Generation of GLA-knockout human embryonic stem cell lines to model peripheral neuropathy in Fabry disease.

Fabry disease is an X-linked glycolipid storage disorder caused by mutations in the gene which result in a deficiency in the lysosomal enzyme alpha galactosidase A (AGA). As a result, the glycolipid substrate Gb3 accumulates in critical tissues and organs producing a progressive debilitating disease. In Fabry disease up to 80% of patients experience life-long neuropathic pain that is difficult to treat and greatly affects their quality of life.

Generation of an model for peripheral neuropathy in Fabry disease using CRISPR-Cas9 in the nociceptive dorsal root ganglion cell line 50B11.

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.

Development of a model system for neuronal dysfunction in Fabry disease.

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.

A Short Synthetic Peptide Mimetic of Apolipoprotein A1 Mediates Cholesterol and Globotriaosylceramide Efflux from Fabry Fibroblasts.

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.

Decreased glucocerebrosidase activity in Gaucher disease parallels quantitative enzyme loss due to abnormal interaction with TCP1 and c-Cbl.

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.

Use of lissamine rhodamine ceramide trihexoside as a functional assay for alpha-galactosidase A in intact cells.

Fabry disease is an X-linked disorder caused by mutations in the GLA gene encoding for alpha-galactosidase A (AGA, EC 3.2.1.

Free sialic acid storage disease without sialuria.

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.

Translational read-through of a nonsense mutation in ATP7A impacts treatment outcome in Menkes disease.

Protein translation ends when a stop codon in a gene's messenger RNA transcript enters the ribosomal A site. Mutations that create premature stop codons (nonsense mutations) typically cause premature translation termination. An alternative outcome, read-through translation (or nonsense suppression), is well known in prokaryotic, viral, and yeast genes but has not been clearly documented in humans except in the context of pharmacological manipulations.

Cerebellar ataxia with elevated cerebrospinal free sialic acid (CAFSA).

In order to identify new metabolic abnormalities in patients with complex neurodegenerative disorders of unknown aetiology, we performed high resolution in vitro proton nuclear magnetic resonance spectroscopy on patient cerebrospinal fluid (CSF) samples. We identified five adult patients, including two sisters, with significantly elevated free sialic acid in the CSF compared to both the cohort of patients with diseases of unknown aetiology (n = 144; P < 0.001) and a control group of patients with well-defined diseases (n = 91; P < 0.

Globotriaosylceramide induces oxidative stress and up-regulates cell adhesion molecule expression in Fabry disease endothelial 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.

Prediction of response of mutated alpha-galactosidase A to a pharmacological chaperone.

Objective: 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).

Methods: 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.

Results: 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.

Time series proteome profiling to study endoplasmic reticulum stress response.

Time series profiling is a powerful approach for obtaining information on protein expression dynamics and prevailing biochemical pathways. To date, such information could only be obtained at the mRNA level using mature and highly parallel technologies such as microarray gene expression profiling. The generation of time series data at the protein level has lagged due to the lack of robust and highly reproducible methodologies.

Isolated ocular disease is associated with decreased mucolipin-1 channel conductance.

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.

Cellular and tissue localization of globotriaosylceramide in Fabry disease.

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.

Establishment and characterization of Fabry disease endothelial cells with an extended lifespan.

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.

Screening for pharmacological chaperones in 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.

The cerebral vasculopathy of Fabry disease.

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.

Peripheral and central hypomyelination with hypogonadotropic hypogonadism and hypodontia.

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.

Myeloperoxidase predicts risk of vasculopathic events in hemizgygous males with Fabry disease.

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.

Improved intracellular delivery of glucocerebrosidase mediated by the HIV-1 TAT protein transduction domain.

Enzyme replacement therapy (ERT) for Gaucher disease designed to target glucocerebrosidase (GC) to macrophages via mannose-specific endocytosis is very effective in reversing hepatosplenomegaly, and normalizing hematologic parameters but is less effective in improving bone and lung involvement and ineffective in brain. Recombinant GCs containing an in-frame fusion to the HIV-1 trans-activator protein transduction domain (TAT) were expressed in eukaryotic cells in order to obtain active, normally glycosylated GC fusion proteins for enzyme uptake studies. Despite the absence of mannose-specific endocytic receptors on the plasma membranes of various fibroblasts, the recombinant GCs with C-terminal TAT fusions were readily internalized by these cells.

Heightened stress response in primary fibroblasts expressing mutant eIF2B genes from CACH/VWM leukodystrophy patients.

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.

The N370S (Asn370-->Ser) mutation affects the capacity of glucosylceramidase to interact with anionic phospholipid-containing membranes and saposin C.

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.

Correlation between enzyme activity and substrate storage in a cell culture model system for Gaucher disease.

Gaucher disease, the most common sphingolipidosis, is caused by a decreased activity of glucosylceramide beta-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 beta-glucosidase, conduritol B-epoxide, and feeding red blood cell ghosts, in order to mimic the disease state.

Transfer of a mitochondrial DNA fragment to MCOLN1 causes an inherited case of mucolipidosis IV.

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.

Clinical features of adult GM1 gangliosidosis: report of three Indian patients and review of 40 cases.

Deficiency of enzyme acid beta-galactosidase causes GM1 gangliosidosis. Patients with adult GM1 gangliosidosis typically present with generalized dystonia. We describe clinical, bone marrow, and radiological features of adult GM1 gangliosidosis to help improve its recognition.