Accessibility of sialo components in a murine tumor cell to extracellular N-acetylneuraminate glycohydrolase (sialidase).

Lipid-bound sialic acid in the murine melanoma cell is not totally inaccessible to an exogenous macromolecular probe, as formerly believed. Roughly 30% of the dialic acid bound to lipid, and an equal proportion of the sialic acid bound to protein is cleaved by the action of Clostridium perfringens N-acetylneuraminate glycohydrolase (neuraminidase, sialidase) when the purified enzyme is added to the suspenion medium of intact murine melanoma cells freshly derived from the tumor. Cleavage of lipid-bound sialic acid is indifferent to the presence of Ca (2+) in the medium. However, maximum release from protein requires a physiological concentration of this divalent cation. Variation in ionic strength has no effect on release of sialic acid. These findings show that restricted portion of the bound sialic acid may be released from the intact murine melanama cell by the extracellularly supplied enzyme acting topographically.

Retroviral transfer of the glucocerebrosidase gene into CD34+ cells from patients with Gaucher disease: in vivo detection of transduced cells without myeloablation.

Retroviral gene transfer of the glucocerebrosidase gene to hematopoietic progenitor and stem cells has shown promising results in animal models and corrected the enzyme deficiency in cells from Gaucher patients in vitro. Therefore, a clinical protocol was initiated to explore the safety and feasibility of retroviral transduction of peripheral blood (PB) or bone marrow (BM) CD34+ cells with the G1Gc vector. This vector uses the viral LTR promoter to express the human glucocerebrosidase cDNA. Three adult patients have been entered with follow-up of 6-15 months. Target cells were G-CSF-mobilized and CD34-enriched PB cells or CD34-enriched steady state BM cells, and were transduced ex vivo for 72 hr. Patient 1 had PB cells transduced in the presence of autologous stromal marrow cells. Patient 2 had PB cells transduced in the presence of autologous stroma, IL-3, IL-6, and SCF. Patient 3 had BM cells transduced in the presence of autologous stroma, IL-3, IL-6, and SCF. At the end of transduction, the cells were collected and infused immediately without any preparative treatment of the patients. The transduction efficiency of the CD34+ cells at the end of transduction was approximately 1, 10, and 1 for patients 1, 2, and 3, respectively, as estimated by semiquantitative PCR on bulk samples and PCR analysis of individual hematopoietic colonies. Gene marking in vivo was demonstrated in patients 2 and 3. Patient 2 had vector-positive PB granulocytes and mononuclear bone marrow cells at 1 month postinfusion and positive PB mononuclear cells at 2 and 3 months postinfusion. Patient 3 had a positive BM sample at 1 month postinfusion but was negative thereafter. These results indicate that gene-marked cells can engraft and persist for at least 3 months postinfusion, even without myeloablation. However, the level of corrected cells (<0.02%) is too low to result in any clinical benefit, and glucocerebrosidase enzyme activity did not increase in any patient following infusion of transduced cells. Modifications of vector systems and transduction conditions, along with partial myeloablation to allow higher levels of engraftment, may be necessary to achieve beneficial levels of correction in patients with Gaucher disease.

Structural organization and expression of the mouse gene encoding alpha-galactosidase A.

alpha-Galactosidase A (alpha-D-galactoside galactohydrolase, EC 3.2.1.22; alpha GalA) is a lysosomal enzyme that hydrolyses the alpha-D-galactosyl residues from glycosphingolipids. Fabry disease, an inhibited X-linked recessive human metabolic disorder, results from a mutation in the alpha GalA gene at Xq22. As a prerequisite for generating a mouse model of Fabry disease by gene targeting, we have isolated and characterized the mouse alpha GalA gene and cDNA. A cloned mouse alpha GalA cDNA encoded a putative precursor protein of 419 amino acids (aa), including a 31-aa signal peptide (SP). The deduced aa sequence showed high homology (79%) with the human alpha GalA protein. Nucleotide sequence analysis of genomic clones revealed that the overall structure and organization of the gene was very similar to that of human alpha GalA. All exon-intron splice junctions conformed to the GT/AG consensus sequence. Comparison of genomic and cDNA sequences revealed the occurrence of two putative polyadenylation signals whose alternative use results in the two mouse alpha GalA transcripts of 1.4 and 3.6 kb. The 5'-flanking region of mouse alpha GalA had no typical TATA box. Several putative promoter-associated elements including Sp1, AP1 and a potential cAMP-responsive element (CRE) were identified. Northern blot analysis revealed the widespread tissue distribution of mouse alpha GalA transcripts. Lower expression levels, however, were observed in some tissues, implying tissue-specific differences in alpha GalA promoter function.

The role of neurogenetics in Gaucher disease.

Gaucher disease is the most prevalent hereditary metabolic storage disorder, and the most common genetic disease in individuals of Ashkenazic Jewish ancestry. Patients with Gaucher disease have been classified into three clinical phenotypes. Patients with type 1 disease exhibit markedly variable hepatosplenomegaly, anemia, thrombocytopenia, skeletal, and, to a lesser extent, pulmonary and kidney involvement. The central nervous system does not appear to be involved. In patients with type 2 Gaucher disease, hepatosplenomegaly and extensive central nervous system damage are apparent in infancy. These patients usually die between 1 and 2 years of age. Patients with type 3 Gaucher disease have been subclassified into types 3a and 3b. Type 3a patients exhibit mild-to-moderate hepatosplenomegaly and slowly progressive neurologic deterioration. Recurrent myoclonic seizures are common. Patients with type 3b Gaucher disease exhibit splenomegaly along with extensive hepatomegaly that is frequently accompanied by esophageal varices. Horizontal supranuclear gaze paresis is the major neurologic sign. Excessive quantities of glucocerebroside accumulate in the organs of patients with Gaucher disease because of a deficiency of the enzyme glucocerebrosidase. In the vast majority of patients, the reduction of glucocerebrosidase activity is caused by mutations in the gene that codes for glucocerebrosidase. In a few instances, glucocerebroside accumulates due to a lack of saposin C, a cohydrolase that is required in addition to glucocerebrosidase for the catabolism of glucocerebroside. Mutations in the glucocerebrosidase gene are discussed in the context of the severity of disease and the presence or absence of nervous system involvement. Enzyme replacement therapy is highly beneficial for patients with type 1 Gaucher disease. Enzyme replacement is also being investigated for patients with type 3b Gaucher disease. Novel procedures must be developed to deliver glucocerebrosidase to the nervous system so that patients with type 2 and type 3a Gaucher disease can be helped. Exploration of gene therapy for Gaucher disease is under way.

Neurologic complications of nonneuronopathic Gaucher's disease.

We describe eight patients with type 1 Gaucher's disease who developed neurologic complications that were secondary to systemic features of the illness. Four patients experienced neurologic difficulties because of coagulopathy, and the other four patients had involvement of the nervous system secondary to skeletal disease. Early recognition of these complications in patients with type 1 Gaucher's disease may lead to improved neurologic outcome.

Pyridoxine-responsive hyper-beta-alaninemia associated with Cohen's syndrome.

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.

Quantitative analysis of epidermal innervation in Fabry disease.

OBJECTIVE: To use skin biopsy specimens to quantitate the cutaneous innervation density of Fabry patients who had preserved renal function. BACKGROUND: The small fiber neuropathy of Fabry disease is difficult to detect and quantitate by conventional methods. Because this neuropathy is a common characteristic of Fabry disease, quantitating changes in this parameter would be helpful in demonstrating the effectiveness of enzyme or gene replacement therapy. METHODS: Patients underwent skin biopsy at the thigh and foot. Innervation density was determined by counting free nerve endings in the epidermis. These data were compared with nerve conduction studies, and in selected patients, fiber quantitation of sural nerve biopsy specimens. RESULTS: The Fabry patients had normal results of nerve conduction studies and large fiber quantitation by sural nerve biopsy. However, the involvement of small cutaneous fibers in these patients was easily demonstrable and quantifiable by skin biopsy. All patients showed severe loss of intraepidermal innervation at the ankle, but fiber loss at the distal thigh was proportionately less severe. CONCLUSIONS: The nerve damage in Fabry patients with preserved renal function involves exclusively small myelinated and unmyelinated fibers, and skin biopsy is a useful in detecting and quantitating such damage. Comparison of cutaneous innervation density with quantitation of sural nerve biopsy specimens demonstrated that skin biopsy specimens were as sensitive in detecting the presence of neuropathy as were the nerve specimens. It is speculated that analysis of cutaneous innervation may provide a useful marker of the nervous system's response to specific therapy for Fabry disease.

Proton magnetic resonance spectroscopic imaging in childhood ataxia with diffuse central nervous system hypomyelination.

The spatial distribution of metabolite signal intensities can be measured within entire sections of the brain by proton magnetic resonance spectroscopic imaging (1H-MRSI). A group of six patients (4 unrelated girls and 2 brothers from 5 families) with childhood ataxia with diffuse CNS hypomyelination (CACH) underwent long-echo-time, single-slice 1H-MRSI. Relative to controls, there was a decrease in the signal intensity of N-acetylaspartate, choline, and creatine throughout the white matter in all six patients. We identified lactate signals in white matter in three of them with advanced disease. The degree of white matter involvement was not homogeneous over the entire patient group, but did correlate with clinical presentation. Deep and posterior white matter tended to be more involved. There were no 1H-MRSI abnormalities in the gray matter. 1H-MRSI findings suggest that this syndrome is secondary to a metabolic defect causing hypomyelination, axonal degeneration, and, in the most compromised cases, accumulation of lactate. This study shows that CACH is not limited to girls.

A clinical staging classification for type C Niemann-Pick disease.

Analysis of the temporal sequence of neurologic events, neurophysiologic abnormalities, and longevity in 36 Niemann-Pick type C patients revealed two clinical subgroups with five stages of severity within each group. Patients with a preschool onset (group I; n = 18) had a higher mortality than did patients with a school-age onset (group II; n = 18). An asymptomatic phase (stage 0) was defined by biochemical and histopathologic evidence of disease. The initial manifestations of stage 1 were a movement disorder (group I) and cognitive difficulties (group II) accompanied by impaired vertical saccadic eye movements and abnormal acoustic reflexes. Stage 2 was characterized by the sequential occurrence of vertical supranuclear gaze palsy (VSGP), cognitive difficulties, and dysarthria in group I and a movement disorder, VSGP, and dysarthria in group II. Pyramidal tract signs and abnormal brainstem auditory evoked responses defined stage 3 in both groups. Stage 4 culminated in a nonambulant, vegetative state.

Quantitative analysis of cerebral vasculopathy in patients with Fabry disease.

OBJECTIVE: This study's purpose was to obtain a quantitative natural history of the cerebrovascular involvement in Fabry disease. BACKGROUND: Fabry disease is an X-linked recessive disorder due to alpha-galactosidase A deficiency. Progressive accumulation of ceramidetrihexoside within the intima and media of cerebral blood vessels causes ischemic lesions in the majority of affected patients. Determination of the natural history of the cerebral vasculopathy in Fabry disease is important to assess the effects of therapeutic intervention in this disorder. METHODS: A longitudinal MRI study of 50 patients who had a total of 129 MRI scans was performed. The burden of cerebrovascular disease was determined using direct linear measurement. RESULTS: On T2-weighted MRI scans, 32% of the patients had no lesions (mean age, 33 years), 16% had gray matter lesions only (mean age, 36 years), 26% had lesions in white matter only (mean age, 43 years), and 26% had lesions in white and gray matter (mean age, 47 years). Disease burden increased with age, but no patient younger than 26 had lesions on MRI. All patients older than 54 had cerebrovascular involvement. The distribution of MRI-detectable lesions was typical of a small-vessel disease. Only 37.5% of patients with cerebral lesions had neurologic symptoms. CONCLUSION: These findings provide a predictable outcome measure to assess the effect of molecular interventions on the cerebrovascular circulation in Fabry disease.

Tetrahydrobiopterin administration in biopterin-deficient progressive dystonia with diurnal variation.

We administered tetrahydrobiopterin (BH4) to 4 patients with progressive dystonia with diurnal variation (PDDV). One patient improved clinically. Deficient CSF concentrations of HVA and 5-HIAA were unchanged despite marked elevation of CSF biopterin concentration. Variable effectiveness of BH4 in PDDV may reflect reduced number or function of biopterin-metabolizing neurons or variable entry of BH4 into these neurons.

Carbamazepine in Fabry's disease: effective analgesia with dose-dependent exacerbation of autonomic dysfunction.

Seven patients with Fabry's disease and severe pain received carbamazepine (CMZ). Five of 7 patients had moderate to complete relief based upon self-assessment of pain levels. Preexisting autonomic dysfunction was exacerbated by CMZ in 2. Complications encountered were ileus, urinary retention, and gastrointestinal disturbance. Although CMZ was useful in treatment of pain, caution should be employed in this disease.

Hypoprebetalipoproteinemia, acanthocytosis, retinitis pigmentosa, and pallidal degeneration (HARP syndrome).

We describe the clinical and laboratory studies of an 11-year-old girl with prominent orofacial dyskinesia, dystonia, and progressive dementia. Investigations revealed hypoprebetalipoproteinemia, acanthocytosis, atypical retinitis pigmentosa, and evidence of iron deposition in the pallidal nuclei. Electroneuromyography and skin and sural nerve biopsies were normal. The "eye-of-the-tiger" sign, used to describe the pallidal nuclei in Hallervorden-Spatz syndrome, was present on T2-weighted MRIs (GE Signa, 1.5 T). Phase-contrast microscopy of whole blood showed 80 to 90% acanthocytes whose morphology was confirmed by electron microscopy. High-resolution lipoprotein electrophoresis demonstrated an absence of the pre-beta fraction. This case differs phenotypically from the previous reports of Hallervorden-Spatz disease with acanthocytosis by the presence of prominent orofacial dyskinesia and abnormal serum lipoproteins.

Gaucher's disease: lack of antibody response in 12 patients following repeated intravenous infusions of mannose terminal glucocerebrosidase.

An amplified ELISA has been employed for monitoring the safety of repeated intravenous infusions of modified human placental glucocerebrosidase. The enzyme infusions consisted of biweekly injections of macrophage targeted glucocerebrosidase over a 6 month duration. Serum samples collected throughout the study were assayed by use of an ELISA using alkaline phosphatase coupled to alcohol dehydrogenase for amplification. Using this protocol, 0.2-5 ng affinity purified immunoglobulin specific for glucocerebrosidase can be detected. Occasional false positives necessitate multiple repeat assays over time to accurately assess immunogenic response. Blinded ELISAs were performed on sera from both infused patients with Gaucher's disease and uninfused control patients and compared with apparent immunoglobulin concentration in 54 normal control sera. Although several samples showed apparently elevated immunoglobulin levels, repeat analyses failed to demonstrate high levels reproducibly. Furthermore, these sera were unable to neutralize enzyme or to precipitate radiolabelled enzyme, confirming the absence of antibody. Problems with high sensitivity ELISA formats are discussed.

Radiopharmacology of inhaled 133Xe in skeletal sites containing deposits of Gaucher cells.

Gaucher's disease is a lysosomal storage disease in which cells of the reticuloendothelial system accumulate the lipid glucocerebroside. It is characterized by slowly progressive visceral and osseous involvement. One of the latter manifestations includes lipid infiltration of bone marrow. We monitored the rate of inhaled 133Xe uptake and wash-out over diseased and normal metaphyseal and epiphyseal areas of the knee. Twenty-two patients (15 adults, 7 children) with various degrees of previously diagnosed Gaucher's disease were positioned supine under a gamma-camera interfaced to a computer system. All patients rebreathed 133Xe gas from a closed system for 10 min followed by 14 min of wash-out. Digitized images of the lung, liver, spleen, bony sites and soft tissue were obtained at 1 min intervals during the wash-in and wash-out phases. Counts for each ROI were normalized per 100 pixels and plotted as a function (time). Maximum uptake was also calculated by relating the counts/ROI/100 pixels to the 10 min integrated lung count during equilibrium (the administered "dose"). There was essentially no 133Xe uptake in liver and spleen involved with Gaucher's disease. Monophasic uptake and biphasic wash-out curves were observed in the limited investigative population. Skeletal Gaucher deposits released the 133Xe at a greater rate relative to soft tissue.

Niemann-Pick disease types C and D.

Patients grouped into categories termed type C Niemann-Pick disease and the Nova Scotia isolate called type D Niemann-Pick disease are characterized by mild to moderate hepatosplenomegaly, sea-blue histiocytes in the bone marrow, supranuclear gaze paresis in the vertical plane, slowly progressing ataxia, and mental deterioration. These signs are caused by abnormal intracellular cholesterol homeostasis. Cholesterol that enters cells from the circulation through the LDL receptor is not processed in a timely, normal manner by cells in parenchymal organs and the CNS. It therefore accumulates in toxic quantities as unesterified cholesterol causing cellular and tissue damage. Knowledge of the primary, consistent disturbance in cholesterol disposition has led to the development of tests to diagnose patients, identify heterozygotes, and assure the prenatal detection of these disorders. Therapeutic strategies include reduction of dietary cholesterol, apheresis techniques designed to reduce LDL cholesterol available to cells, and reduction of formation of LDL and increase of synthesis of HDL to lower cellular uptake of cholesterol and enhance egress of this lipid from intracellular storage sites. The development of procedures that block cholesterol formation but do not up-regulate LDL receptors on plasma cell membranes is considered to be highly important for the therapy of types C and D Niemann-Pick disease.

Fabry's disease presenting with stroke.

An atypical patient with Fabry's disease is presented. This patient developed a left internal capsule lacunar stroke at the age of 25. The etiology of the stroke was unclear. At the age of 29 he was discovered to have corneal lesions suggestive of Fabry's disease but had no other clinical features typical of Fabry's disease. The diagnosis of Fabry's disease was confirmed biochemically. Fabry's disease should be included in the differential diagnosis of stroke of unclear etiology in young male patients.

Enzyme replacement and gene therapy for Gaucher's disease.

The lipid storage disorders have long been considered primary candidates for enzyme replacement therapy. This goal has been achieved with a remarkable degree of success in Gaucher's disease. Among the accomplishments that were important to obtain clinical benefit were the development of a large-scale procedure to purify human placental glucocerebrosidase and a method to target this enzyme to lipid-storing macrophages through glycoform modification. In addition, the effectiveness of recombinantly produced macrophage-targeted glucocerebrosidase has recently been demonstrated. Because macrophages originate from stem cells in the bone marrow, ex vivo transduction of these cells with retroviral vectors containing the cDNA for human glucocerebrosidase is being explored for the genetic therapy of Gaucher's disease.

Vascular involvement in type 3 neuronopathic Gaucher's disease.

A 6 3/4-year-old boy with neuronopathic Gaucher's disease died unexpectedly. Autopsy demonstrated unusual vascular findings with the presence of Gaucher's cells and intimal-medial fibrosis in the ascending aorta and widespread acute hemorrhagic necrosis of the left ventricular myocardium. The combination of eccentric, intimal fibrosis of coronary arteries, left ventricular hypertrophy, pulmonary involvement, and anemia probably accounted for this child's death.

Gastrointestinal symptoms and delayed gastric emptying in Fabry's disease: response to metoclopramide.

The pathophysiology of gastrointestinal symptoms in patients with Fabry's disease is uncertain, despite the demonstration of histological and radiographic abnormalities of the gastrointestinal tract. The aims of this study were to determine if the gastrointestinal symptoms reported by patients with Fabry's disease are associated with abnormal gastric emptying and, if they are, whether prokinetic drug therapy would be beneficial. Ten patients with Fabry's disease had radionuclide gastric emptying studies performed to determine if gastrointestinal symptoms correlated with objective evidence of abnormal gastric emptying. A second study was performed in seven patients who received 5 days of therapy with oral metoclopramide. The mean percent solid gastric emptying for Fabry's patients was significantly less than that of a normal control group (40 vs 67%, P < 0.005). Five of seven patients with symptoms had abnormal gastric emptying. Six of seven symptomatic patients reported clinical improvement with metoclopramide. Of four symptomatic patients who had a repeat study after treatment, two showed improved emptying. In conclusion, our results suggest that gastrointestinal symptoms in Fabry's disease are frequently associated with delayed gastric emptying and that treatment with metoclopramide produces symptomatic and sometimes functional improvement.