A case of Sandhoff disease caused by a novel ß-hexosaminidase B (HEXB) mutation c.118delG (p.A40fs*24): A case report from China.

BACKGROUND: Sandhoff disease (SD, Online Mendelian Inheritance in Man: 268800) is an autosomal recessive lysosomal storage disorder caused by variants of the ß-hexosaminidase B (HEXB) gene (Online Mendelian Inheritance in Man: 606873). The HEXB gene has been mapped to chromosome 5q13 and contains 14 exons. The symptoms of SD include progressive weakness, intellectual disability, visual and hearing impairment, exaggerated startle response, and seizures; the patients usually die before the age of 3 years.[1]. CASE SUMMARY: We present a case of SD caused by a homozygous frameshift mutation in the HEXB gene, c.118delG (p.A40fs*24). The male child, aged 2 years 7 months, showed movement retrogression with orbital hypertelorism at age 2 years, accompanied by seizures. Magnetic resonance imaging of the head showed cerebral atrophy and delayed myelination of the white matter of the brain. CONCLUSION: A novel homozygous frameshift c.118delG (p.A40fs*24) variant of HEXB has caused SD in the child. The major symptoms are intellectual disability, visual and hearing impairment, and seizures. Investigation will be continued in the future to comprehensively describe the genotype/phenotype and gain information on other associated features to understand the variable expressivity of this condition.

Saposin C, Key Regulator in the Alpha-Synuclein Degradation Mediated by Lysosome.

Lysosomal dysfunction has been proposed as one of the most important pathogenic molecular mechanisms in Parkinson disease (PD). The most significant evidence lies in the GBA gene, which encodes for the lysosomal enzyme ß-glucocerebrosidase (ß-GCase), considered the main genetic risk factor for sporadic PD. The loss of ß-GCase activity results in the formation of α-synuclein deposits. The present study was aimed to determine the activity of the main lysosomal enzymes and the cofactors Prosaposin (PSAP) and Saposin C in PD and healthy controls, and their contribution to α-synuclein (α-Syn) aggregation. 42 PD patients and 37 age-matched healthy controls were included in the study. We first analyzed the ß-GCase, ß-galactosidase (ß-gal), ß-hexosaminidase (Hex B) and Cathepsin D (CatD) activities in white blood cells. We also measured the GBA, ß-GAL, ß-HEX, CTSD, PSAP, Saposin C and α-Syn protein levels by Western-blot. We found a 20% reduced ß-GCase and ß-gal activities in PD patients compared to controls. PSAP and Saposin C protein levels were significantly lower in PD patients and correlated with increased levels of α-synuclein. CatD, in contrast, showed significantly increased activity and protein levels in PD patients compared to controls. Increased CTSD protein levels in PD patients correlated, intriguingly, with a higher concentration of α-Syn. Our findings suggest that lysosomal dysfunction in sporadic PD is due, at least in part, to an alteration in Saposin C derived from reduced PSAP levels. That would lead to a significant decrease in the ß-GCase activity, resulting in the accumulation of α-syn. The accumulation of monohexosylceramides might act in favor of CTSD activation and, therefore, increase its enzymatic activity. The evaluation of lysosomal activity in the peripheral blood of patients is expected to be a promising approach to investigate pathological mechanisms and novel therapies aimed to restore the lysosomal function in sporadic PD.

[Clinical characteristics and genetic analysis of a child with infantile Sandhoff disease and eosinophilia].

OBJECTIVE: To explore the genetic basis for a girl featuring epilepsy, developmental delay and regression. METHODS: Clinical data of the patient was collected. Activities of hexosaminidase A (Hex A) and hexosaminidase A&B (Hex A&B) in blood leukocytes were determined by using a fluorometric assay. Peripheral blood samples were collected from the proband and six members from her pedigree. Following extraction of genomic DNA, whole exome sequencing was carried out. Candidate variants were verified by Sanger sequencing. RESULTS: Enzymatic studies of the proband have shown reduced plasma Hex A and Hex A&B activities. Genetic testing revealed that she has carried c.1260_1263del and c.1601G>C heterozygous compound variants of the HEXB gene. Her mother, brother and sister were heterozygous carriers of c.1260_1263del, while her father, mother, three brothers and sister did not carry the c.1601G>C variant, suggesting that it has a de novo origin. Increased eosinophils were discovered upon cytological examination of peripheral blood and bone marrow samples. CONCLUSION: The compound heterozygous variants of c.1260_1263del and c.1601G>C of the HEXB gene probably underlay the Sandhoff disease in this child. Eosinophilia may be noted in infantile Sandhoff disease.

Atypical presentation of late-onset Sandhoff disease: a case report.

BACKGROUND AND PURPOSE: Sandhoff disease is a rare type of hereditary (autosomal recessive) GM2-gangliosidosis, which is caused by mutation of the HEXB gene. Disruption of the ß subunit of the hexosaminidase (Hex) enzyme affects the function of both the Hex-A and Hex-B isoforms. The severity and the age of onset of the disease (infantile or classic; juvenile; adult) depends on the residual activity of the enzyme. The late-onset form is characterized by diverse symptomatology, comprising motor neuron disease, ataxia, tremor, dystonia, psychiatric symptoms and neuropathy. METHODS: A 36-year-old female patient has been presenting progressive, symmetrical lower limb weakness for 9 years. Detailed neurological examination revealed mild symmetrical weakness in the hip flexors without the involvement of other muscle groups. The patellar reflex was decreased on both sides. Laboratory tests showed no relevant alteration and routine electroencephalography and brain MRI were normal. Nerve conduction studies and electromyography revealed alterations corresponding to sensory neuropathy. Muscle biopsy demonstrated signs of mild neurogenic lesion. Her younger brother (32-year-old) was observed with similar symptoms. RESULTS: Detailed genetic study detected a known pathogenic missense mutation and a 15,088 base pair long known pathogenic deletion in the HEXB gene (NM_000521.4:c.1417G>A; NM_000521:c.-376-5836_669+1473del; double heterozygous state). Segregation analysis and hexosaminidase enzyme assay of the family further confirmed the diagnosis of late-onset Sandhoff disease. CONCLUSION: The purpose of this case report is to draw attention to the significance of late-onset Sandhoff disease amongst disorders presenting with proximal predominant symmetric lower limb muscle weakness in adulthood.

Infantile onset Sandhoff disease: clinical manifestation and a novel common mutation in Thai patients.

BACKGROUND: Sandhoff disease (SD) is an autosomal recessive lysosomal storage disorder, resulting in accumulation of GM2 ganglioside, particular in neuronal cells. The disorder is caused by deficiency of ß-hexosaminidase B (HEX-B), due to pathogenic variant of human HEXB gene. METHOD: This study describes clinical features, biochemical, and genetic defects among Thai patients with infantile SD during 2008-2019. RESULTS: Five unrelated Thai patients presenting with developmental regression, axial hypotonia, seizures, exaggerated startle response to noise, and macular cherry red spot were confirmed to have infantile SD based on deficient HEX enzyme activities and biallelic variants of the HEXB gene. In addition, an uncommon presenting feature, cardiac defect, was observed in one patient. All the patients died in their early childhood. Plasma total HEX and HEX-B activities were severely deficient. Sequencing analysis of HEXB gene identified two variants including c.1652G>A (p.Cys551Tyr) and a novel variant of c.761T>C (p.Leu254Ser), in 90 and 10% of the mutant alleles found, respectively. The results from in silico analysis using multiple bioinformatics tools were in agreement that the p.Cys551Tyr and the p.Leu254Ser are likely pathogenic variants. Molecular modelling suggested that the Cys551Tyr disrupt disulfide bond, leading to protein destabilization while the Leu254Ser resulted in change of secondary structure from helix to coil and disturbing conformation of the active site of the enzyme. Genome-wide SNP array analysis showed no significant relatedness between the five affected individuals. These two variants were not present in control individuals. The prevalence of infantile SD in Thai population is estimated 1 in 1,458,521 and carrier frequency at 1 in 604. CONCLUSION: The study suggests that SD likely represents the most common subtype of rare infantile GM2 gangliosidosis identified among Thai patients. We firstly described a potential common variant in HEXB in Thai patients with infantile onset SD. The data can aid a rapid molecular confirmation of infantile SD starting with the hotspot variant and the use of expanded carrier testing.

Intravenous administration of scAAV9-Hexb normalizes lifespan and prevents pathology in Sandhoff disease mice.

Sandhoff disease (SD) is a rare inherited disorder caused by a deficiency of ß-hexosaminidase activity which is fatal because no effective treatment is available. A mouse model of Hexb deficiency reproduces the key pathognomonic features of SD patients with severe ubiquitous lysosomal dysfunction, GM2 accumulation, neuroinflammation and neurodegeneration, culminating in death at 4 months. Here, we show that a single intravenous neonatal administration of a self-complementary adeno-associated virus 9 vector (scAAV9) expressing the Hexb cDNA in SD mice is safe and sufficient to prevent disease development. Importantly, we demonstrate for the first time that this treatment results in a normal lifespan (over 700 days) and normalizes motor function assessed by a battery of behavioral tests, with scAAV9-treated SD mice being indistinguishable from wild-type littermates. Biochemical analyses in multiple tissues showed a significant increase in hexosaminidase A activity, which reached 10-15% of normal levels. AAV9 treatment was sufficient to prevent GM2 and GA2 storage almost completely in the cerebrum (less so in the cerebellum), as well as thalamic reactive gliosis and thalamocortical neuron loss in treated Hexb-/- mice. In summary, this study demonstrated a widespread protective effect throughout the entire CNS after a single intravenous administration of the scAAV9-Hexb vector to neonatal SD mice.

Murine Sialidase Neu3 facilitates GM2 degradation and bypass in mouse model of Tay-Sachs disease.

Tay-Sachs disease is a severe lysosomal storage disorder caused by mutations in Hexa, the gene that encodes for the α subunit of lysosomal ß-hexosaminidase A (HEXA), which converts GM2 to GM3 ganglioside. Unexpectedly, Hexa-/- mice have a normal lifespan and show no obvious neurological impairment until at least one year of age. These mice catabolize stored GM2 ganglioside using sialidase(s) to remove sialic acid and form the glycolipid GA2, which is further processed by ß-hexosaminidase B. Therefore, the presence of the sialidase (s) allows the consequences of the Hexa defect to be bypassed. To determine if the sialidase NEU3 contributes to GM2 ganglioside degradation, we generated a mouse model with combined deficiencies of HEXA and NEU3. The Hexa-/-Neu3-/- mice were healthy at birth, but died at 1.5 to 4.5months of age. Thin-layer chromatography and mass spectrometric analysis of the brains of Hexa-/-Neu3-/- mice revealed the abnormal accumulation of GM2 ganglioside. Histological and immunohistochemical analysis demonstrated cytoplasmic vacuolation in the neurons. Electron microscopic examination of the brain, kidneys and testes revealed pleomorphic inclusions of many small vesicles and complex lamellar structures. The Hexa-/-Neu3-/- mice exhibited progressive neurodegeneration with neuronal loss, Purkinje cell depletion, and astrogliosis. Slow movement, ataxia, and tremors were the prominent neurological abnormalities observed in these mice. Furthermore, radiographs revealed abnormalities in the skeletal bones of the Hexa-/-Neu3-/- mice. Thus, the Hexa-/-Neu3-/- mice mimic the neuropathological and clinical abnormalities of the classical early-onset Tay-Sachs patients, and provide a suitable model for the future pre-clinical testing of potential treatments for this condition.

Canine GM2-Gangliosidosis Sandhoff Disease Associated with a 3-Base Pair Deletion in the HEXB Gene.

BACKGROUND: GM2-gangliosidosis is a fatal neurodegenerative lysosomal storage disease (LSD) caused by deficiency of either ß-hexosaminidase A (Hex-A) and ß-hexosaminidase B (Hex-B) together, or the GM2 activator protein. Clinical signs can be variable and are not pathognomonic for the specific, causal deficiency. OBJECTIVES: To characterize the phenotype and genotype of GM2-gangliosidosis disease in an affected dog. ANIMALS: One affected Shiba Inu and a clinically healthy dog. METHODS: Clinical and neurologic evaluation, brain magnetic resonance imaging (MRI), assays of lysosomal enzyme activities, and sequencing of all coding regions of HEXA, HEXB, and GM2A genes. RESULTS: A 14-month-old, female Shiba Inu presented with clinical signs resembling GM2-gangliosidosis in humans and GM1-gangliosidosis in the Shiba Inu. Magnetic resonance imaging (MRI) of the dog's brain indicated neurodegenerative disease, and evaluation of cerebrospinal fluid (CSF) identified storage granules in leukocytes. Lysosomal enzyme assays of plasma and leukocytes showed deficiencies of Hex-A and Hex-B activities in both tissues. Genetic analysis identified a homozygous, 3-base pair deletion in the HEXB gene (c.618-620delCCT). CONCLUSIONS AND CLINICAL IMPORTANCE: Clinical, biochemical, and molecular features are characterized in a Shiba Inu with GM2-gangliosidosis. The deletion of 3 adjacent base pairs in HEXB predicts the loss of a leucine residue at amino acid position 207 (p.Leu207del) supporting the hypothesis that GM2-gangliosidosis seen in this dog is the Sandhoff type. Because GM1-gangliosidosis also exists in this breed with almost identical clinical signs, genetic testing for both GM1- and GM2-gangliosidosis should be considered to make a definitive diagnosis.

Suppression of NK and CD8+ T cells reduces astrogliosis but accelerates cerebellar dysfunction and shortens life span in a mouse model of Sandhoff disease.

Sandhoff disease is an inherited lysosomal storage disease, resulting from the deficiency of lysosomal ß-hexosaminidase A and B enzyme activity. The Hexb-/- mouse model recapitulates human disease and leads to fatal neurodegeneration and neuroinflammation. IL-15 is important for the proliferation of NK, NK T, and CD8+ cytotoxic/memory T cells. In order to determine how changes to IL-15-dependent immune cell populations would alter the course of Sandhoff disease in mice, we generated a Hexb-/-Il-15-/- double knockout mouse and used motor behaviour tests, analyzed peripheral blood and brain leukocyte immunophenotypes, cytokine secretion, as well as examined markers of microgliosis, astrogliosis and apoptosis. Hexb-/-Il-15-/- mice had an accelerated neurodegenerative phenotype, and reached the humane endpoint at 118±3.5d, compared to Hexb-/- mice (127±2.2d). The performance of Hexb-/-Il-15-/- mice declined earlier than Hexb-/- mice on the rotarod and righting reflex motor behaviour tests. Hexb-/- mice had a significantly higher prevalence of pro-inflammatory monocytes in the blood relative to C57BL/6 mice, but this was unaltered by IL-15 deficiency. The prevalence of NK cells and CD8+ T cells in Il-15-/- and Hexb-/-Il-15-/- mice was decreased compared to wild type and Hexb-/- mice. While Hexb-/- mice displayed an increase in the prevalence of CD4+ and CD8+ T cells in brain leukocytes compared to C57BL/6 mice, there was a decrease in CD8+ T cells in Hexb-/-Il-15-/- compared to Hexb-/- mice. In addition, circulating IL-17 and IL-10 levels were significantly higher in Hexb-/-Il-15-/- mice, suggesting heightened inflammation compared to Hexb-/- mice. Interestingly, astrogliosis levels were significantly reduced in the cerebellum of Hexb-/-Il-15-/- mice compared to Hexb-/- mice while microgliosis was not affected in brains of Hexb-/-Il-15-/- mice. Our study demonstrated that IL-15 depletion dramatically reduced numbers of NK and CD8+ T cells as well as astrocytes but accelerated disease progression in Sandhoff mice. These results pointed to interactions between NK/CD8+ T cells and astrogliosis and potentially a protective role for NK/CD8+ T cells and/or astrocytes during disease progression. This observation supports the notion that expanding the IL-15-dependent NK and CD8+ T cells populations with IL-15 therapy may have therapeutic benefits for Sandhoff disease.

Lysosomal Proteins as a Therapeutic Target in Neurodegeneration.

Several proteins that are mutated in lysosomal storage diseases are linked to neurodegenerative disease. This review focuses on some of these lysosomal enzymes and transporters, as well as current therapies that have emerged from the lysosomal storage disease field. Given the deeper genetic understanding of lysosomal defects in neurodegeneration, we explore why some of these orphan disease drug candidates are also attractive targets in subpopulations of individuals with neurodegenerative disease.

Early cardiac involvement in an infantile Sandhoff disease case with novel mutations.

INTRODUCTION: Hepatosplenomegaly is often present in infantile Sanshoff disease. However, cardiac involvement is extremely uncommon. CASE REPORT: We describe a 14-month-old female baby who exhibited mitral regurgitation and cardiomegaly at the age of 2months, dilation of the left atrium and left ventricle at age of 6months, followed by regression of developmental milestones after an episode of minor infection at age of 14months. Brain magnetic resonance imaging revealed signal changes over the bilateral thalami, bilateral cerebral white matter and left putamen. An examination of the fundus showed presence of cherry-red spots in both macular areas. The lysosomal enzymatic activities showed a marked reduction of ß-hexosaminidase B (HEXB) activity. Two novel mutations of HEXB gene were identified. One of the mutations was a c.1538 T>C mutation, which predicted a p.L513P amino acid substitution of leucine to proline; the other was a c.299+5 G>A mutation, which was a splice site mutation. CONCLUSION: Cardiac involvement might occur prior to neurological symptoms in infantile Sandhoff disease, and it should be included in the differential diagnoses of metabolic cardiomyopathies in the infantile stage.

GM2 gangliosidosis associated with a HEXA missense mutation in Japanese Chin dogs: a potential model for Tay Sachs disease.

GM2 gangliosidosis is a fatal lysosomal storage disease caused by a deficiency of ß-hexosaminidase (EC 3.2.1.52). There are two major isoforms of the enzyme: hexosaminidase A composed of an α and a ß subunit (encoded by HEXA and HEXB genes, respectively); and, hexosaminidase B composed of two ß subunits. Hexosaminidase A requires an activator protein encoded by GM2A to catabolize GM2 ganglioside, but even in the absence of the activator protein, it can hydrolyze the synthetic substrates commonly used to assess enzyme activity. GM2 gangliosidosis has been reported in Japanese Chin dogs, and we identified the disease in two related Japanese Chin dogs based on clinical signs, histopathology and elevated brain GM2 gangliosides. As in previous reports, we found normal or elevated hexosaminidase activity when measured with the synthetic substrates. This suggested that the canine disease is analogous to human AB variant of G(M2) gangliosidosis, which results from mutations in GM2A. However, only common neutral single nucleotide polymorphisms were found upon sequence analysis of the canine ortholog of GM2A from the affected Japanese Chins. When the same DNA samples were used to sequence HEXA, we identified a homozygous HEXA:c967G>A transition which predicts a p.E323K substitution. The glutamyl moiety at 323 is known to make an essential contribution to the active site of hexosaminidase A, and none of the 128 normal Japanese Chins and 92 normal dogs of other breeds that we tested was homozygous for HEXA:c967A. Thus it appears that the HEXA:c967G>A transition is responsible for the GM2 gangliosidosis in Japanese Chins.

A frameshift mutation in the canine HEXB gene in toy poodles with GM2 gangliosidosis variant 0 (Sandhoff disease).

GM2 gangliosidosis variant 0 (Sandhoff disease, SD) is a fatal, progressive neurodegenerative lysosomal storage disease caused by mutations in the HEXB gene. Toy poodles recently were reported as the second breed of dog with SD. The present paper describes the molecular defect of this canine SD as the first identification of a pathogenic mutation in the canine HEXB gene. Genomic and complementary DNA sequences covering exonic regions of the canine HEXB gene, except exon 1, were analysed using DNA and RNA in an affected dog. A homozygous single base pair deletion of guanine in exon 3 was identified at nucleotide position 283 of the putative open reading frame (c.283delG). This mutation has the potential to cause a frameshift resulting in the alteration of valine at amino acid position 59 to a stop codon (p.V59fsX). Genotyping using the mutagenically separated PCR method demonstrated a correlation between phenotype and genotype in dogs with a pedigree related to the disease and that the mutation was rare in a randomly-selected population of toy poodles. These results strongly suggest that the deletion is pathogenic.

GM2 gangliosidoses in Spain: analysis of the HEXA and HEXB genes in 34 Tay-Sachs and 14 Sandhoff patients.

The GM2 gangliosidoses are autosomal recessive lysosomal storage diseases caused by a deficiency of the ß-hexosaminidase A enzyme. This enzyme is composed of two polypeptide chains designated the α- and ß- subunits and it interacts with the GM2 activator protein. The HEXA and HEXB genes encode the α-subunit and the ß-subunit, respectively. Mutations in these genes are causative of Tay-Sachs disease (HEXA) and Sandhoff disease (HEXB). We analyzed the complete HEXA gene in 34 Spanish patients with Tay-Sachs disease and the HEXB gene in 14 Spanish patients with Sandhoff disease. We identified 27 different mutations, 14 of which were novel, in the HEXA gene and 14 different mutations, 8 of which unreported until now, in the HEXB gene, and we attempted to correlate these mutations with the clinical presentation of the patients. We found a high frequency of c.459+5G>A (IVS4+5G>A) mutation in HEXA affected patients, 22 of 68 alleles, which represent the 32.4%. This is the highest percentage found of this mutation in a population. All patients homozygous for mutation c.459+5G>A presented with the infantile form of the disease and, as previously reported, patients carrying mutation p.R178H in at least one of the alleles presented with a milder form. In HEXB affected patients, the novel deletion c.171delG accounts for 21.4% of the mutant alleles (6/28). All patients with this deletion showed the infantile form of the disease. The Spanish GM2 gangliosidoses affected patients show a great mutational heterogeneity as seen in other inherited lisosomal diseases in this country.

Lysosomal dysfunction in a mouse model of Sandhoff disease leads to accumulation of ganglioside-bound amyloid-ß peptide.

Alterations in the lipid composition of endosomal-lysosomal membranes may constitute an early event in Alzheimer's disease (AD) pathogenesis. In this study, we investigated the possibility that GM2 ganglioside accumulation in a mouse model of Sandhoff disease might be associated with the accumulation of intraneuronal and extracellular proteins commonly observed in AD. Our results show intraneuronal accumulation of amyloid-ß peptide (Aß)-like, α-synuclein-like, and phospho-tau-like immunoreactivity in the brains of ß-hexosaminidase knock-out (HEXB KO) mice. Biochemical and immunohistochemical analyses confirmed that at least some of the intraneuronal Aß-like immunoreactivity (iAß-LIR) represents amyloid precursor protein C-terminal fragments (APP-CTFs) and/or Aß. In addition, we observed increased levels of Aß40 and Aß42 peptides in the lipid-associated fraction of HEXB KO mouse brains, and intraneuronal accumulation of ganglioside-bound Aß (GAß) immunoreactivity in a brain region-specific manner. Furthermore, α-synuclein and APP-CTFs and/or Aß were found to accumulate in different regions of the substantia nigra, indicating different mechanisms of accumulation or turnover pathways. Based on the localization of the accumulated iAß-LIR to endosomes, lysosomes, and autophagosomes, we conclude that a significant accumulation of iAß-LIR may be associated with the lysosomal-autophagic turnover of Aß and fragments of APP-containing Aß epitopes. Importantly, intraneuronal GAß immunoreactivity, a proposed prefibrillar aggregate found in AD, was found to accumulate throughout the frontal cortices of postmortem human GM1 gangliosidosis, Sandhoff disease, and Tay-Sachs disease brains. Together, these results establish an association between the accumulation of gangliosides, autophagic vacuoles, and the intraneuronal accumulation of proteins associated with AD.

Gene transfer corrects acute GM2 gangliosidosis--potential therapeutic contribution of perivascular enzyme flow.

The GM2 gangliosidoses are fatal lysosomal storage diseases principally affecting the brain. Absence of ß-hexosaminidase A and B activities in the Sandhoff mouse causes neurological dysfunction and recapitulates the acute Tay-Sachs (TSD) and Sandhoff diseases (SD) in infants. Intracranial coinjection of recombinant adeno-associated viral vectors (rAAV), serotype 2/1, expressing human ß-hexosaminidase α (HEXA) and ß (HEXB) subunits into 1-month-old Sandhoff mice gave unprecedented survival to 2 years and prevented disease throughout the brain and spinal cord. Classical manifestations of disease, including spasticity-as opposed to tremor-ataxia-were resolved by localized gene transfer to the striatum or cerebellum, respectively. Abundant biosynthesis of ß-hexosaminidase isozymes and their global distribution via axonal, perivascular, and cerebrospinal fluid (CSF) spaces, as well as diffusion, account for the sustained phenotypic rescue-long-term protein expression by transduced brain parenchyma, choroid plexus epithelium, and dorsal root ganglia neurons supplies the corrective enzyme. Prolonged survival permitted expression of cryptic disease in organs not accessed by intracranial vector delivery. We contend that infusion of rAAV into CSF space and intraparenchymal administration by convection-enhanced delivery at a few strategic sites will optimally treat neurodegeneration in many diseases affecting the nervous system.

Highly phosphomannosylated enzyme replacement therapy for GM2 gangliosidosis.

OBJECTIVE: Novel recombinant human lysosomal ß-hexosaminidase A (HexA) was developed for enzyme replacement therapy (ERT) for Tay-Sachs and Sandhoff diseases, ie, autosomal recessive GM2 gangliosidoses, caused by HexA deficiency. METHODS: A recombinant human HexA (Om4HexA) with a high mannose 6-phosphate (M6P)-type-N-glycan content, which was produced by a methylotrophic yeast strain, Ogataea minuta, overexpressing the OmMNN4 gene, was intracerebroventricularly (ICV) administered to Sandhoff disease model mice (Hexb⁻/⁻ mice) at different doses (0.5-2.5 mg/kg), and then the replacement and therapeutic effects were examined. RESULTS: The Om4HexA was widely distributed across the ependymal cell layer, dose-dependently restored the enzyme activity due to uptake via cell surface cation-independent M6P receptor (CI-M6PR) on neural cells, and reduced substrates, including GM2 ganglioside (GM2), asialo GM2 (GA2), and oligosaccharides with terminal N-acetylglucosamine residues (GlcNAc-oligosaccharides), accumulated in brain parenchyma. A significant inhibition of chemokine macrophage inflammatory protein-1 α (MIP-1α) induction was also revealed, especially in the hindbrain (< 63%). The decrease in central neural storage correlated with an improvement of motor dysfunction as well as prolongation of the lifespan. INTERPRETATION: This lysosome-directed recombinant human enzyme drug derived from methylotrophic yeast has the high therapeutic potential to improve the motor dysfunction and quality of life of the lysosomal storage diseases (LSDs) patients with neurological manifestations. We emphasize the importance of neural cell surface M6P receptor as a delivery target of neural cell-directed enzyme replacement therapy (NCDERT) for neurodegenerative metabolic diseases.

Rapid and simple polymerase chain reaction-based diagnostic assays for GM2 gangliosidosis variant 0 (Sandhoff-like disease) in Japanese domestic cats.

Polymerase chain reaction (PCR)-based assays combined with microchip electrophoresis were developed and evaluated for diagnosis and genotyping of GM2 gangliosidosis variant 0 (Sandhoff-like disease) in Japanese domestic cats. A preliminary genotyping survey was carried out in the population of Japanese domestic cats (1,015 cats in total) in southern Japan. Three kinds of assays including PCR primer-induced restriction analysis (PIRA) and mutagenically separated (MS)-PCR were carried out using blood-stained Flinders Technology Associates filter papers (FTA cards) as templates. The PCR products were analyzed by both agarose gel and microchip electrophoreses. All assays were sufficient to determine the genotypes of this disease, but MS-PCR offered the most rapid and simplest test, as it does not need the restriction enzyme step required in PCR-PIRA. The use of microchip electrophoresis in combination with FTA cards for sampling could shorten the time required for genotyping and simplify the procedure as well. The genotyping survey in the current study did not find any cats that possessed the mutant allele, suggesting that the prevalence of this allele is low (<0.1%) in southern Japan.

Substrate deprivation therapy in juvenile Sandhoff disease.

Substrate deprivation therapy has been successfully applied in a number of lysosomal storage diseases, such as Gaucher disease. So far only limited experience is available in Sandhoff disease. We initiated substrate deprivation therapy in one male patient, who initially presented at the age of 3.5 years with epilepsy and regression in motor skills and speech development. Juvenile Sandhoff disease was diagnosed on the basis of a decreased hexosaminidase activity in leukocytes and a homozygous HEXB gene mutation. After the epilepsy was controlled, the clinical course remained stable for years, defined by a mild proximal myopathy and stable mental retardation. At 14 years of age the patient experienced a second episode with progressively worsening general condition with diminishing muscle power and progressive ataxia. Treatment was started with the N-alkylated imino sugar miglustat, inhibiting the glucosylceramide synthase, an essential enzyme for the synthesis of glycosphingolipids. Diarrhoea was treated with lactose restriction. We performed detailed biochemical investigations, motor and mental development analysis, brain imaging, organ function studies and quality of life score prior to and at different time points after start of the treatment. Two years after the initiation of therapy the patient has a stable neurological picture without further regression in his motor development, ataxia or intelligence. There is a subjective improvement in the fine motor skills and walking up the stairs but no change in the quality of life score. Under treatment with miglustat the clinical course in our patient with Sandhoff disease did not further deteriorate.