by dysostosis multiplex, ligament laxity, mildly coarse facies and heart valve defects due to keratan sulphate accumulation, predominantly in the cartilage. Morquio B patients have normal neurological development, setting them apart from those with the more severe GM1 gangliosidosis. Morquio B disease, with an incidence of 1:250.000 to 1:1.000.000 live births, is very rare. Here we report the clinical-biochemical data of nine patients. High amounts of keratan sulfate were detected using LC-MS/MS in the patients' urinary samples, while electrophoresis, the standard procedure of qualitative glycosaminoglycans analysis, failed to identify this metabolite in any of the patients' samples. We performed molecular analyses at gene, gene expression and protein expression levels, for both isoforms of the GLB1 gene, lysosomal GLB1, and the cell-surface expressed Elastin Binding Protein. We characterised three novel GLB1 mutations [c.75 + 2 T > G, c.575A > G (p.Tyr192Cys) and c.2030 T > G (p.Val677Gly)] identified in three heterozygous patients. We also set up a copy number variation assay by quantitative PCR to evaluate the presence of deletions/ insertions in the GLB1 gene. We propose a diagnostic plan, setting out the specific clinical- biochemical and molecular features of Morquio B, in order to avoid misdiagnoses and improve patients' management.

The natural history of Type 1 infantile GM1 gangliosidosis: A literature-based meta-analysis.

INTRODUCTION: Type 1 GM1 gangliosidosis is an ultra-rare, rapidly fatal lysosomal storage disorder, with life expectancy of <3 years of age. To date, only one prospective natural history study of limited size has been reported. Thus, there is a need for additional research to provide a better understanding of the progression of this disease. We have leveraged the past two decades of medical literature to conduct the first comprehensive retrospective study characterizing the natural history of Type 1 GM1 gangliosidosis. OBJECTIVES: The objectives of this study were to establish a large sample of patients from the literature in order to identify: 1) clinically distinguishing factors between Type 1 and Type 2 GM1 gangliosidosis, 2) age at first symptom onset, first hospital admission, diagnosis, and death, 3) time to onset of common clinical findings, and 4) timing of developmental milestone loss. METHODS: PubMed was searched with the keyword "GM1 Gangliosidosis" and for articles from the year 2000 onwards. A preliminary review of these results was conducted to establish subtype classification criteria for inclusion of only Type 1 patients, resulting in 44 articles being selected to generate the literature dataset of 154 Type 1 GM1 gangliosidosis patients. Key clinical events of these patient cases were recorded from the articles. RESULTS: Comprehensive subtyping criteria for Type 1 GM1 gangliosidosis were created, and clinical events, including onset, diagnosis, death, and symptomology, were mapped over time. In this dataset, average age of diagnosis was 8.7 months, and average age of death was 18.9 months. DISCUSSION: This analysis demonstrates the predictable clinical course of this disease, as almost all patients experienced significant multi-organ system dysfunction and neurodevelopmental regression, particularly in the 6- to 18-month age range. Patients were diagnosed at a late age relative to disease progression, indicating the need for improved public awareness and screening. CONCLUSION: This study highlights the significant burden of illness in this disease and provides critical natural history data to drive earlier diagnosis, inform clinical trial design, and facilitate family counseling.

Infantile gangliosidoses: Mapping a timeline of clinical changes.

BACKGROUND: Infantile gangliosidoses include GM1 gangliosidosis and GM2 gangliosidosis (Tay-Sachs disease, Sandhoff disease). To date, natural history studies in infantile GM2 (iGM2) have been retrospective and conducted through surveys. Compared to iGM2, there is even less natural history information available on infantile GM1 disease (iGM1). There are no approved treatments for infantile gangliosidoses. Substrate reduction therapy using miglustat has been tried, but is limited by gastrointestinal side effects. Development of effective treatments will require identification of meaningful outcomes in the setting of rapidly progressive and fatal diseases. OBJECTIVES: This study aimed to establish a timeline of clinical changes occurring in infantile gangliosidoses, prospectively, to: 1) characterize the natural history of these diseases; 2) improve planning of clinical care; and 3) identify meaningful future treatment outcome measures. METHODS: Patients were evaluated prospectively through ongoing clinical care. RESULTS: Twenty-three patients were evaluated: 8 infantile GM1, 9 infantile Tay-Sachs disease, 6 infantile Sandhoff disease. Common patterns of clinical change included: hypotonia before 6months of age; severe motor skill impairment within first year of life; seizures; dysphagia and feeding-tube placement before 18months of age. Neurodevelopmental testing scores reached the floor of the testing scale by 20 to 28months of age. Vertebral beaking, kyphosis, and scoliosis were unique to patients with infantile GM1. Chest physiotherapy was associated with increased survival in iGM1 (p=0.0056). Miglustat combined with a low-carbohydrate ketogenic diet (the Syner-G regimen) in patients who received a feeding-tube was associated with increased survival in infantile GM1 (p=0.025). CONCLUSIONS: This is the first prospective study of the natural history of infantile gangliosidoses and the very first natural history of infantile GM1. The homogeneity of the infantile gangliosidoses phenotype as demonstrated by the clinical events timeline in this study provides promising secondary outcome measure candidates. This study indicates that overall survival is a meaningful primary outcome measure for future clinical trials due to reliable timing and early occurrence of this event. Combination therapy approaches, instead of monotherapy approaches, will likely be the best way to optimize clinical outcomes. Combination therapy approaches include palliative therapies (e.g., chest physiotherapy) along with treatments that address the underlying disease pathology (e.g. miglustat or future gene therapies).

The GM1 and GM2 Gangliosidoses: Natural History and Progress toward Therapy.

The gangliosidoses are lysosomal storage disorders caused by accumulation of GM1 or GM2 gangliosides. GM1 gangliosidosis has both central nervous system and systemic findings; while, GM2 gangliosidosis is restricted primarily to the central nervous system. Both disorders have autosomal recessive modes of inheritance and a continuum of clinical presentations from a severe infantile form to a milder, chronic adult form. Both are devastating diseases without cure or specific treatment however, with the use of supportive aggressive medical management, the lifespan and quality of life has been extended for both diseases. Naturally occurring and engineered animal models that mimic the human diseases have enhanced our understanding of the pathogenesis of disease progression. Some models have shown significant improvement in symptoms and lifespan with enzyme replacement, substrate reduction, and anti-inflammatory treatments alone or in combination. More recently gene therapy has shown impressive results in large and small animal models. Treatment with FDA-approved glucose analogs to reduce the amount of ganglioside substrate is used as off-label treatments for some patients. Therapies also under clinical development include small molecule chaperones and gene therapy.

Effects of cyclodextrins on GM1-gangliosides in fibroblasts from GM1-gangliosidosis patients.

OBJECTIVES: GM1-gangliosidosis is an inherited disorder characterized by the accumulation of GM1-gangliosides in many tissues and organs, particularly in the brain. Currently, there is no treatment available for patients with ganglioside storage diseases. Therefore, we investigated the effects of cyclodextrins (CyDs) on the GM1-ganglioside level in EA1 cells, fibroblasts from patients with GM1-gangliosidosis. METHODS: The concentrations of cholesterol and phospholipids in supernatants were determined by Cholesterol E-test Wako and Phospholipid C-test Wako, respectively. The effects of CyDs on GM1-ganglioside levels in EA1 cells using fluorescence-labelled cholera toxin B-subunit, which can bind to GM1-gangliosides specifically, were investigated by flow cytometry and confocal laser scanning microscopy. KEY FINDINGS: The treatment with methylated CyDs, hydroxypropylated CyDs and branched CyDs decreased GM1-ganglioside levels in EA1 cells at 1 mm for 24 h. Unexpectedly, there was no significant change in the efflux of cholesterol or phospholipids from the cells after treatment with CyDs under the same experimental conditions, indicating that the efflux of membrane components is not associated with down-regulation of GM1-ganglioside levels in EA1 cells upon CyDs treatment. CONCLUSIONS: CyDs may have the potential as drugs for GM1-gangliosidosis, although the mechanism should be thereafter clarified.

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.

1-Deoxy-D-galactonojirimycins with dansyl capped N-substituents as ß-galactosidase inhibitors and potential probes for GM1 gangliosidosis affected cell lines.

Two simple and reliably accessible intermediates, N-carboxypentyl- and N-aminohexyl-1-deoxy-D-galactonojirimycin were employed for the synthesis of a set of terminally N-dansyl substituted derivatives. Reaction of the terminal carboxylic acid of N-carboxypentyl-1-deoxy-D-galactonojirimycin with N-dansyl-1,6-diaminohexane provided the chain-extended fluorescent derivative. Employing bis(6-dansylaminohexyl)amine, the corresponding branched di-N-dansyl compound was obtained. Partially protected N-aminohexyl-1-deoxy-D-galactonojirimycin served as intermediate for two additional chain-extended fluorescent 1-deoxy-D-galactonojirimycin (1-DGJ) derivatives featuring terminal dansyl groups in the N-alkyl substituent. These new compounds are strong inhibitors of d-galactosidases and may serve as leads en route to pharmacological chaperones for GM1-gangliosidosis.

Three novel beta-galactosidase gene mutations in Han Chinese patients with GM1 gangliosidosis are correlated with disease severity.

BACKGROUND: GM1 gangliosidosis (GM1) is an autosomal recessive lysosomal storage disease caused by deficiency of acid beta-galactosidase (GLB1; EC3.2.1.23). Here, we identify three novel mutations in the GLB1 gene from two Han Chinese patients with GM1 that appear correlated with clinical phenotype. METHODS: One of the two Han Chinese patients with GM1 presented with the juvenile form, and the other with the infantile form with cardiac involvement. Sequencing of the entire GLB1 gene revealed three novel mutations (p.H102 D, p.G494V, c.495_497delTCT), which were absent in 94 normal controls. Transient expression of cDNA encoding these variants was performed in COS-1 cells to evaluate ß-galactosidase activities. RESULTS: The first case (patient 1) with the juvenile form contained two missense mutations, p.H102 D and p.A301V. Patient 2 diagnosed with the infantile form of the disease with cardiac involvement was compound heterozygous for p.G494V and c.495_497delTCT mutations. All mutant beta-galactosidases exhibited significantly reduced activity (12%, 0%, 0%, and 0% for p.H102 D, p.A301V, p.G494V, and c.495_497delTCT), compared with the wild-type beta-galactosidase cDNA clone. The mutations identified in patient 2 with cardiomyopathy were localized in the GLB1 gene region common to both lysosomal beta-galactosidase and elastin binding protein (EBP), and caused a deletion in the elastin-binding domain of EBP. CONCLUSIONS: All four mutations identified in Han Chinese patients induce significant suppression of ß-galactosidase activity, correlating with severity of disease and presence of cardiomyopathy.

Critical role of iron in the pathogenesis of the murine gangliosidoses.

Neurodegeneration is a prominent feature of the gangliosidoses, a group of lysosomal storage diseases. Here we show altered iron homeostasis in mouse models of both GM1 and GM2 gangliosidoses, which are characterized by progressive depletion of iron in brain tissue. This finding contrasts with the findings in many other neurological disorders, where excess iron deposition has been reported. We found that key regulators of iron homeostasis, hepcidin and IL-6, were increased in gangliosidoses mice. In the brain, the principal iron transport and delivery protein transferrin was reduced, accompanied by a progressive inability of the brain to acquire iron from the circulation. Expression of the transferrin receptor was up-regulated reciprocally. Despite the deregulation of iron homeostasis administration of iron prolonged survival in the diseased mice by up to 38%, with onset of disease delayed and motor function preserved.

Beneficial effects of substrate reduction therapy in a mouse model of GM1 gangliosidosis.

GM1 gangliosidosis is an inherited neurodegenerative disorder caused by lysosomal beta-galactosidase deficiency, resulting in the storage of GM1 and GA1, primarily in the central nervous system. This disease typically afflicts infants and young children and there is currently no effective therapy. Substrate reduction therapy (SRT) could be of potential benefit. The imino sugars N-butyldeoxynojirimycin (NB-DNJ, miglustat, Zavesca) and N-butyldeoxygalactonojirimycin (NB-DGJ) used for SRT inhibit glucosylceramide synthase (GlcCerS) that catalyses the first committed step in glycosphingolipid biosynthesis. We have compared the efficacy and tolerability of NB-DNJ and NB-DGJ in the beta-galactosidase knockout mouse. NB-DGJ was better tolerated than NB-DNJ, due to intrinsic gastrointestinal tract dysfunction that was exacerbated by NB-DNJ. However, functional improvement was greatest with NB-DNJ treatment which may potentially be caused by novel anti-inflammatory properties of NB-DNJ.

Brain proton magnetic resonance spectroscopy and neuromuscular pathology in a patient with GM1 gangliosidosis.

The authors report the clinical, neuroradiologic, and neuromuscular pathological findings in a patient with GM1 gangliosidosis. The proton magnetic resonance spectroscopy, previously reported in a single patient with GM1 gangliosidosis, detected a mild reduction of N-acetylaspartate, consistent with relative paucity of axons and neurons and increased levels of myoinositol suggestive of gliotic white matter changes along with the accumulation of an additional compound that could represent either guanidinoacetate or Gal beta 1-6Gal beta 1-4)GlcNAc, an oligosaccharide previously isolated from the urine of GM1 gangliosidosis patients. Although these findings will have to be further confirmed in more patients with GM1 gangliosidosis, they suggest that proton magnetic resonance spectroscopy may provide useful end points to assess the efficacy of novel treatments that could soon become clinically available. Histologically, no significant alterations were found in axons, but there was evidence of redundant and inappropriately folded myelin, which is a feature attributed to disturbed axon-glial interactions.

Chemical chaperone therapy: clinical effect in murine G(M1)-gangliosidosis.

Certain low-molecular-weight substrate analogs act both as in vitro competitive inhibitors of lysosomal hydrolases and as intracellular enhancers (chemical chaperones) by stabilization of mutant proteins. In this study, we performed oral administration of a chaperone compound N-octyl-4-epi-beta-valienamine to G(M1)-gangliosidosis model mice expressing R201C mutant human beta-galactosidase. A newly developed neurological scoring system was used for clinical assessment. N-Octyl-4-epi-beta-valienamine was delivered rapidly to the brain, increased beta-galactosidase activity, decreased ganglioside G(M1), and prevented neurological deterioration within a few months. No adverse effect was observed during this experiment. N-Octyl-4-epi-beta-valienamine will be useful for chemical chaperone therapy of human G(M1)-gangliosidosis.

Motor and reflex testing in GM1-gangliosidosis model mice.

A large number of genetic disease model mice have been produced by genetic engineering. However, phenotypic analysis is not sufficient, particularly for brain dysfunction in neurogenetic diseases. We tried to develop a new assessment system mainly for motor and reflex functions in G(M1)-gangliosidosis model mice. Two genetically engineered model mouse strains were used for this study: the beta-galactosidase-deficient knockout mouse representing infantile G(M1)-gangliosidosis (severe form), and transgenic mouse representing juvenile G(M1)-gangliosidosis (mild form). We modified human child neurology techniques, and selected eleven tests for motor assessment and reflex testing. The test results were scored in four grades: 0 (normal), 1 (slightly abnormal), 2 (moderately abnormal), and 3 (severely abnormal). Both disease model mouse strains showed high scores even at the apparently pre-symptomatic stage of the disease, particularly with abnormal tail and hind limb postures. Individual and total test scores were well correlated with the progression of the disease. This method is simple, quick, and reproducible. The testing is sensitive enough to detect early neurological abnormalities, and will be useful for monitoring the natural clinical course and effect of therapeutic experiments in various neurogenetic disease model mice, such as chemical chaperone therapy for G(M1)-gangliosidosis model mice.

The Arg482His mutation in the beta-galactosidase gene is responsible for a high frequency of GM1 gangliosidosis carriers in a Cypriot village.

GM1 gangliosidosis is a lysosomal storage disorder caused by deficiency of beta-galactosidase. It is mainly characterized by progressive neurodegeneration, and in its most severe infantile form, it leads to death before the age of 4. The GLB1 gene gives rise to two alternatively spliced mRNAs that encode the beta-galactosidase and the elastin binding protein (EBP). The diagnosis of two patients with the infantile form of GM1 gangliosidosis and 11 carriers in a small mountainous village in Cyprus prompted us to carry out a study in order to establish the frequency of carriers in the village and identify the mutations involved. Carrier detection was initially based on the measurement of beta-galactosidase activity in leucocytes. Among 85 random samples from the village, 10 were classified as carriers. Sequencing of the GLB1 gene in a Cypriot patient identified the missense mutation c.1445G>A (p.Arg482His) in the homozygous state. Seven of the 10 carriers identified using the enzyme assay were found to carry the same mutation by NspI restriction enzyme analysis. The three individuals who were negative for the c.1445G>A had borderline enzyme results and were probably wrongly classified as carriers. The frequency of GM1 gangliosidosis carriers in this village is approximately 8% (1:12). Western blot analysis showed a marked decrease of the 64-kDa mature form of the enzyme protein and a similar reduction of the 67-kDa EBP. Our results indicate that the c.1445G>A mutation, which appears to be responsible for all GM1 gangliosidosis alleles in this Cypriot village, affects protein conformation.

GM1-ganglioside-mediated activation of the unfolded protein response causes neuronal death in a neurodegenerative gangliosidosis.

GM1-ganglioside (GM1) is a major sialoglycolipid of neuronal membranes that, among other functions, modulates calcium homeostasis. Excessive accumulation of GM1 due to deficiency of lysosomal beta-galactosidase (beta-gal) characterizes the neurodegenerative disease GM1-gangliosidosis, but whether the accumulation of GM1 is directly responsible for CNS pathogenesis was unknown. Here we demonstrate that activation of an unfolded protein response (UPR) associated with the upregulation of BiP and CHOP and the activation of JNK2 and caspase-12 leads to neuronal apoptosis in the mouse model of GM1-gangliosidosis. GM1 loading of wild-type neurospheres recapitulated the phenotype of beta-gal-/- cells and activated this pathway by depleting ER calcium stores, which ultimately culminated in apoptosis. Activation of UPR pathways did not occur in mice double deficient for beta-gal and ganglioside synthase, beta-gal-/-/GalNAcT-/-, which do not accumulate GM1. These findings suggest that the UPR can be induced by accumulation of the sialoglycolipid GM1 and this causes a novel mechanism of neuronal apoptosis.

The leukoencephalopathy of infantile GM1 gangliosidosis: oligodendrocytic loss and axonal dysfunction.

A myelin deficit in the cerebral white matter in infantile GM1 gangliosidosis is well established. Some have proposed this deficit to be secondary to axonal loss, while others argue for delayed or arrested myelination. We compared the frontal white and gray matter of two infants with GM1 gangliosidosis with four age-matched controls, using light microscopy with a quantitative analysis, immunohistochemistry, terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL), and electron microscopy (EM). In the GM1 cases, we found a marked decrease in the number of oligodendrocytes (85% in case 1 and 50% in case 2) and myelin sheaths (80% and 40%), with a mild decrease in axons (20% and 10%). Ultrastructurally, some naked axons and dilated cisterns of rough endoplasmic reticulum (RER) in oligodendrocytes were observed. There was no appreciable storage in remaining oligodendrocytes, nor obvious neocortical neuronal loss. An immunohistochemical decrease in proteolipid protein (PLP) and a more profound deficiency of myelin basic protein (MBP) indicate that this lesion is not simply the result of a delay or arrest in myelination and suggests a "dying-back" oligopathy. TUNEL-positive oligodendrocytes correlated with activated caspase-3 immunoreactivity. Amyloid precursor protein (APP)-immunoreactive aggregates were observed in proximal axons and meganeurites as well as in white matter axons. These data suggest that the myelin deficit results from a loss of oligodendrocytes and abnormal axoplasmic transport, perhaps consequent to massive neuronal storage of GM1.

Clinical and clinico-pathologic characteristics of Shiba dogs with a deficiency of lysosomal acid beta-galactosidase: a canine model of human GM1 gangliosidosis.

The present study was conducted to determine the clinical and clinico-pathologic characteristics of Shiba dogs with GM1 gangliosidosis, which is due to an autosomal recessively inherited deficiency of lysosomal acid beta-galactosidase activity. Clinical and clinico-pathological features were investigated in 10 homozygous Shiba dogs with GM1 gangliosidosis. The age at onset was 5 to 6 months and the dogs manifested progressive neurologic signs including loss of balance, intermittent lameness, ataxia, dysmetria and intention tremor of the head. The dogs were unable to stand by 10 months of age due to a progression of ataxia and spasticity in all limbs. Corneal clouding, a visual defect, generalized muscle rigospasticity, emotional disorder and a tendency to be lethargic were observed at 9 to 12 months. The dogs became lethargic from 13 months of age. The survival period seemed to be 14 to 15 months. As a clinico-pathologic feature, lymphocytes with abnormally large vacuoles were observed in peripheral blood (30 to 50% of total lymphocytes) through the lifetime of the dogs. The clinical and clinico-pathologic characteristics of this animal model are useful for not only the development and testing of potential methods of therapy, but also the diagnosis of affected homozygous Shiba dogs in veterinary clinics.

Galactonojirimycin derivatives restore mutant human beta-galactosidase activities expressed in fibroblasts from enzyme-deficient knockout mouse.

Ten low molecular compounds analogous to galactose were screened for inhibition of human beta-galactosidase activity. Among them, 1-deoxy-galactonojirimycin and N-(n-butyl)-deoxy-galactonojirimycin showed an inhibitory effect at high concentrations. However, they restored mutant enzyme activities expressed in enzyme-deficient knockout mouse fibroblasts and human beta-galactosidosis fibroblasts at lower intracellular concentrations. This effect was more remarkable on G(M1)-gangliosidosis mutations (R201C, I51T, R201H, R457Q) than Morquio B disease mutations (W273L, Y83H). These low molecular compounds pass though the blood-brain barrier in mice. We hope that this new therapeutic approach will become clinically applicable in the near future.

Myelination arrest demonstrated using magnetic resonance imaging in a child with type I GM1 gangliosidosis.

An 18-month-old girl was diagnosed as having GM1 gangliosidosis, on the basis of the clinical symptoms of muscle stiffness, developmental retardation, hepatosplenomegaly, and kyphoscoliosis and a laboratory study that revealed a deficiency in the lysosomal degradative enzyme beta-galactosidase. Magnetic resonance T1-weighted images showed persistent hyperintensity in the bilateral thalami, brainstem, and deep cerebellum at 14 and 18 months of age, indicating arrest of the myelination process in these areas, and that the arrest had occurred at the newborn stage. There was no myelination in the basal ganglia and diffuse leukomalacia developed in the cerebral hemispheres. Only supportive treatment was given; the patient died at 2 years of age. Myelination arrest at the newborn stage associated with progressive leukomalacia is a possible characteristic of GM1 gangliosidosis.