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Fabry disease is a glycosphingolipid storage disorder that is caused by a genetic deficiency of the lysosomal enzyme alpha-galactosidase A (AGA, EC 3.2.1.22). As a result, the glycolipid substrate, globotriaosylceramide (Gb3) accumulates in various cell types throughout the body producing a multisystem disease that affects the vascular, cardiac, renal, and nervous systems. A hallmark of this disorder is neuropathic pain that occurs in up to 80% of Fabry patients and has been characterized as a small fiber neuropathy. The molecular mechanism by which changes in AGA activity produce neuropathic pain is not clear, in part due to a lack of relevant model systems. Using 50B11 cells, an immortalized dorsal root ganglion neuron with nociceptive characteristics derived from rat, we used CRISPR-Cas9 gene editing of the galactosidase alpha (

Fabry disease is a glycosphingolipid storage disorder that is caused by a genetic deficiency of the enzyme alpha-galactosidase A (AGA, EC 3.2.1.22). It is a multisystem disease that affects the vascular, cardiac, renal, and nervous systems. One of the hallmarks of this disorder is neuropathic pain and sympathetic and parasympathetic nervous dysfunction. The exact mechanism by which changes in AGA activity result in change in neuronal function is not clear, partly due to of a lack of relevant model systems. In this study, we report the development of an in vitro model system to study neuronal dysfunction in Fabry disease by using short-hairpin RNA to create a stable knock-down of AGA in the human cholinergic neuronal cell line, LA-N-2. We show that gene-silenced cells show specifically reduced AGA activity and store globotriaosylceramide. In gene-silenced cells, release of the neurotransmitter acetylcholine is significantly reduced, demonstrating that this model may be used to study specific neuronal functions such as neurotransmitter release in Fabry disease.

Fabry disease is an X-linked disorder caused by mutations in the GLA gene encoding for alpha-galactosidase A (AGA, EC 3.2.1.22). Measurement of AGA enzyme activity using cell homogenates can easily identify men with Fabry disease, but in women, the degree of X-inactivation in the tested tissue may produce activities in homogenates that are indistinguishable from normal. Monti et al. developed a series of lissamine rhodamine-labeled glycosphingolipid substrates that can be used to measure clearance of these lipids in intact cells (1). We report here that one of these substrates, lissamine rhodamine ceramide trihexoside (LR-CTH), can be used as a probe for functional activity of AGA in intact fibroblasts, endothelial cells, and T-lymphocytes from patients with Fabry disease. By utilizing standard detection techniques, such as microscopic imaging, fluorescence microplate spectrophotometry, and flow cytometry, cells with impaired AGA activity can easily be distinguished from wild-type (WT) cells, and these two cell types can be isolated into separate populations using fluorescence-activated cell sorting (FACS). The assay we report here can be adapted to evaluate new therapies by high-throughput screening, can aid in the study of AGA activity in living cells, and can assist in the diagnosis of women with the Fabry trait.

Contains fulltext : 80646.pdf (Publisher’s version ) (Closed access) We performed high-resolution in vitro proton nuclear magnetic resonance spectroscopy on cerebrospinal fluid and urine samples of 44 patients with leukodystrophies of unknown cause. Free sialic acid concentration was increased in cerebrospinal fluid of two siblings with mental retardation and mild hypomyelination. By contrast, urinary excretion of free sialic acid in urine was normal on repeated testing by two independent methods. Both patients were homozygous for the K136E mutation in SLC17A5, the gene responsible for the free sialic acid storage diseases. Our findings demonstrate that mutations in the SLC17A5 gene have to be considered in patients with hypomyelination, even in the absence of sialuria.

To examine the relationship between types and locations of mutations of the enzyme alpha-galactosidase (Gal) A in Fabry disease and the response to the pharmacological chaperone 1-deoxygalactonojirimycin (DGJ).T cells grown from normal individuals or from patients with Fabry disease were tested for response to treatment with DGJ by increased activity of alpha-Gal A.Cells from normal controls responded with a 28% increase in alpha-Gal A activity, whereas response in Fabry individuals was mutation dependent ranging from no increase to fully normal activity. Nine truncation mutations (all nonresponsive) and 31 missense mutations were tested. Three groups of missense mutations were categorized: responders with activity more than 25% of normal, nonresponders, with less than 7% and an intermediate response group. In normal cells and in responders an increase in the mature lysosomal form of alpha-Gal A was observed after DGJ treatment. Nonresponders showed little or no protein with or without DGJ. The intermediate response group showed an increase in band intensity but incomplete processing of the enzyme to the mature form.Mapping the missense mutations to the structure of alpha-Gal A identified several factors that may influence response. Mutations in regions that are not in alpha-helix or beta-sheets, neither involved in disulfide bonds nor with an identified functional or structural role were more likely to respond. Predictability is, however, not precise and testing of each mutation for response to pharmacological chaperone therapy is necessary for Fabry disease and related lysosomal storage disorders.

Fabry disease is an inborn error of glycosphingolipid catabolism resulting from a deficiency of lysosomal enzyme alpha-galactosidase A. The major clinical manifestations of the disease, such as stroke, cardiac dysfunction, and renal impairment, are thought to be caused by vasculopathy due to progressive accumulation of globotriaosylceramide in vascular endothelial cells. The pathogenesis of the vasculopathy has not been elucidated. Since in vitro studies using primary endothelial cells are hampered by the limited lifespan of these cells, the availability of cultured endothelial cells with an extended lifespan is critical for the study of the vasculopathy of Fabry disease. We therefore generated an endothelial cell line from a Fabry hemizygote by introduction of human telomerase reverse transcriptase gene. The cell line has markedly extended lifespan compared to parental primary cells. The cells stably express many key markers of endothelial cells such as von Willebrand factor, CD31, CD34, and endothelial nitric oxide synthase (eNOS) and retain functional characteristics such as uptake of acetylated low-density lipoprotein, responsiveness to angiogenic growth factors, up-regulation of eNOS production upon extracellular stimuli, and formation of tube-like structures on Matrigel basement membrane matrix. The cells show significantly reduced activity of alpha-galactosidase A compared with primary endothelial cells from normal individuals and accumulate globotriaosylceramide in lysosomes. This cell line will provide a useful in vitro model of Fabry disease and will facilitate systematic studies to investigate pathogenic mechanisms and explore new therapeutic approaches for Fabry disease.

As a prerequisite for clinical trials of pharmacological chaperone therapy (PCT) for Fabry disease, we developed a rapid screening assay for enhancement of endogenous alpha-galactosidase A (alpha-Gal A) in patient-derived cells. We used a T-cell based system to screen 11 mutations causing Fabry disease for enhanceability using 1-deoxygalactonojirimycin (DGJ). When patient-derived T-cells were grown in the presence of DGJ, alpha-Gal A activity increased to more than 50% of normal in several mutations but was unaffected in others. In addition to the mutation R301Q, reported previously, A97V, R112H, R112C, A143T, and L300P were enhanceable, but R356W, G132R, A143P, R220X, and 30delG were not. The level of alpha-Gal A activity achieved provides a basis for the therapeutic trial of DGJ in patients with similarly enhanceable enzyme. This assay method has general utility in other disorders in assessing the degree of enhancement of activity of mutated proteins by PCT.

Fabry disease is an X-linked disorder affecting both males and females. It is associated with an increased risk of stroke in up to 4% of patients below 55 years of age in the general population. The cerebral vasculopathy consists of ischemic strokes involving large and small vessels. The neuronal accumulation of glycosphingolipids appears to have no clinical effect on the natural history of Fabry disease with the possible exception of some reported mild cognitive abnormalities. The pathogenesis of Fabry vasculopathy remains poorly understood but is associated with abnormal functional control of the vessel secondary to endothelial dysfunction, cerebral hyper-perfusion and a prothrombotic state with likely increased production of reactive oxygen species. These abnormalities are further modified by genetic and possibly other vascular risk factors. This vasculopathy illustrates the role of glycolipids in this and possibly other types of cerebral vasculopathies. Therapy is preventive relying on standard medical care and in particular on anti-platelet agents such as clopidogrel.

We identified four unrelated patients (three female, one male) aged 20 to 30 years with hypomyelination, pituitary hypogonadotropic hypogonadism, and hypodontia. Electron microscopy and myelin protein immunohistochemistry of sural nerves showed granular debris-lined clefts, expanded abaxonal space, outpocketing with vacuolar disruption, and loss of normal myelin periodicity. Reduced galactocerebroside, sphingomyelin, and GM1-N-acetylglucosamine and increased esterified cholesterol were found. This is a clinically homogeneous progressive hypomyelinating disorder. The term 4H syndrome is suggested.

The properties of the endolysosomal enzyme GCase (glucosylceramidase), carrying the most prevalent mutation observed in Gaucher patients, namely substitution of an asparagine residue with a serine at amino acid position 370 [N370S (Asn370→Ser) GCase], were investigated in the present study. We previously demonstrated that Sap (saposin) C, the physiological GCase activator, promotes the association of GCase with anionic phospholipid-containing membranes, reconstituting in this way the enzyme activity. In the present study, we show that, in the presence of Sap C and membranes containing high levels of anionic phospholipids, both normal and N370S GCases are able to associate with the lipid surface and to express their activity. Conversely, when the amount of anionic phospholipids in the membrane is reduced (∼20% of total lipids), Sap C is still able to promote binding and activation of the normal enzyme, but not of N370S GCase. The altered interaction of the mutated enzyme with anionic phospholipid-containing membranes and Sap C was further demonstrated in Gaucher fibroblasts by confocal microscopy, which revealed poor co-localization of N370S GCase with Sap C and lysobisphosphatidic acid, the most abundant anionic phospholipid in endolysosomes. Moreover, we found that N370S Gaucher fibroblasts accumulate endolysosomal free cholesterol, a lipid that might further interfere with the interaction of the enzyme with Sap C and lysobisphosphatidic acid-containing membranes. In summary, our results show that the N370S mutation primarily affects the interaction of GCase with its physiological activators, namely Sap C and anionic phospholipid-containing membranes. We thus propose that the poor contact between N370S GCase and its activators may be responsible for the low activity of the mutant enzyme in vivo.

Background: Recessive mutations in the five eucaryotic initiation factor 2B (eIF2B) subunits have been found in leukodystrophies of variable age at onset and severity.Objectives: To evaluate the clinical spectrum of eIF2B-related disorders and search for a phenotype-genotype correlation.Methods: Ninety-three individuals (78 families) with an undetermined leukodystrophy were selected on MRI-based criteria of childhood ataxia with central hypomyelination/vanishing white matter (CACH/VWM) for EIF2B genes analysis.Results: Eighty-nine percent of individuals with MRI criteria of CACH/VWM have a mutation in one of the eIF2B beta to epsilon subunits. For 83 individuals (68 families), 46 distinct mutations (90% missense) in four of the five eIF2B subunits (beta, gamma, delta, epsilon) were identified. Sixty-four percent were in the epsilon subunit, a R113H substitution was found in 71% of eIF2B epsilon-mutated families. A large clinical spectrum was observed from rapidly fatal infantile to asymptomatic adult forms. Disease severity was correlated with age at onset (p < 0.0001) but not with the type of the mutated subunit nor with the position of the mutation within the protein. Mutations R113H in the epsilon subunit and E213G in the beta subunit were significantly associated with milder forms.Conclusions: The degree of eIF2B dysfunction, which is involved in the regulation of protein synthesis during cellular stress, may play a role in the clinical expression of eIF2B-related disorders.

A patient with mucolipidosis-IV heterozygous for two mutations in MCOLN1 expressed only her father's cDNA mutation c.1207C>T predicting an R403C change in mucolipin. She inherited a 93bp segment from mitochondrial NADH dehydrogenase 5 (MTND5) from her mother that was inserted in-frame prior to the last nucleotide of exon 2 of MCOLN1 (c.236_237ins93). This alteration abolished proper splicing of MCOLN1. The splice site at the end of the exon was not used due to an inhibitory effect of the inserted segment, resulting in two aberrant splice products containing stop codons in the downstream intron. These products were eliminated via nonsense-mediated decay. This is the first report of an inherited transfer of mitochondrial nuclear DNA causing a genetic disease. The elimination of the splice site by the mitochondrial DNA requires a change in splicing prediction models.

Two brothers with a leukodystrophy, progressive spastic diplegia, and peripheral neuropathy were found to have proteinaceous aggregates in the peripheral nerve myelin sheath. The patients' mother had only subclinical peripheral neuropathy, but the maternal grandmother had adult-onset leukodystrophy. Sequencing of the proteolipid protein (PLP) gene showed a point mutation IVS4 + 1 G-->A within the donor splice site of intron 4. We identified one transcript with a deletion of exon 4 (Deltaex4, 169bp) encoding for PLP and DM20 proteins and lacking two transmembrane domains, and a second transcript with exon 4 + 10bp encoding three transmembrane domains. Immunohistochemistry showed abnormal aggregation in the myelin sheath of MBP and P0. Myelin-associated glycoprotein was present in the Schmidt-Lanterman clefts but significantly reduced in the periaxonal region. Using immunogold electron microscopy, we demonstrated the presence of mutated PLP/DM20 and the absence of the intact protein in the patient peripheral myelin sheath. We conclude that insertion of mutant PLP/DM20 with resulting aberrant distribution of other myelin proteins in peripheral nerve may constitute an important mechanism of dysmyelination in disorders associated with PLP mutations.

Lentiviral vectors are prime candidate vectors for gene transfer into dividing and non-dividing cells, including neuronal cells and stem cells. For safety, HIV-2 lentiviral vectors may be better suited for gene transfer in humans than HIV-1 lentiviral vectors. HIV-2 vectors cross-packaged in HIV-1 cores may be even safer. Demonstration of the efficacy of these vectors in disease models will validate their usefulness. Parkinson's disease and Fabry disease provide excellent models for validation. Parkinson's disease is a focal degeneration of dopaminergic neurons in the brain with progressive loss of ability to produce the neurotransmitter dopamine. Current treatment entails administration of increasing doses of L-dopa, with attendant toxicity. We explore here the hypothesis that gene transfer of aromatic acid decarboxylase (AADC), a key enzyme in the pathway, will make neuronal cells more efficiently convert L-dopa into dopamine. Fabry disease on the other hand is a monogenic inherited disease, characterized by α-galactosidase A (AGA) deficiency, resulting in glycolipid accumulation in several cell types, including fibroblasts. Animal models for preclinical investigations of both of these diseases are available. We have designed monocistronic HIV-1 and HIV-2 vectors with the AADC transgene and monocistronic and bicistronic HIV-2 vectors with the AGA and puromycin resistance transgenes. They were packaged with either HIV-2 cores or HIV-1 cores (hybrid vectors). Gene transfer of AADC gene in neuronal cells imparted the ability on the transduced cells to efficiently convert L-dopa into dopamine. Similarly, the AGA vectors induced Fabry fibroblasts to produce high levels of AGA enzyme and caused rapid clearance of the glycolipids from the cells. Both monocistronic and bicistronic vectors were effective. Thus, the insertion of a second gene downstream in the bicistronic vector was not deleterious. In addition, both the self-packaged vectors and the cross-packaged hybrid vectors were effective in gene transfer. J. Med. Virol. 71:173–182, 2003. © 2003 Wiley-Liss, Inc.

Fabry disease is an X-linked sphingolipid storage disorder caused by a deficiency of the lysosomal enzyme α-galactosidase A (AGA, EC 3.2.1.22) resulting in the intracellular accumulation of globotriaosylceramide (Gb3). We found that Gb3 storage also correlates with accumulation of endosomal-lysosomal cholesterol in Fabry fibroblasts. This cholesterol accumulation may contribute to the phenotypic pathology of Fabry disease by slowing endosomal-lysosomal trafficking. We found that LDL receptor expression is not downregulated in Fabry fibroblasts resulting in accumulation of both cholesterol and Gb3. 5A-Palmitoyl oleoyl-phosphatidylcholine (5AP) is a phospholipid complex containing a short synthetic peptide that mimics apolipoprotein A1, the main protein component of high-density lipoprotein (HDL) that mediates the efflux of cholesterol from cells via the ATP-binding cassette transporter. We used 5AP and HDL to remove cholesterol from Fabry fibroblasts to examine the fate of accumulated cellular Gb3. Using immunostaining techniques, we found that 5AP is highly effective for depleting cholesterol and Gb3 in these cells. 5AP restores the ApoA-1-mediated cholesterol efflux leading to mobilization of cholesterol and reduction of Gb3 in Fabry fibroblasts.

Background Fabry disease is an X-linked lysosomal deficiency of α-galactosidase A that results in cellular accumulation of galacto-conjugates such as globotriosylceramide, particularly in blood vessels. It is associated with early-onset stroke and kidney and heart failure. Methods and Results Using [ 15 O] H 2 O and PET, we found increased resting regional cerebral blood flow in Fabry disease without evidence of occlusive vasculopathy or cerebral hypoperfusion. Because nitric oxide is known to play an important role in vascular tone and reactivity, we studied plasma nitrate, nitrite, and low-molecular-weight S -nitrosothiol levels by chemiluminescence. Skin biopsy specimens and archived brain tissue were also examined immunohistochemically for nitrotyrosine. Plasma nitrate, nitrite, and low-molecular-weight S -nitrosothiol were in the normal range; however, enhanced nitrotyrosine staining was observed in dermal and cerebral blood vessels. After a double-blind, placebo-controlled trial of α-galactosidase A therapy, the resting regional cerebral blood flow in the treated group was significantly reduced, with a notable decrease of nitrotyrosine staining in dermal blood vessels. Conclusions These findings suggest a chronic alteration of the nitric oxide pathway in Fabry disease, with critical protein nitration that is reversible with enzyme replacement therapy.

Mucolipidosis type IV (MLIV) is a developmental neurodegenerative disorder characterized by severe neurologic and ophthalmologic abnormalities. The MLIV gene, ML4 (MCOLN1), has recently been localized to chromosome 19p13.2-13.3 by genetic linkage. Here we report the cloning of a novel transient receptor potential cation channel gene and show that this gene is mutated in patients with the disorder. ML4 encodes a protein, which we propose to call mucolipin, which has six predicted transmembrane domains and is a member of the polycystin II subfamily of the Drosophila transient receptor potential gene family. The role of a potential receptor-stimulated cation channel defect in the pathogenesis of mucolipidosis IV is discussed.

Mucolipidosis IV (MLIV) is an autosomal recessive disorder of unknown etiology characterized by severe visual impairment and psychomotor retardation. Recently, there has been considerable interest in positional cloning of the MLIV gene. It is unknown whether MLIV is a genetically homogenous disorder. In this paper, we present experiments that determined whether the MLIV phenotype in fibroblasts could be corrected by fusing normal cells to MLIV cells and fusing fibroblasts from pairs of patients. All of our MLIV patients fulfilled several diagnostic criteria that we developed. In addition, we found high sensitivity to chloroquine in cultured fibroblasts from MLIV patients. We found that normal cells corrected the MLIV phenotype. Fusion products of normal and MLIV fibroblasts, but not MLIV fibroblasts themselves, were relatively protected against chloroquine selection. In addition, 74% of the normal-to-patient fusion products had reduced levels or total loss of MLIV characteristic autofluorescence. However, there was no complementation of the phenotype in fibroblast cultures from any of our MLIV patients, including those of non-Jewish ancestry. In fusion products of MLIV cultures from 24 patients, 90–100% of the cells remained autofluorescent. These results indicate that all of our known MLIV patients, regardless of ancestry or severity of the developmental defect, have a single mutated gene.

Fabry disease results in a global vasculopathy leading to early-onset stroke and renal and cardiac failure. We found that random myeloperoxidase in serum and plasma was significantly elevated in 73 consecutive male patients with Fabry disease. Random serum myeloperoxidase level in men predicted the risk of a Fabry vasculopathy–related event in subsequent years. Long-term enzyme replacement therapy did not reduce myeloperoxidase level or eliminate the risk of vasculopathic events.

Niemann-Pick type C (NP-C) disease, a fatal neurovisceral disorder, is characterized by lysosomal accumulation of low density lipoprotein (LDL)–derived cholesterol. By positional cloning methods, a gene (NPC1)with insertion, deletion, and missense mutations has been identified in NP-C patients. Transfection of NP-C fibroblasts with wild-typeNPC1cDNA resulted in correction of their excessive lysosomal storage of LDL cholesterol, thereby defining the critical role of NPC1 in regulation of intracellular cholesterol trafficking. The 1278–amino acid NPC1 protein has sequence similarity to the morphogen receptor PATCHED and the putative sterol-sensing regions of SREBP cleavage-activating protein (SCAP) and 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase.

We describe clinical, biochemical, pathological, and spectroscopic findings in 4 women, aged 15 to 29 years, from three unrelated families who had a unique combination of a central nervous system white matter disease and primary ovarian failure. All had normal initial development but 3 had borderline low IQ and academic difficulties in primary school. Puberty did not develop in 2 patients and was arrested in a third patient. The fourth patient had premature ovarian failure at the age of 13 years. Head magnetic resonance imaging showed diffuse white matter disease, with frontal cortical atrophy in the most clinically advanced patient. All patients had normal karyotype and normal findings on extensive evaluations for known leukodystrophies, for other metabolic diseases, and for causes of ovarian failure. Proton magnetic resonance spectroscopic imaging showed reduction of choline-containing compounds in the affected white matter in all patients and reduction of N-acetylaspartate in the unaffected frontal white matter of 2 patients. All patients had evidence of primary gonadal insufficiency with a normal hypothalamic-hypophyseal axis. Pathological analysis showed streak ovaries in 1 patient and signs of hypomyelination, and gliosis on brain biopsy in another patient. In conclusion, we present a novel group of patients who have in common leukodystrophy, primary ovarian dysfunction, and magnetic resonance spectroscopic abnormalities.

With the goal of improving the detection of lysosomal sphingolipid hydrolases within intact cells, we have recently synthesized a new fluorophor, O-[4-(1-imidazolyl)butyl]-2,3-dicyano-1,4-hydroquinonyl beta-D-galactopyranoside (Im-DCH-beta-Gal). In the present study, we evaluated the interaction of Im-DCH-beta-Gal and its tetraacetate derivative, Im-DCH-beta-Gal(OAc)4, with living human fibroblasts. Im-DCH-beta-Gal was shown to be a specific substrate for human lysosomal beta-galactosidase in cell homogenates. Im-DCH-beta-Gal(OAc)4 was taken up and hydrolyzed by normal fibroblasts under physiological culture conditions. Very little hydrolysis of Im-DCH-beta-Gal(OAc)4 was observed in fibroblasts genetically deficient in lysosomal acid beta-galactosidase or in normal cells pretreated with the lysosomal inhibitors chloroquine and ammonium chloride. Analysis of substrate processing by cells indicated that normal and acid beta-galactosidase-deficient cells showed similar rates of uptake and deacetylation of Im-DCH-beta-Gal(OAc)4, with an 80% decrease in the rate of deglycosylation of substrate by beta-galactosidase-deficient fibroblasts. However, under our conditions, the fluorescent product was not well retained by cells. Our results indicate that this novel class of compounds may be useful in measuring lysosomal enzyme function in intact cells and may have application as a fluorescent marker for genetically altered cells.

Gaucher disease (GD), the most common lysosomal storage disorder of humans, is caused by mutations in the gene coding for the enzyme glucocerebrosidase (GCase). Clinical manifestations vary among patients with the three types of GD, and phenotypic heterogeneity occurs even among patients with identical mutations. To gain insight into why phenotypic heterogeneity occurs in GD, we investigated mechanisms underlying the net loss of GCase catalytic activity in cultured skin fibroblasts derived from patients with the three types of GD. The findings indicate that the loss of catalytic activity of GCase correlates with its quantitative reduction, rather than a decrease in functional capacity of mutant enzyme. Use of a proteasome inhibitor, lactacystin, resulted in increased expression of GCase, suggesting a mechanism of protein degradation in GD. Furthermore, reduced binding of GCase to TCP1 ring complex (TRiC), a regulator of correct protein folding, may result in defective maturation of nascent GCase in GD cells. Additionally, increased interaction between GCase and c-Cbl, an E3 ubiquitin ligase, may be involved in the degradation and loss of GCase in GD. The findings suggest that specific molecular mediators involved in GCase maturation and degradation could be responsible for phenotypic variation among patients with the same genotypes and that these mediators could be therapeutically targeted to increase GCase activity in patients with GD.

Fluorogenic substrates that can be used to detect the activity of enzymes within intact cells are needed for biochemical investigations of human lysosomal storage disorders and identification of gene transfer recombinants. The first member of a new series of lysosomotropic fluorescent probes has been synthesized and characterized. This compound, O-[4-(1-imidazolyl) butyl]-2,3-dicyano-1,4-hydroquinonyl β-D-galactopyranoside (Im-DCH-β-Gal), and its aglycon, Im-DCH, contain an N-alkylimidazole as a lysosomotropic substituent. The concentration of such moderately basic amines within acidic cellular organelles should provide increased specificity toward lysosomal hydrolases. The physiochemical properties of Im-DCH, in particular the intensity of its fluorescence at acidic pH's, make its glycosides well suited for the measurement of enzymatic activity within intact cells

Fluorogenic substrates that can be used to detect the activity of enzymes within intact cells are needed for biochemical investigations of human lysosomal storage disorders and identification of gene transfer recombinants. The first member of a new series of lysosomotropic fluorescent probes has been synthesized and characterized. This compound, O-[4-(1-imidazolyl) butyl]-2,3-dicyano-1,4-hydroquinonyl β-D-galactopyranoside (Im-DCH-β-Gal), and its aglycon, Im-DCH, contain an N-alkylimidazole as a lysosomotropic substituent. The concentration of such moderately basic amines within acidic cellular organelles should provide increased specificity toward lysosomal hydrolases. The physiochemical properties of Im-DCH, in particular the intensity of its fluorescence at acidic pH's, make its glycosides well suited for the measurement of enzymatic activity within intact cells

Fabry disease, an X-linked systemic vasculopathy, is caused by a deficiency of alpha-galactosidase A resulting in globotriaosylceramide (Gb(3)) storage in cells. The pathogenic role of Gb(3) in the disease is not known. Based on previous work, we tested the hypothesis that accumulation of Gb(3) in the vascular endothelium of Fabry disease is associated with increased production of reactive oxygen species (ROS) and increased expression of cell adhesion molecules. Gb(3)-loading resulted in increased intracellular ROS production in cultured vascular endothelial cells in a dose-dependent manner. Increased Gb(3) also induced expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin. Reduction of endogenous Gb(3) by treatment of the cells with an inhibitor of glycosphingolipid synthase or alpha-galactosidase A led to decreased expression of adhesion molecules. Plasma from Fabry patients significantly increased ROS generation in endothelial cells when compared with plasma from non-Fabry controls. This effect was not influenced by reduction of intracellular Gb(3). This study provided direct evidence that excess intracellular Gb(3) induces oxidative stress and up-regulates the expression of cellular adhesion molecules in vascular endothelial cells. In addition, other factors in patient's plasma may also contribute to oxidative stress in Fabry vascular endothelial cells.

PURPOSE. To evaluate a 15-year-old boy with MLIV (mucolipidosis type IV) and clinical abnormalities restricted to the eye who also had achlorhydria with elevated blood gastrin levels. METHODS. In addition to a detailed neuro-ophthalmic and electrophysiological assessment, his mutant mucolipin-1 was experimentally expressed in liposomes and its channel properties studied in vitro. RESULTS. The patient was a compound heterzygote for c.920delT and c.1615delG. Detailed neuro-ophthalmic examination including electroretinography showed him to have a typical retinal dystrophy predominantly affecting rod and bipolar cell function. In vitro expression of MCOLN1 in liposomes showed that the c.1615delG mutated channel had significantly reduced conductance compared with wild-type mucolipin-1, whereas the inhibitory effect of low pH and amiloride remained intact. CONCLUSIONS. These findings suggest that reduced channel conductance is relatively well tolerated by the brain during development, whereas retinal cells and stomach parietal cells require normal protein function. MLIV should be considered in patients with retinal dystrophy of unknown cause and screened for using blood gastrin levels. (Invest Ophthalmol Vis Sci. 2008;49:3134‐3142) DOI:10.1167/iovs.07-1649

*Developmental and Metabolic Neurology Branch, National Institute of Neurological Disorders, t National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda Maryland. **Correspondence to: Michele R. Filling-Katz, MD, Bldg.103C103, Section on Genetic Studies, Laboratory of Clinical Investigations, N1AAA, NIH, Bethesda, Maryland 20892. Niemann-Pick Type B disease (NPB) is an autosomal

The pathogenesis of Fabry disease is poorly understood. We used a variety of immunohistological techniques to localize globotriaosylceramide, the main glycolipid that accumulates in Fabry disease. Globotriaosylceramide immunoreactivity in a heterogenous pattern was present in all organs examined of a patient on long-term enzyme replacement therapy. In the brain, immmunopositivity was found only in the parahippocampal region. Globotriaosylceramide immunostaining was present in the cell membrane and cytoplasm of endothelial cells, even in the absence of lysosomal inclusions. In kidney tissue, globotriaosylceramide colocalized with lysosomal, endoplasmic reticulum, and nuclear markers. Pre- and postembedding immunogold electron microscopy of skin biopsies and untreated patient cultured skin fibroblasts confirmed the presence of globotriaosylceramide in the cell membrane, in various cytoplasmic structures, and in the nucleus. Control organ tissues and cultured fibroblasts from five unaffected subjects were uniformly negative for globotriaosylceramide by immunohistochemistry and immunogold electron microscopy. We conclude that a substantial amount of lysosomal and extralysosomal globotriaosylceramide immunoreactivity remains in cells and tissues even after years of enzyme replacement therapy in Fabry disease. These findings are crucial for the understanding of the disease mechanism and suggest the usefulness of immunostaining for globotriaosylceramide as a means to assess response to novel, specific therapies.

Childhood ataxia with central nervous system hypomyelination (CACH), also called vanishing white matter (VWM) leukoencephalopathy, is a fatal genetic disease caused by mutations in eukaryotic initiation factor 2B (eIF2B) genes. The five subunits eIF2B factor is critical for translation initiation under normal conditions and regulates protein synthesis in response to cellular stresses. Primary fibroblasts from CACH/VWM patients and normal individuals were used to measure basal eIF2B activity as well as global protein synthesis and ATF4 induction in response to stress in the endoplasmic reticulum. We show that although the cells expressing mutant eIF2B genes respond normally to stress conditions by reduced global translation rates, they exhibit significantly greater increase in ATF4 induction compared to normal controls despite equal levels of stress and activity of the upstream eIF2alpha kinase. This heightened stress response observed in primary fibroblasts that suffer from minor loss of basal eIF2B activity may be employed as an initial screening tool for CACH/VWM leukodystrophy.

Summary: Gaucher disease, the most common sphingolipidosis, is caused by a decreased activity of glucosylceramide β-glucosidase, resulting in the accumulation of glucosylceramide in macrophage-derived cells known as Gaucher cells. Much of the storage material is thought to originate from the turnover of cell membranes, such as phagocytosed red and white blood cells. In this study, an in vitro model of Gaucher disease was developed by treating the murine macrophage cell line J774 with a specific inhibitor of glucosylceramide β-glucosidase, conduritol B-epoxide, and feeding red blood cell ghosts, in order to mimic the disease state. It was found in this model system that glucosylceramide β-glucosidase activity could be reduced to about 11–15! of the normal control level before increased storage of glucosylceramide occurred. This in vitro system allows insight into the correlation between enzyme activity and lipid storage as predicted by the theory of residual enzyme activity that was proposed by Conzelmann and Sandhoff.

Gaucher disease, the inherited deficiency of lysosomal glucocerebrosidase, presents with a wide spectrum of manifestations. Although Gaucher disease has been divided into three clinical types, patients with atypical presentations continue to be recognized. A careful phenotypic and genotypic assessment of patients with unusual symptoms may help define factors that modify phenotype in this disorder. One such example is a rare subgroup of patients with type 3 Gaucher disease who develop progressive myoclonic epilepsy. We evaluated 16 patients with myoclonic epilepsy, nine of whom were diagnosed by age 4 y with severe visceral involvement and myoclonus, and seven with a more chronic course, who were studied between ages 22 and 40. All of the patients had abnormal horizontal saccadic eye movements. Fourteen different genotypes were encountered, yet there were several shared alleles, including V394L (seen on two alleles), G377S (seen on three alleles), and L444P, N188S, and recombinant alleles (each found on four alleles). V394L, G377S, and N188S are mutations that have previously been associated with non-neuronopathic Gaucher disease. The spectrum of genotypes differed significantly from other patients with type 3 Gaucher disease, where genotypes L444P/L444P and R463C/null allele predominated. Northern blot studies revealed a normal glucocerebrosidase transcript, whereas Western studies showed that the patients studied lacked the processed 56 kD isoform of the enzyme, consistent with neuronopathic Gaucher disease. Brain autopsy samples from two patients demonstrated elevated levels of glucosylsphingosine, a toxic glycolipid, which could contribute to the development of myoclonus. Thus, although there were certain shared mutant alleles found in these patients, both the lack of a shared genotype and the variability in clinical presentations suggest that other modifiers must contribute to this rare phenotype.

Background: Mucolipidosis type IV (MLIV) is an autosomal recessive disease caused by mutations in the MCOLN1 gene that codes for mucolipin, a member of the transient receptor potential (TRP) gene family. Objective: To comprehensively characterize the clinical and genetic abnormalities of MLIV. Methods: Twenty-eight patients with MLIV, aged 2 to 25 years, were studied. Ten returned for follow-up every 1 to 2 years for up to 5 years. Standard clinical, neuroimaging, neurophysiologic, and genetic techniques were used. Results: All patients had varying degrees of corneal clouding, with progressive optic atrophy and retinal dystrophy. Twenty-three patients had severe motor and mental impairment. Motor function deteriorated in three patients and remained stable in the rest. All had a constitutive achlorhydria with elevated plasma gastrin level, and 12 had iron deficiency or anemia. Head MRI showed consistent characteristic findings of a thin corpus callosum and remained unchanged during the follow-up period. Prominent abnormalities of speech, hand usage, and swallowing were also noted. Mutations in the MCOLN1 gene were present in all patients. Correlation of the genotype with the neurologic handicap and corpus callosum dysplasia was found. Conclusions: MLIV is both a developmental and a degenerative disorder. The presentation as a cerebral palsy-like encephalopathy may delay diagnosis.

The beneficial effects of macrophage-targeted glucocerebrosidase (alglucerase, Ceredase) in patients with Gaucher disease are well established. A minority of recipients develop transient non-neutralizing antibodies to the exogenous enzyme. A 7-year-old patient with type 3 Gaucher disease, whose clinical course began to deteriorate while receiving alglucerase developed a progressively increasing titer of IgG antibody, that blocked the catalytic activity of alglucerase. We investigated the acid beta-glucosidase genotype in this patient. Direct sequencing of both cDNA and genomic PCR products was used to characterize the mutations underlying acid beta-glucosidase deficiency. The patient was shown to be a compound heterozygote for a previously reported missense mutation (G377S), and a novel single nucleotide deletion (g5255delT). The transcript originating from the latter allele was undetectable in RT-PCR experiments. We report the first characterization of a GBA genotype associated with the development of neutralizing antibody to alglucerase, in a patient affected with type 3 Gaucher disease. Our results may help to shed light on the mechanisms underlying this phenomenon which, in the rare instances where it occurs, hampers the efficacy of enzyme replacement therapy.

A significant number of patients with progressive leukodystrophy do not have a definitive diagnosis. This report describes the clinical, morphological, and biochemical characteristics of 4 unrelated girls with progressive ataxic diplegia of unknown etiology. These patients had normal development until the ages of 1.5 to 5 years. A diffuse confluent abnormality of the white matter of the central nervous system was present on computed tomography and magnetic resonance scans obtained early in the course of the illness. Dementia was not present and peripheral nerves were normal. All patients were evaluated for known metabolic and degenerative diseases and no abnormalities were observed. Light and electron microscopy of open-brain biopsy specimens from 2 girls showed selective white matter abnormalities including hypomyelination, demyelination, and gliosis. Myelin-specific proteins in the subcortical white matter were examined immunocytochemically and by Western blot analysis. They were of normal molecular size but were markedly reduced in quantity in both patients compared to control subjects. Lipid analysis revealed decreased levels of characteristic myelin lipids. When examined by magnetic resonance spectroscopic imaging, all patients showed a marked decrease of N-acetylaspartic acid, choline, and creatine in white matter only. The magnetic resonance spectroscopic imaging profile is a unique diagnostic feature of this clinicopathological syndrome.

Mucolipidosis IV (ML IV) (McKusick 252650) is an autosomal recessive metabolic disorder that displays signs of both lipid and mucopolysaccharide (glycosaminoglycan) storage. It has been reported that fibroblasts from ML IV patients exhibit abnormally high synthesis of hyaluronic acid in culture. In our search for a biochemical marker that will enable positive identification of ML IV, we studied glycosaminoglycan synthesis in fibroblast cultures from patients with this disease. ML IV and normal control fibroblasts were incubated with [3H]glucosamine and [35S]sulphate. Labelled glycosaminoglycans were extracted from the cell layer and medium. Chondroitin sulphate and hyaluronic acid were determined by analysis of disaccharides after digestion with chondroitinase ABC. Synthesis of neither of these two glycosaminoglycans differed significantly between control and ML IV fibroblasts. Synthesis of hyaluronic acid was nearly linear for 24 h, with mean calculated values of 11.7 +/- 1.4 and 14.4 +/- 1.6 pg/cell per 24 h in control and ML IV cultures respectively. The variability within the two groups is attributed primarily to population variability and possibly to culture density. These experiments exclude the possibility that a general metabolic defect in hyaluronic acid synthesis is responsible for the ML IV phenotype, nor can such a defect be used as a diagnostic tool for the disease.

Fabry disease is an X-linked disorder caused by a deficiency of the lysosomal enzyme alpha-galactosidase A. The mutations responsible for Fabry disease are diverse and include large rearrangements as well as single base substitutions, and they are dispersed throughout the seven exons of the gene. In this study, we found five novel mutations in four different exons. We have detected the mutations by the PCR-SSCP method and then analysed them by direct sequencing. Three of the novel mutations were deletions: 1205delA, 1238del26 and 5236del18. We also found one novel nonsense mutation: W162X. The final novel mutation was an insertion combined with a deletion: 10995ins24del4.

Type C Niemann-Pick disease (NPC) is an autosomal recessive neurovisceral storage disorder in which defective intracellular cholesterol processing has been demonstrated in fibroblasts from NPC patients and obligate heterozygotes. In the present paper, the ability to esterify LDL-cholesterol was examined in cultured lymphocytes from 8 NPC patients, 8 obligate heterozygotes and 8 controls. Cholesteryl ester synthesis was 8% (±5%) and 45% (±16%) of controls in homozygous and heterozygous cell lines, respectively. Histochemical and electron microscopic examinations confirmed that this biochemical lesion was associated with abnormal intracellular accumulation of unesterified cholesterol in mutant lymphocytes. These results demonstrate that measurement of cholesterol esterification in cultured lymphocytes offers a quick and reliable means of confirming the diagnosis of NPC and that these cells may be useful for probing the primary molecular lesion of NPC.

Molecular, Clinical and Biochemical Characterization of a Mild Menkes Phenotype Associated with a Novel Missense Mutation (Ala1362Asp) in the ATP7A Copper Transport Gene. † 612

We report intermittent seizures, lethargy, and Cohen's syndrome in a 4-year-old girl with hyper-beta-alaninemia and a partial deficiency of beta-alanyl-alpha-ketoglutarate transaminase (AKT). To examine the role of beta-alanine (beta ALA) in cellular metabolism, we cultured her skin fibroblasts in medium containing increasing amounts of beta ALA. At concentrations of 10 to 25 mM, beta ALA caused more than a 50% reduction in the growth of her cells compared with normal control skin fibroblasts. The addition of 0.1 mM of pyridoxine to the culture medium abolished these toxic effects and increased her skin fibroblast AKT enzyme activity more than twofold. During a 2-year period of clinical observation, there were no further episodes of seizures or somnolence in our patient while she received oral pyridoxine therapy.

Type 3 neuronopathic Gaucher's disease (GD3) is phenotypically heterogeneous. In many GD3 patients, progressive myoclonus and dementia dominate the illness, with death secondary to progressive CNS disease. We have designated this group as GD3a. We studied 14 children with Gaucher's disease, isolated horizontal supranuclear gaze palsy, and aggressive systemic disease, and designated this group as GD3b. In comparison with 13 children with type 1 non-neuronopathic Gaucher's disease, the GD3b children presented earlier, and were shorter, underweight, and more prone to cardiopulmonary, hepatic, and skeletal complications. One-half of the children died in childhood or adolescence of systemic complications. Patients with at least one copy of the mutation that causes substitution of asparagine for serine at amino acid 370 of glucocerebrosidase did not develop neurologic signs. Patients homoallelic for the mutation causing substitution of leucine for proline at position 444 had severe systemic disease; neurologic signs were frequently, but not invariably, present. Early diagnosis and timely enzyme replacement therapy promise to improve the prognosis in GD3b.

We describe the unique clinical and histopathologic features of a child with biochemical and immunocytochemical features of Niemann-Pick disease type C (NPC). Clinically, she was found to have multiple xanthomas of the upper aerodigestive tract with dysphagia and expressive language delay, splenomegaly, bony infarcts, and type IIb hyperlipidemia. Neurologic examination was otherwise normal. Microscopy revealed foam cells in her bone marrow, liver, tongue, tonsils, glottis, and in normal-appearing peritonsillar mucosa. Lipid analysis of a liver biopsy specimen showed a small increase in phospholipids, a twofold increase in sphingomyelin, a fivefold increase in cholesterol, and a marked (25-fold) increase in bis(monoacylglycerol) phosphate. Lysosomal acid hydrolase activities in cultured skin fibroblasts were nondiagnostic. Biochemical and immunocytochemical studies of cultured fibroblasts demonstrated lysosomal accumulation of unesterified LDL-derived cholesterol as well as delayed induction of homeostatic responses to endogenous cholesterol consistent with a diagnosis of NPC. Based upon these observations, we speculate that this patient could have a new phenotypic expression of NPC or represents a new cholesterol lipidosis biochemically resembling NPC. The chance occurrence of two separate lipid disorders seems less likely.

Ovarian failure (OF) at age40 years occurs in approximately 1% of all women. Other than karyotype abnormalities, very few genes are known to be associated with this ovarian dysfunction. We studied eight patients who presented with premature OF and white-matter abnormalities on magnetic resonance imaging. Neurological signs may be absent or present after OF. In seven patients, we report for the first time mutations in three of the five EIF2B genes (EIF2B2, -4, and -5) that were recently shown to cause childhood ataxia with central nervous system hypomyelination/vanishing white-matter disease leukodystrophy. The correlation we observed between the age at onset of the neurological deterioration and the severity of OF suggests a common pathophysiological pathway.