Pathology of GM2 gangliosidosis in Jacob sheep.

The G(M2) gangliosidoses are a group of lysosomal storage diseases caused by defects in the genes coding for the enzyme hexosaminidase or the G(M2) activator protein. Four Jacob sheep from the same farm were examined over a 3-year period for a progressive neurologic disease. Two lambs were 6-month-old intact males and 2 were 8-month-old females. Clinical findings included ataxia in all 4 limbs, proprioceptive deficits, and cortical blindness. At necropsy, the nervous system appeared grossly normal. Histologically, most neurons within the brain, spinal cord, and peripheral ganglia were enlarged, and the cytoplasm was distended by foamy to granular material that stained positively with Luxol fast blue and Sudan black B stains. Other neuropathologic findings included widespread astrocytosis, microgliosis, and scattered spheroids. Electron microscopy revealed membranous cytoplasmic bodies within the cytoplasm of neurons. Biochemical and molecular genetic studies confirmed the diagnosis of G(M2) gangliosidosis. This form of G(M2) gangliosidosis in Jacob sheep is very similar to human Tay-Sachs disease and is potentially a useful animal model.

Vitamin B12-responsive severe leukoencephalopathy and autonomic dysfunction in a patient with "normal" serum B12 levels.

Leukoencephalopathy and autonomic dysfunction have been described in individuals with very low serum B(12) levels (<200 pg/ml), in addition to psychiatric changes, neuropathy, dementia and subacute combined degeneration. Elevated homocysteine and methylmalonic acid levels are considered more sensitive and specific for evaluating truly functional B(12) deficiency. A previously healthy 62-year-old woman developed depression and cognitive deficits with autonomic dysfunction that progressed over the course of 5 years. The patient had progressive, severe leukoencephalopathy on multiple MRI scans over 5 years. Serum B(12) levels ranged from 267 to 447 pg/ml. Homocysteine and methylmalonic acid levels were normal. Testing for antibody to intrinsic factor was positive, consistent with pernicious anaemia. After treatment with intramuscular B(12) injections (1000 µg daily for 1 week, weekly for 6 weeks, then monthly), she made a remarkable clinical recovery but remained amnesic for major events of the last 5 years. Repeat MRI showed partial resolution of white matter changes. Serum B(12), homocysteine and methylmalonic acid levels are unreliable predictors of B(12)-responsive neurologic disorders, and should be thoroughly investigated and presumptively treated in patients with unexplained leukoencephalopathy because even long-standing deficits may be reversible.

Neurovisceral and skeletal GM1-gangliosidosis in dogs with beta-galactosidase deficiency.

Beta-galactosidase-deficient siblings in two litters of English springer spaniel puppies showed a progressive neurological impairment, dwarfism, orbital hypertelorism, and dysostosis multiplex. An excess of GM1-ganglioside was found in the brain. Three abnormal oligosaccharides were present in samples of urine, brain, liver, and cartilage. Light microscopy of selected tissue specimens revealed cytoplasmic vacuoles in neurons, circulating blood cells, macrophages, and chondrocytes. Ultrastructural studies demonstrated that these membrane-bound vacuoles were of two types--one containing lamellated membranes and the other, finely granular material. These clinical and pathological findings are similar to those observed in human patients affected by the infantile form of GM1-gangliosidosis.

Spontaneous appearance of Tay-Sachs disease in an animal model.

Tay-Sachs disease (TSD) is a progressive neurodegenerative disorder due to an autosomal recessively inherited deficiency of beta-hexosaminidase A (Hex A). Deficiency of Hex A in TSD is caused by a defect of the alpha-subunit resulting from mutations of the HEXA gene. To date, there is no effective treatment for TSD. Animal models of genetic diseases, similar to those known to exist in humans, are valuable and essential research tools for the study of potentially effective therapies. However, there is no ideal animal model of TSD available for use in therapeutic trials. In the present study, we report an animal model (American flamingo; Phoenicopterus ruber) of TSD with Hex A deficiency occurring spontaneously in nature, with accumulation of G(M2)-ganglioside, deficiency of Hex A enzymatic activity, and a homozygous P469L mutation in exon 12 of the hexa gene. In addition, we have isolated the full-length cDNA sequence of the flamingo, which consists of 1581 nucleotides encoding a protein of 527 amino acids. Its coding sequence indicates approximately 71% identity at the nucleotide level and about 72.5% identity at the amino acid level with the encoding region of the human HEXA gene. This animal model, with many of the same features as TSD in humans, could represent a valuable resource for investigating therapy of TSD.

Very long chain acyl-CoA dehydrogenase deficiency in a pair of mildly affected monozygotic twin sister in their late fifties.

Very long-chain acyl-CoA dehydrogenase (VLCAD) catalyses the initial step of mitochondrial beta-oxidation of long-chain fatty acids with a chain length of 14 to 20 carbons. Deficiency of VLCAD activity has been associated with a range of phenotypes, including a severe lethal form presenting in the infantile period and a milder variant with onset in childhood. Varying rates of residual enzyme activity partly explain the heterogeneity in presentations. Here we report the course of disease in a pair of monozygotic twin sisters who were diagnosed in their late forties during an evaluation for rhabdomyolysis and fatigue. Interestingly, the patients' complaints were most severe during puberty and declined significantly after the menopause. The basis for this observation is uncertain, but may be related to hormonally-mediated changes in lipid metabolism that may occur at these times. As metabolic decompensation can be associated with significant morbidity, timely diagnosis of VLCAD deficiency is important. The introduction of appropriate dietary measures (i.e. avoidance of fasting, long-chain fat restriction and supplementation with medium-chain triglycerides) greatly reduces the likelihood of complications.

Metabolic activities in human skin fibroblasts preloaded with labeled GM2-ganglioside.

Confluent cultures of human skin fibroblasts were maintained for 10 days with sphingosine labeled [3H]GM2. Labeled medium was then replaced with normal medium and the cells maintained for 42 days with weekly medium changes. Cells were harvested at regular intervals and cells, medium, and trypsin digest supernatant analyzed for [3H]GM2 and its metabolic products. The ganglioside can be membrane associated and removed by trypsin, or membrane incorporated and trypsin insensitive. The membrane incorporated material is apparently transported to the lysosomes slowly by membrane flow, where 80% of the cellular GM2 can be metabolized by day 42. [3H]GM2 as well as its metabolic products in control cells is continuously released into the medium, during which it can also become associated with the cell surface membrane. There is no detectable metabolism of the [3H]GM2 in GM2 gangliosidosis cell lines over the extended post-labeling period, indicating that there is no residual enzyme activity in these cells. Undegraded GM2 is continuously released into the medium and remains associated with the cell surface membrane as well.

GM2-ganglioside metabolism in cultured human skin fibroblasts: unambiguous diagnosis of GM2-gangliosidosis.

The metabolism of GM2-ganglioside was studied in situ using cultured skin fibroblasts from normal individuals and patients with different forms of GM2-gangliosidosis. [3H]Sphingosine-labeled GM2 was provided in the culture medium to confluent cells in 6-cm petri dishes. After 10 days, the cells were washed free of radioactivity and harvested by trypsinization. The cellular lipids were extracted and analyzed for radioactivity in GM2 and its metabolic products. In fibroblasts from healthy subjects, 50-60% of the total cellular radioactivity was found in the neutral glycosphingolipids, ceramide, sphingomyelin and fatty acids. Degradation of the labeled GM2 progressed rapidly via GM3, ceramide dihexoside and ceramide monohexoside with a build-up of radioactivity mainly in the ceramide pool of the cell. The labeled ceramide is also reutilized for the synthesis of ceramide trihexoside, globoside and sphingomyelin or is converted to fatty acid and incorporated in ester linkages. In contrast, cells from patients with GM2-gangliosidosis representing Tay-Sachs, Sandhoff and AB variant forms of the disease did not metabolize the ingested labeled GM2-like controls. Nearly all of the radioactivity was present in the ganglioside fraction in the lipid extracts from these cells and consisted of unhydrolyzed GM2. High-performance liquid chromatographic analysis of monosialogangliosides from cells grown without added labeled GM2 in the medium indicated accumulation of endogenously synthesized GM2 in cell lines from all patients with GM2 gangliosidosis compared to healthy controls. This approach provides a reliable tool for pre- and post-natal diagnosis of all forms of GM2-gangliosidosis without ambiguity.

Occurrence of novel branched-chain fatty acids in Refsum's disease.

Two novel branched-chain fatty acids, which appear to be unsaturated analogs of phytanic acid, have been observed in sera and urine of patients with Refsum's disease. They occur in both phospholipids and neutral lipids, and have been isolated and characterized.

The Gaucher registry: demographics and disease characteristics of 1698 patients with Gaucher disease.

BACKGROUND: The Gaucher Registry, the largest database of patients with Gaucher disease (GD) worldwide, was initiated to better delineate the progressive nature of the disorder and determine optimal therapy. This report describes the demographic and clinical characteristics of 1698 patients with GD before they received enzyme replacement therapy. METHODS: Physicians worldwide who treat patients with GD were invited to submit prospective and retrospective data for an ongoing registry, using standardized data collection forms, for central processing and review. RESULTS: Most patients were from the United States (45%) and Israel (17%), but patients are from 38 countries. Most (94%) had type 1 GD, fewer than 1% had type 2, and 5% had type 3. Mutant allele frequency data, available for 45% of patients, showed the most common alleles to be N370S (53%), L444P (18%), 84GG (7%), and IVS2+1 (2%). Twenty-five percent of L444P homozygotes (13 of 52 patients) had type 1 GD phenotype. Mean age at diagnosis in patients with the N370S/N370S genotype was 27.2 years (SD, 19.7 years); in L444P/L444P patients, 2. 3 years (SD, 3.2 years). Histories of bone pain and radiological bone disease were reported by 63% and 94% of patients, respectively; both were more likely in asplenic patients than in patients with spleens. Mean spleen and liver volumes were 19.8 and 2.0 multiples of normal, respectively. Anemia and thrombocytopenia were present in 64% and 56%, respectively. Thrombocytopenia was present in 13% of asplenic patients. CONCLUSIONS: The Gaucher Registry permits a comprehensive understanding of the clinical spectrum of GD because of the uniquely large sample size. The Registry will be useful in evaluating the effects of specific therapies in GD and the possible influences of environment, ethnicity, and genotype on the natural history of the disorder.

Gaucher disease: recommendations on diagnosis, evaluation, and monitoring.

BACKGROUND: Timely diagnosis and continued monitoring of patients with type I Gaucher disease is critical because skeletal involvement can permanently disable patients and visceral organ involvement can lead to abdominal pain and secondary hematologic and biochemical complications. OBJECTIVE: To seek clinical consensus for minimum recommendations for effective diagnosis and monitoring of patients with type I Gaucher disease. PARTICIPANTS, EVIDENCE, AND CONSENSUS PROCESS: Contributing authors collaborated in quarterly meetings over a 2-year period to synthesize recommendations from peer-reviewed publications and their own medical experiences. These physicians care for most patients with Gaucher disease in the United States and serve as the US Regional Coordinators for the International Collaborative Gaucher Group Registry, the world's largest database for this disorder. CONCLUSIONS: The definitive method of diagnosis is enzyme assay of beta-glucocerebrosidase activity. Schedules differ for monitoring complications of type I Gaucher disease, depending on symptoms and whether enzyme replacement therapy is used. Hematologic and biochemical involvement should be assessed by complete blood cell count, including platelets, acid phosphatase, and liver enzymes, at baseline and every 12 months in untreated patients and every 3 months and at enzyme replacement therapy changes in treated patients. Visceral involvement should be assessed at diagnosis using magnetic resonance imaging or computed tomographic scans. Skeletal involvement should be assessed at diagnosis using T1- and T2-weighted magnetic resonance imaging of the entire femora and plain radiography of the femora, spine, and symptomatic sites. Follow-up skeletal and visceral assessments are recommended every 12 to 24 months in untreated patients, and every 12 months and at enzyme replacement therapy changes in treated patients.

Bone marrow-derived mesenchymal stem cells remain host-derived despite successful hematopoietic engraftment after allogeneic transplantation in patients with lysosomal and peroxisomal storage diseases.

Human bone marrow contains mesenchymal stem cells (MSCs) that can differentiate into various cells of mesenchymal origin. We developed an efficient method of isolating and culture expanding a homogenous population of MSCs from bone marrow and determined that MSCs express alpha-L-iduronidase, arylsulfatase-A and B, glucocerebrosidase, and adrenoleukodystrophy protein. These findings raised the possibility that MSCs may be useful in the treatment of storage disorders. To determine if donor derived MSCs are transferred to the recipients with lysosomal or peroxisomal storage diseases by allogeneic hematopoietic stem cell (HSC) transplantation, we investigated bone marrow derived MSCs of 13 patients 1-14 years after allogeneic transplantation. Highly purified MSCs were genotyped either by fluorescence in situ hybridization using probes for X and Y-chromosomes in gender mis-matched recipients or by radiolabeled PCR amplification of polymorphic simple sequence repeats. Phenotype was determined by the measurement of disease specific protein/enzyme activity in purified MSCs. We found that MSCs isolated from recipients of allogeneic HSC transplantation are not of donor genotype and have persistent phenotypic defects despite successful donor type hematopoietic engraftment. Whether culture expanded normal MSCs can be successfully transplanted into patients with storage diseases and provide therapeutic benefit needs to be determined.

In vivo and in vitro glioma cell killing induced by an adenovirus expressing both cytosine deaminase and thymidine kinase and its association with interferon-alpha.

An adenovirus, AdCDTK, expressing both bacterial cytosine deaminase (CD) and herpes simplex virus thymidine kinase (HSVTK) was constructed and introduced into glioma cells. AdCDTK selectively rendered glioma cells sensitive to both 5-fluorocytosine (5-FCyt) and ganciclovir (GCV) (termed AdCDTK/5-FCyt-GCV). AdCDTK/5-FCyt-GCV not only potently mediated apoptosis and the arrest of glioma cell growth in vitro, but also significantly increased the survival time of glioma-bearing rats as compared with controls. The 90-day survival time was observed in 50% of rats. Interferon-alpha (IFN-alpha) further enhanced the tumor cell killing of AdCDTK/5-FCyt-GCV. In the group of AdCDTK/5-FCyt-GCV/IFN-alpha, the average survival time was significantly increased, and the average tumor size was smaller than that in the group of AdCDTK/5-FCyt-GCV. Ninety-day survival increased from 50% in the group of AdCDTK/5-FCyt-GCV to 75% in the group of AdCDTK/5-FCyt-GCV/IFN-alpha. Complete tumor regression was observed in 50% of rats in the group of AdCDTK/5-FCyt-GCV/IFN-alpha. The data indicate that AdCDTK/5-FCyt-GCV induces glioma cell killing greater than that induced by either CD/5-FCyt or HSVTK/GCV alone. IFN-alpha synergistically enhances this effect by increasing apoptosis.

Mucolipidosis IV: morphology and histochemistry of an autopsy case.

Mucolipidosis Type IV is a rare, autosomal recessive disorder characterized by corneal opacification, mental retardation, and delayed motor milestones. Whereas lysosomal storage material has been demonstrated in biopsied tissues and leukocytes, the complete autopsy pathology, including neuropathology, is unknown. The metabolic defect remains speculative. We report the general and neuropathologic findings of the only known autopsy. In the central nervous system, neuronal loss in the cerebral cortex, basal ganglia, deep cerebellar nuclei, and brainstem nuclei was marked by astrocytosis; the cytoplasm of residual neurons had brown granules. These granules were positive with periodic acid-Schiff, Concanavalia ensiformis, and Sudan black, but not with Luxol-fast blue. Ultrastructurally, neurons contained lysosomes laden with osmiophilic, amorphous and granular material, and few lamellated membrane structures. Hepatocytes, epithelia, endothelia, chondrocytes, and tissue macrophages also stained positively with Datura stramonium and Ricinus communis-I agglutinins, with renal glomeruli also staining with peanut agglutinin; most non-neural cells contained osmiophilic granules on toluidine blue-stained, plastic embedded sections, corresponding to lamellated membrane structures. These findings complement the previously reported ocular morphology and brain and liver biochemistry performed in the same patient, and suggest that the storage material in neurons differs from that in non-neural cells. Furthermore, the underlying defect is not likely to be a deficiency of a single enzyme (i.e. a lysosomal hydrolase).

The pituitary-thyroid axis in adults with phenylketonuria.

Serum thyroxine and triiodothyronine levels in 15 adult phenylketonuric patients on an unrestricted diet were normal despite reduced circulating tyrosine levels. Serum thyrotropin levels were normal in the basal state or in response to thyrotropin-releasing hormone in selected patients tested. These results support and extend previous observations of normal thyroid function in phenylketonuria.

Oligosaccharides from placenta: early diagnosis of feline mannosidosis.

High-pressure liquid chromatography analysis of oligosaccharides from placentas allowed the diagnosis of alpha-mannosidosis in three litters of kittens. The chromatography also afforded a detailed comparison of the oligosaccharide pattern and levels in placenta, liver, brain, urine and ocular fluid of the affected animals. In all cases, two series of compounds were observed, with one or two residues of N-acetylglucosamine at the reducing terminus, respectively, and between two and nine mannose residues. This pattern is unlike that of human mannosidosis, and resembles that of ruminants, except that the major oligosaccharide contains three mannose residues instead of two.

Pathologic findings in fetal GM1 gangliosidosis.

A 24-week fetus with GM1 gangliosidosis (type 1) was studied using biochemical and histopathologic methods. Foam cells in viscera and placenta demonstrated widespread accumulation of a lipidlike material. By microscopy, central nervous system storage appeared confined to the retina and dorsal root ganglia, but the brain ganglioside content was measurably elevated compared with that of age-matched controls. These data, along with those of others, imply that, if the observed pathologic findings are irreversible, any attempts at intrauterine therapy must commence prior to the middle of the second trimester.

Communicating hydrocephalus and lysosomal inclusions in mannosidosis.

A 32-year-old man with mannosidosis had a gait disorder develop that was associated with communicating hydrocephalus. The gait disorder improved with ventriculoperitoneal shunting, but proximal muscle weakness remained. Biopsy specimens of muscle and nerve disclosed typical lysosomal inclusions in both tissues, as well as selective loss of unmyelinated axons.

Mannosidosis. New clinical presentation, enzyme studied, and carbohydrate analysis.

Mannosidosis is a rare inborn error of metabolism characterized by deficiency of the lysosomal enzyme alpha-mannosidase and widespread storage of complex carbohydrate, which is enriched in mannose. Two affected unrelated males, aged 6 and 26 years, are reported. Both had a nonprogressive encephalopathy with moderately severe mental retardation. The older patient showed several unique features, including massive gingival hyperplasia associated with histiocytes containing large amounts of a material with the staining characteristics of glycoprotein. The best determinant of mannose storage proved to be the ratio of mannose to other carbohydrates in urinary polysaccharides. The enzyme deficiency in this disease is most convincingly demonstrated at pH values below 4.0. The ability of zinc to activate the mutant enzyme in vitro offers a possible mode of therapy for this disease. Retarded individuals with a Hurler-like appearance and gum hyperplasia of unknown cause should be screened for alpha-mannosidase deficiency.

Microcephaly vera, progressive motor neuron disease, and nigral degeneration.

A patient is described who was microcephalic at birth. After attaining early motor milestones, she developed progressive lower motor neuron dysfunction of the limb and bulbar musculature, combined with dystonia and an action tremor. The pathology and possible pathogenesis are discussed.

Fabry disease: impaired autonomic function.

Previous reports of extensive lipid accumulation within neurons of the autonomic nervous system in Fabry disease suggest an anatomicopathologic basis for the peculiar pain, diminished sweating, and gastrointestinal symptoms experienced in this disorder. To further assess autonomic function in Fabry disease, noninvasive clinical tests were performed on 10 patients. Diminished sweating was found in each; the loss was approximately uniform proximally and distally, suggesting sweat gland dysfunction rather than autonomic neuropathy. Impaired pupillary constriction with pilocarpine, and reduced saliva and tear formation were found in half the patients. Disordered intestinal mobility was demonstrated in the oldest patients. In all cases, the cutaneous flare response to scratch and intradermal histamine was diminished, and pruritus was not experienced. Signs of autonomic dysfunction are present in Fabry disease and correlate with the known lipid deposition in autonomic neurons.