Prenatal diagnosis of Gaucher disease Assay of the ß-glucosidase activity in amniotic fluid cells cultivated in two laboratories with different cultivation conditions

Sixteen pregnancies at risk for Gaucher disease - six with the Norrbottnian form, one with a juvenile form with a similar clinical course to the patients from Norrbotten and nine with the infantile form - have been monitored by the assay of β-glucosidase activity in cultivated amniotic fluid cells with natural labelled glycosylceramide as substrate. Two methods of cultivation were compared in respect of their effect on the activity of lysosomal enzymes. No significant difference was found between the two marker enzymes, β-galactosidase and N -acetyl-β-glucosaminidase, but the β-glucosidase activity was significantly higher in the cells cultivated with one of the methods. In four of the pregnancies at risk, the β-glucosidase activity in the cultivated amniotic fluid cells was less than 5 % of that in the two control materials. These fetuses were regarded as affected with Gaucher disease and were aborted. Differentiation between controls and Gaucher heterozygotes was not possible in cultivated amniotic fluid cells. The diagnosis of Gaucher disease in the amniotic fluid cells was confirmed in three of the four cases by the assay of the β-glucosidase activity in the liver and brain of the aborted fetuses. The glucosylceramide content of the liver from two aborted fetuses was not augmented. The β-glucosidase activity was examined in seven placentas from pregnancies at risk for Gaucher disease and found to be in agreement with that in the cultivated amniotic fluid cells.     

Gauchev's Disease Type I: Report of a Case with Prominent Deposition of Ceroid in Splenic Endothelial Cells and Intestinal Smooth Muscle Fibers

A case of type I Gaucher's disease in a 39 year old male is reported. Autopsy showed marked enlargement of the spleen (3,070g) and infiltration of typical Gaucher's cells in the spleen, liver, bone marrow, gastrointestinal tract, lymph nodes and adrenal glands. The diagnosis of Gaucher's disease was ascertained by the very low β-glucosidase activity of cultured subcutaneous fibroblasts and the high content of glucocerebroside in the spleen tissue. A peculiar finding in this case was prominent deposition of brown pigment in endothelial cells of the spleen and smooth muscle fibers of the gastrointestinal tract, urinary bladder and prostate. Histochemical examination revealed that the granules in endothelial cells and smooth muscle fibers were ceroid. Such deposition of ceroid has never been reported previously in Gaucher's disease. Ceroid deposition in generalized smooth muscle fibers is known as brown bowel syndrome, and is highly suggestive of severe vitamin E deficiency. Although other symptoms of vitamin E deficiency were not noticed in this case, some malnutritional condition might play a role in prominent deposition of ceroid in lysosomes, possibly together with deficient activity of a Jysosomal enzyme.     

Gaucher's disease: a paradigm for interventional genetics

Gaucher's disease (GD) is one of the most prevalent lysosomal storage disorders (LSDs) and a rare genetic disease for which specific therapy is now available. GD is an autosomal, recessive, inborn error of glycosphingolipid metabolism, due to a deficiency in the enzyme acid β-glucosidase. Partial deficiency of acid β-glucosidase is associated with parenchymal disease of the liver, spleen, and bone marrow with concomittant anemia and thrombocytopenia in non-neuronopathic, type 1 GD. Severe deficiency of glucocerebrosidase caused by severe mutations is additionally associated with neurological manifestations in the less common type 2 and type 3 GD subtypes. Outside of the Ashkenazi Jewish community, a high molecular diversity is observed. Clarification of genotype/phenotype relationship and the identification of modifier loci that impact on GD phenotypes remains a critical area for research. Enzyme replacement therapy (ERT) is proven to be safe and effective in the treatment of type 1 GD, establishing imiglucerase as the current standard of care. Amelioration of hepatosplenomegaly and of hematological manifestations is usually apparent within 6–12 months, whereas the bone disease responds more slowly. ERT cannot reverse the neurological deficits in type 2 or type 3 GD. Small molecule inhibitors of glucosylceramide synthase are being developed for substrate reduction therapy. Other potential therapeutic options such as chaperon-mediated enzyme enhancement therapy and gene therapy are being explored.     

Assay of the β-glucosidase activity with natural labelled and artificial substrates in cultivated skin fibroblasts from homozygotes and heterozygotes with the Norrbottnian type of Gaucher disease

Fibroblasts from 13 homozygotes and 27 obligate heterozygotes with the Norrbottnian type of Gaucher disease and 17 controls were cultivated and assayed with five β-glucosidase methods, two with D- [glucose- U-¹⁴C] glucosylceramide and three with the artificial substrate 4-methylumbelliferyl-β-glucoside. Two marker enzymes were assayed on the same cell samples, 4-methylumbelliferyl-β-galactosidase and N -acetyl-β-glucosaminidase. The β-glucosidase activity of cultured fibroblasts, as measured with all five β-glucosidase methods, was significantly lower (P < 0.001) for Gaucher homozygotes than heterozygotes. There was no overlap between fibroblasts from Gaucher homozygotes and the others with any of the β-glucosidase methods used. The β-glucosidase activity was also significantly lower ( P < 0.001) for Gaucher heterozygotes than controls. However, none of the five β-glucosidase assays differentiated between all Gaucher heterozygotes and controls, as several overlaps occurred in each assay.     

Activator protein deficient Gaucher's disease

Summary A report is presented based on the biochemical and immunochemical studies of various tissues from a 15-year-old boy with a neuronopathic form of Gaucher's disease. Qualitative and quantitative lipid analyses revealed a storage of glucosylceramide. The striking feature was that, employing the usual assay methods, a normal activity of the lysosomal enzyme glucosylceramidase was revealed, despite massive lipid accumulation. Immunochemical assays of hepatic and splenic tissue extracts from this atypical Gaucher's patient disclosed the absence of A₁ activator protein, which is necessary for the enzymic degradation of glucosylceramide in vivo. This is the second documented case of a patient presenting with glucosylceramide activator protein deficiency.     

Activity of glucocerebrosidase in extracts of different cell types from type 1 Gaucher disease patients

Glucocerebrosidase activity in extracts of leukocytes, Epstein-Barr virus transformed lymphocytes and fibroblasts from Portuguese Type 1 Gaucher disease patients was studied. The residual glucocerebrosidase activity in all extracts from patients was less than 25% if measured in the presence of bile salt taurocholate. However, if measured in the absence of bile salt the residual enzyme activity in extracts from patients was cell type specific: it was severely reduced in the case of fibroblasts, mildly reduced in the case of lymphoblasts and not significantly reduced in the case of leukocytes. The glucocerebrosidase activity in extracts from all control cell types was stimulated by taurocholate. In the patients the enzyme activity in fibroblasts extracts was also stimulated but that in lymphoblasts and leukocytes was inhibited by the bile salt. The differences in glucocerebrosidase activity (in the absence of taurocholate) in extracts from different cell types from Gaucher disease patients are attributable to differences in the proportion of glucocerebrosidase present as a monomer with low activity (form I) and as a highly active aggregate (form II) that may also contain sphingolipid activator protein 2 (SAP-2). In extracts from leukocytes and lymphocytes from Type 1 Gaucher disease patients, but not in those from fibroblasts, a relatively high proportion of enzyme is present in aggregated form with near normal specific activity.     

Synthetic substrate ß-glucosidase activity in leukocytes: A reproducible method for the identification of patients and carriers of Gaucher''s disease

A method is described for the identification of patients and carriers of Gaucher's disease, using leukocytes from a small volume of blood. The fluorogenic substrate, 4-methylumbelliferyl-beta-D-glucopyranoside, was assayed in the presence of pure sodium taurocholate (2.5 mg/ml) and Triton X-100 (2.0 mg/ml). Some commercial brands of pure sodium taurocholate were satisfactory for this purpose. The pH optimum for controls, Gaucher disease carriers and Gaucher disease patients was 5.4 using citrate-phosphate buffer. Although leukocytes prepared from only a small amount of blood (2-8 ml) are required, there is sufficient quantity for measuring other lysosomal enzymes as controls. Using this method, 12 patients with all types of Gaucher's disease and 12 obligate heterozygotes were identified. Carrier status was predicted in six other family members and ruled out in six others. Eight unaffected people married to Gaucher carriers or Gaucher patients were predicted to be non-carriers of Gaucher's disease, thereby ruling out children affected with Gaucher's disease in that mating.     

GAUCHER-LIKE CELLS IN JUVENILE GM1-GANGLIOSIDOSIS AND IN β-THALASSEMIA–A Histochemical and Ultrastructural Observation–

Peculiar storage cells appearing in bone marrow aspirates from a patient with juvenile GM₁-gangliosidosis and from one with β-thalassemia were examined light microscopically, histochemically and electron microscopically. Light microscopically, most of the storage cells closely resembled Gaucher cells pathognomonic for Gaucher's disease. The cytoplasm of the Gaucher-like cells contained numerous variable-shaped, membrane-bound inclusions mostly arranged in a mosaic pattern and filled with fibrillar materials. Intermingled tubular structures were usually narrow as compared to those of the Gaucher cells. These ultrastructural differences of the stored materials between the Gaucher-like cells and Gaucher cells were more clearly substantiated by the high resolution electron microscopy with negative staining technique. Enzyme cytochemically, acid phosphatase activity was proved in or around the storage inclusions, suggesting their lysosomal origin. Histochemically, it might be suggested that the stored materials of the Gaucher-like cells in juvenile GM₁-gangliosidosis were non-sulfated acid mucopolysaccharides and glycopeptides, whereas glycoproteins were the major component of the storage cells in β-thalassemia. Possible mechanisms of storage in the Gaucher-like cells were discussed in both disorders.     

Intracellular transport of acid β-glucosidase and lysosome-associated membrane proteins is affected in Gaucher's disease (G202R mutation)

Gaucher's disease (GD) is caused by an inherited deficiency of acid β-glucosidase with storage of glucosylceramides in the lysosomes of macrophages. This study identifies a G202R mutation in the acid β-glucosidase gene in an infant with severe neuronopathic (type 2) GD and only slightly reduced acid β-glucosidase activity. Western blot analysis, pulse chase experiments, and the thin frozen section immunogold method were used to analyse the implications of this mutation on the pathogenesis, clinical heterogeneity and diagnostic evaluation of GD. The results show that acid β-glucosidase persists in the patient's fibroblasts as a mannose-rich polypeptide in the endoplasmic reticulum and is not transported to the lysosomes. By contrast, high expression of the lysosome-associated membrane proteins LAMP-1 and LAMP-2, saposin C, and cathepsin D was observed in the patient's lysosomes. Immunogold labelling of the integral membrane proteins LAMP-1 and LAMP-2 increases significantly at the cell surface of Kupffer cells and fibroblasts as well as at the apical membrane of hepatocytes. In addition, LAMP-1 and LAMP-2 associate with the bilayer of stored glucosylceramide. It is concluded that defective intracellular transport of mutant acid β-glucosidase from the endoplasmic reticulum to lysosomes leads to a more severe clinical phenotype than the residual enzyme activity may indicate. Furthermore, the detection of LAMP in the tubular bundles of undigested glucosylceramides, as well as their increased concentration at the surfaces of the affected cells, suggests that these proteins play a role in the storage or removal of substrate in GD. Intracellular targeting of acid β-glucosidase and LAMP contributes to the broad phenotypic heterogeneity of GD. Copyright © 1999 John Wiley & Sons, Ltd.     

Gaucher's disease type I. Report of a case with prominent deposition of ceroid in splenic endothelial cells and intestinal smooth muscle fibers

A case of type I Gaucher's disease in a 39-year-old male is reported. Autopsy showed marked enlargement of the spleen (3,070 g) and infiltration of typical Gaucher's cells in the spleen, liver, bone, marrow, gastrointestinal tract, lymph nodes and adrenal glands. The diagnosis of Gaucher's disease was ascertained by the very low beta-glucosidase activity of cultured subcutaneous fibroblasts and the high content of glucocerebroside in the spleen tissue. A peculiar finding in this case was prominent deposition of brown pigment in endothelial cells of the spleen and smooth muscles fibers of the gastrointestinal tract, urinary bladder and prostate. Histochemical examination revealed that the granules in endothelial cells and smooth muscle fibers were ceroid. Such deposition of ceroid has never been reported previously in Gaucher's disease. Ceroid deposition in generalized smooth muscle fibers is known as brown bowel syndrome, and is highly suggestive of severe vitamin E deficiency. Although other symptoms of vitamin E deficiency were not noticed in this case, some malnutritional condition might play a role in prominent deposition of ceroid in lysosomes, possibly together with deficient activity of a lysosomal enzyme.     

Biochemical genetics of the Lapland dog model of glycogen storage disease type II (acid α-glucosidase deficiency)

A recently described canine model (Lapland dog) of glycogen storage disease type II (GSD II, Pompe disease, acid α-glucosidase deficiency) was identified with several biochemical genetic methods. Complementation studies in which fibroblasts from a GSD II dog were fused with fibroblasts derived from control dogs and from human patients with different clinical forms of the disease did not lead to restoration of acid α-glucosidase activity in the heterokaryon cell populations. These results indicate that acid α-glucosidase deficiency is the primary defect in canine GSD II and that there is a close genetic parallelism with human GSD II. Immunotitration analysis of the residual acid α-glucosidase activity in the canine GSD II fibroblasts and liver demonstrated that this residual activity was not due to acid α-glucosidase enzyme, in which respect canine GSD II was similar to the infantile form of the human disease. Double immunodiffusion studies showed the presence of catalytically inactive acid α-glucosidase enzyme protein in canine GSD II. This is consistent with a structural gene mutation. It is concluded that canine GSD II in the Lapland dog is a homologous model of the infantile form of human GSD II, a conclusion in concordance with clinical and pathological studies.     

Immunological cell type characterization and Th1-Th17 cytokine production in a mouse model of Gaucher disease

Gaucher disease is a lysosomal storage disease resulting from insufficient acid β-glucosidase (glucocerebrosidase, GCase, EC 4.2.1.25) activity and the resultant accumulation of glucosylceramide. Macrophage (M?) lineage cells are thought to be the major disease effectors because of their secretion of numerous cytokines and chemokines that influence other poorly defined immunological cell populations. Increases in several such populations were identified in a Gba1 mouse model (D409V/null; 9V/null) of Gaucher disease including antigen presenting cells (APCs), i.e., M?, dendritic cells (DCs), neutrophils (PMNs), and CD4(+) T cells. FACS analyses showed increases in these cell types in 9V/null liver, spleen lung, and bone marrow. T-cells or APCs enhanced activations were evident by positivity of CD40L, CD69, as well as CD40, CD80, CD86, and MHCII on the respective cells. M?, and, unexpectedly, DCs, PMNs, and T cells, from 9V/null mice showed excess glucosylceramides as potential bases for activation of APCs and T cells to induce Th1 (IFNγ, IL12, TNFα,) and Th17 (IL17A/F) cytokine production. These data imply that excess glucosylceramides in these cells are pivotal for activation of APCs and T cell induction of Th1 and Th17 responses and PMN recruitment in multiple organs of this model of Gaucher disease.     

Type 2 Gaucher's disease in a Malian family

Gaucher's disease is a recessive autosomal disorder caused by an inherited deficiency of betaglucocerebrosidase. We report here the case of an 8 month old child, fourth in a family of four children, who presents the neuropathic form of the disease. The dosages of betaglucosidase activity using C (14 ) techniques have confirmed the diagnosis, and allowed the detection of the disease in the elder brother. Both parents were considered as responsible for the transmission of this disease to their progeny. The type 2 Gaucher's disease is rare in black population, and may be associated with phenotypes heterogeneity.     

A modified method for the identification of heterozygotes for Gaucher''s disease using differential thermal inactivation

We have found heat inactivation of leukocyte 4-methylumbelliferyl-β-D-glucosidase to be useful in identifying heterozygotes for adult (chronic) Gaucher's disease. The overlap between high heterozygote values and low normal values is reduced by the heat inactivation step, compared to assays without heating; that is from an overlap of 23 % to 6 % . Although preliminary, our results indicate that this procedure may be useful in families with adult (chronic) Gaucher's disease.     

Accumulation and distribution of α-synuclein and ubiquitin in the CNS of Gaucher disease mouse models

Gaucher disease, a prevalent lysosomal storage disease, is caused by insufficient activity of acid β-glucosidase (GCase) and resultant glucosylceramide accumulation. Recently in Parkinson disease (PD) patients, heterozygous mutations in GCase have been associated with earlier onset and more progressive PD. To understand the pathogenic relationships between GCase variants and Parkinsonism, α-synuclein and ubiquitin distributions and levels in the brains of several mouse models containing GCase variants were evaluated by immunohistochemistry. Progressive α-synuclein and ubiquitin aggregate accumulations were observed in the cortex, hippocampus, basal ganglia, brainstem, and some cerebellar regions between 4 and 24 weeks in mice that were homozygous for GCase [D409H (9H) or V394L (4L)] variants and also had a prosaposin hypomorphic (PS-NA) transgene. In 4L/PS-NA and 9H/PS-NA mice, this was coincident with progressive neurological manifestations and brain glucosylceramide accumulation. Ultrastructural studies showed electron dense inclusion bodies in neurons and axons of 9H/PS-NA brains. α-synuclein aggregates were also observed in ventricular, brainstem, and cerebellar regions of older mice (>42-weeks) with the GCase variant (D409H/D409H) without overt neurological disease. In a chemically induced GCase deficiency, α-synuclein aggregates and glucosylceramide accumulation also occurred. These studies demonstrate a relationship between glucosylceramide accumulation and α-synuclein aggregates, and implicate glucosylceramide accumulation as risk factor for the α-synucleinopathies.     

Glycosphingolipid studies of visceral tissues and brain from type 1 Gaucher disease variants

Glucosylceramide and glucosylsphingosine isolated from spleen, liver and brain were quantitated and characterized in two unrelated patients with Gaucher disease, neither of whom had clinical or neuropathologic evidence of neuronal involvement. Visceral glucosylceramide accumulation did not differ in the two patients. Hepatic glucosylsphingosine content was 2-fold greater in a young severely affected 3-year-old American Black patient compared to that in a 56-year-old Ashkenazi Jewish patient. In contrast, significant differences in glycosphingolipid content and composition were observed in the brains of these two cases. Cerebral and cerebellar cortical glucosylceramide accumulated to a greater extent (3-fold) in the severely affected 3-year-old patient compared to that in the older case. The compositions of the acyl and sphingosyl base residues of glucosylceramide in the cerebral and cerebellar cortices from the Ashkenazi Jewish patient were similar to those in normal individuals. In comparison, the gray matter glucosylceramide in the severely affected patient had increased percentages of stearic acid (18:0) and eicosasphingenine (d20:1), suggesting that the accumulated substrate was derived from the brain ganglioside pool. Glucosylsphingosine was found in large amounts only in cerebral and cerebellar cortices from the severely affected patient. The glycolipid content and composition in this patient was similar to that found in the Norrbottnian (Type 3) form of Gaucher disease. The differences in glucosylceramide acyl and sphingosyl base composition in gray matter from the severely affected patient and that in the Ashkenazi Jewish patient suggested that the accumulated substrates were metabolized differently by the residual enzymes in each case.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acquired pseudo-pseudo Bernard-Soulier syndrome complicating Gaucher's disease.

AIMS--To investigate the abnormality in platelet function in two patients with type I Gaucher's disease causing a chronic bleeding tendency despite normalisation of the platelet count after spleen removal. METHODS--Routine laboratory methods were used to assess baseline coagulation. Platelet aggregometry was used to assess platelet responses to a range of agonists, and abnormalities were further assessed in mixing experiments using washed platelets and patients' plasma. RESULTS--Platelets from both patients with Gaucher's disease failed to agglutinate to ristocetin, despite normal platelet surface glycoprotein (GP) Ib and plasma von Willebrand factor activity. The agglutination of normal washed platelets was abolished by incubation in patient plasma. The inhibitory activity did not lie in the IgG fraction of patient plasma, and was found to be loosely associated with the patient platelet surface. CONCLUSIONS--The inhibition of ristocetin induced platelet agglutination in patients with Gaucher's disease causes a prolonged skin bleeding time. This could be due to the accumulated glucocerebroside in the plasma coating the platelet membrane. It is suggested that the term pseudo-pseudo Bernard-Soulier syndrome would be appropriate, as on initial screening, the abnormality has the features of Bernard-Soulier syndrome, but further investigation shows normal plasma von Willebrand activity and platelet surface GP Ib concentrations. The inhibitory activity is not due to a platelet specific antibody as is the case in pseudo-Bernard Soulier syndrome.     

Stimulation of β-adrenoceptors inhibits store-operated channel currents via a cAMP-dependent protein kinase mechanism in rabbit portal vein myocytes

Previously we have described the properties of store-operated channel currents (SOCs) in freshly dispersed rabbit portal vein smooth muscle cells. In addition to Ca²⁺ store depletion these SOCs could also be activated by α-adrenoceptor stimulation and diacylglycerol (DAG) via a protein kinase C (PKC)-dependent mechanism. In the present study we have investigated the effect of β-adrenoceptor stimulation on SOCs in rabbit portal vein myocytes. With whole-cell recording the selective β-adrenoceptor agonist isoprenaline reduced the current evoked by cyclopiazonic acid (CPA, sarcoplasmic/endoplasmic reticulum ATPase inhibitor) by over 85%. With cell-attached patch recording, bath application of isoprenaline produced a pronounced inhibition of SOC activity evoked by either CPA or the acetoxymethyl ester form of BAPTA (BAPTA-AM). SOC activity evoked by CPA, the DAG analogue, 1-oleoyl-acetyl- sn -glycerol (OAG) or the phorbol ester, phorbol-12,13-dibutyrate (PDBu) was also markedly inhibited by the adenylate cyclase activator, forskolin, and the cell-permeable non-hydrolysable analogue of cyclic adenosine monophosphate (cAMP), 8-Br-cAMP. With inside-out patches, bath application of PDBu evoked channel currents with similar properties to SOCs which were inhibited by over 90% by a catalytic subunit of protein kinase A (PKA) and by 8-Br-cAMP. Moreover bath application of PKA inhibitors, H-89, KT5720 and an inhibitory peptide to quiescent cell-attached or inside-out patches, activated channel currents with similar properties to SOCs. These data suggest that in rabbit portal vein myocytes, stimulation of β-adrenoceptors inhibits SOC activity via a cAMP-dependent protein kinase signal transduction cascade. In addition it is concluded that constitutive PKA activity has a profound inhibitory effect on SOC activity in this vascular preparation.     

Electrophoretic analysis of glycoprotein enzymes in the sialidoses and mucolipidoses

Ten enzymes, all known to be glycoproteins, were examined by electrophoresis or gel isoelectric focusing in 12 different patients with primary or secondary sialidase deficiency. Aberrant electrophoretic mobilities of many of the enzymes attributable to abnormal sialylation were found in all the patients. In ten of the patients seven of the enzymes were affected. The unaffected enzymes were β -galactosidase, alkaline phosphatase and β -glucuronidase. In the cells from the two patients with I cell disease (mucolipidosis II) in which sialidase is one of many deficient enzymes, β -galactosidase, α -galactosidase, α -fucosidase and α -mannosidase were undetectable, alkaline phosphatase showed a normal electrophoretic mobility and acid phosphatase, adenosine deaminase, α -glucosidase and β -D- N -acetylhexosaminidase showed aberrant mobilities.     

High antibody titer in an adult with Pompe disease affects treatment with alglucosidase alfa

Clinical trials have demonstrated beneficial effects of enzyme replacement therapy (ERT) with alglucosidase alfa in infants, children and adults with Pompe disease. Recent studies have shown that high antibody titers can occur in patients receiving ERT and counteract the effect of treatment. This particularly occurs in those patients with classic-infantile Pompe disease that do not produce any endogenous acid α-glucosidase (CRIM-negative). It is still unclear to what extent antibody formation affects the outcome of ERT in adults with residual enzyme activity.We present the case of a patient with adult-onset Pompe disease. He was diagnosed at the age of 39 years by enzymatic testing (10.7% residual activity in fibroblasts) and DNA analysis (genotype: c.-32-13T>G/p.Trp516X). Infusion-associated reactions occurred during ERT and the patient's disease progressed. Concurrently, the antibody titer rose to a similarly high level as reported for some CRIM-negative patients with classic-infantile Pompe disease.Using newly developed immunologic-assays we could calculate that approximately 40% of the administered alglucosidase alfa was captured by circulating antibodies. Further, we could demonstrate that uptake of alglucosidase alfa by cultured fibroblasts was inhibited by admixture of the patient's serum.This case demonstrates that also patients with an appreciable amount of properly folded and catalytically active endogenous acid α-glucosidase can develop antibodies against alglucosidase alfa that affect the response to ERT.