Analysis and classification of 304 mutant alleles in patients with type 1 and type 3 Gaucher disease

Gaucher disease results from the inherited deficiency of the enzyme glucocerebrosidase (EC 3.2.1.45). Although >100 mutations in the gene for human glucocerebrosidase have been described, most genotype-phenotype studies have focused upon screening for a few common mutations. In this study, we used several approaches-including direct sequencing, Southern blotting, long-template PCR, restriction digestions, and the amplification refraction mutation system (ARMS)-to genotype 128 patients with type 1 Gaucher disease (64 of Ashkenazi Jewish ancestry and 64 of non-Jewish extraction) and 24 patients with type 3 Gaucher disease. More than 97% of the mutant alleles were identified. Fourteen novel mutations (A90T, N117D, T134I, Y135X, R170C, W184R, A190T, Y304X, A341T, D399Y, c.153-154insTACAGC, c.203-204insC, c.222-224delTAC, and c.1122-1123insTG) and many rare mutations were detected. Recombinant alleles were found in 19% of the patients. Although 93% of the mutant alleles in our Ashkenazi Jewish type 1 patients were N370S, c.84-85insG, IVS2+1G-->A or L444P, these four mutations accounted for only 49% of mutant alleles in the non-Jewish type 1 patients. Genotype-phenotype correlations were attempted. Homozygosity or heterozygosity for N370S resulted in type 1 Gaucher disease, whereas homozygosity for L444P was associated with type 3. Genotype L444P/recombinant allele resulted in type 2 Gaucher disease, and homozygosity for a recombinant allele was associated with perinatal lethal disease. The phenotypic consequences of other mutations, particularly R463C, were more inconsistent. Our results demonstrate a high rate of mutation detection, a large number of novel and rare mutations, and an accurate assessment of the prevalence of recombinant alleles. Although some genotype-phenotype correlations do exist, other genetic and environmental factors must also contribute to the phenotypes encountered, and we caution against relying solely upon genotype for prognostic or therapeutic judgements.     

In situ radiation-inactivation size of fibroblast membrane-bound acid beta-glucosidase in Gaucher type 1, type 2 and type 3 disease

The radiation-inactivation size of membrane-bound acid beta-glucosidase in cultured skin fibroblasts of four normal individuals, five Gaucher type 1 (non-neuropathic), four Gaucher type 2 (acute neuropathic) and three Gaucher type 3 (sub-acute neuropathic) patients was determined using the radiation-inactivation method. The radiation-inactivation size of the enzyme in the control, Gaucher type 2 and Gaucher type 3 fibroblasts ranged from 94 000 to 128 800, and no statistical significant difference was found in the enzyme size between the normal and Gaucher cells nor among the Gaucher type 2 and type 3 cells. Contrary to the normal, Gaucher type 2 and Gaucher type 3 enzyme, the radiation-inactivation size of membrane-bound acid beta-glucosidase in all of the Gaucher type 1 fibroblasts tested is significantly higher, ranging from 158 400 to 235 300. The size of the control lysosomal enzyme, sphingomyelinase, also determined by the radiation-inactivation method in fibroblasts of normal individuals and patients with the three Gaucher subtypes, was between 70 000 and 74 500 and indistinguishable from each other. Since the molecular weight of acid beta-glucosidase subunit determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis was about 60 000 (Pentchev, P.G., Brady, R.O., Hibbert, S.P., Gal, A.E. and Shapiro, C. (1973) J. Biol. Chem. 248, 5256-5261), the above data suggest that: (i) the normal fibroblast enzyme, as well as the Gaucher type 2 and type 3 mutant enzyme, in the membrane-bound form, exists as a dimer; (ii) the underlying biochemical and genetic defect in non-neuropathic (type 1) and neuropathic (type 2 and type 3) Gaucher disease is very different from each other; and (iii) subunit interaction of the mutant enzyme may be present in Gaucher type 1 fibroblasts, resulting in the formation of a higher-molecular-weight aggregate.     

TNF-alpha levels and TNF-alpha gene polymorphism in type I Gaucher disease

The objective of this pilot study was to determine the levels of Tumor Necrosis Factor (TNF)-alpha and TNF-alpha gene polymorphism as a marker of inflammation among patients with type I Gaucher disease as well as to ascertain the relationship between this cytokine and parameters of disease severity and other measures of inflammation. Levels of TNF-alpha and genotyping for the -308 G-->A polymorphism in the promoter of the TNF-alpha gene were performed in 17 patients with type I Gaucher disease. TNF-alpha levels were compared with the promoter gene polymorphism, and with hematological and other clinical parameters of Gaucher disease. Eight patients (47.1%) were homozygotes (A/A) for the TNF-alpha polymorphism, six patients (35.3%) had the wild type (G/G), and three patients (17.6%) were heterozygotes (G/A). A significant correlation was found between serum TNF-alpha levels and TNF-alpha genotypes for homozygotes versus heterozygotes patients (p = 0.02), with patients homozygous for the polymorphism having the lower levels of serum TNF-alpha relative to heterozygotes with the highest levels. No correlation was found between TNF-alpha genotypes and chitotriosidase levels, a putative biochemical marker for Gaucher disease severity. Because a significant correlation was found between homozygosity for a common promoter polymorphism of TNF-alpha and milder expression i.e. non-neuronopathic form, of Gaucher disease (versus the neuronopathic forms), this may be suggestive of an association between genetic variability in TNF-alpha and phenotypic expression in Gaucher disease. Larger studies will be required.     

Substrate reduction therapy: clinical evaluation in type 1 Gaucher disease

Glycosphingolipid (GSL) lysosomal storage disorders are inherited enzyme deficiencies that result in pathological lysosomal accumulation of glycolipids, with widespread clinical consequences. Type 1 Gaucher disease is the commonest of these; the deficient enzyme in this condition is glucocerebrosidase. Clinical manifestations include hepatosplenomegaly, thrombocytopenia, anaemia, recurrent infections and skeletal lesions. The condition can be treated with intravenous enzyme replacement therapy (ERT). Substrate reduction therapy is a new approach in which glycolipid accumulation is counteracted not by replacing the deficient enzyme but by reducing the substrate level to better balance residual activity of the deficient enzyme. Miglustat is an inhibitor of glucosylceramide synthase, a key enzyme in GSL synthesis. Oral administration of miglustat to patients with type 1 Gaucher disease attenuates the synthesis of glucocerebroside, the substrate of the deficient glucocerebrosidase. In the first clinical study, patients with type 1 Gaucher disease who had enlargement of the liver or spleen and (if present) the spleen at baseline received 12 months treatment with oral miglustat. There were mean decreases in liver and spleen volumes of 12% (7.9-16.4, p < 0.001) and 19% (14.3-23.7, p < 0.001), respectively. Mean haemoglobin increased by 0.26 g dl(-1) (-0.5-0.57, not statistically significant) and platelet count by 8.3 x 10(9) l(-1) (1.9-14.7, p = 0.014).     

Gaucher disease: The effects of phosphatidylserine on glucocerebrosidase from normal and Gaucher fibroblasts

Summary Glucocerebroside -glucosidase (glucocerebrosidase) activity was assayed from cultured fibroblasts of normal individuals, and patients with type 1 (non-neuropathic), type 2 (acute neuropathic), and type 3 (subacute neuropathic) form of Gaucher disease. Residual glucocerebrosidase activity of patients was 8.9 to 17.4% of normal controls, and there was no clear correlation between the level of residual enzyme activity and the different clinical subtypes of the disease. When membrane-bound glucocerebrosidase activity was assayed in the presence of crude brain lipid extracts or purified phosphatidylserine, enzyme from both the normal and type 1 Gaucher fibroblasts was stimulated dramatically (35–60% by crude extracts, 85–90% by phosphatidylserine). This stimulation was not observed with fibroblast glucocerebrosidase of an infantile type 2 and two juvenile type 3 Gaucher patients. The presence of inhibitors of glucocerebrosidase in these type 2 and type 3 Gaucher cells was not detected. Contrary to the mutant enzyme from these Gaucher fibroblasts, glucocerebrosidase from fibroblasts of two adult type 3 Gaucher patients with cerebral involvement was stimulated substantially (72–85%) by phosphatidylserine. When membrane-bound glucocerebrosidase from fibroblasts of the infantile type 2 and juvenile type 3 patients was solubilized with sodium cholate (1% w/v) and delipidated, the phospholipid stimulation of enzyme activity was restored. These findings suggest that considerable clinical and biochemical heterogeneity exists among patients with neuropathic Gaucher disease and that phosphatidylserine activation cannot be used as a reliable indicator in predicting future onset of neurodegeneration in Gaucher patients. The possibility of an aberrant binding of mutant glucocerebrosidase to the lysosomal membrane in juvenile type 3 form of Gaucher disease is discussed.     

Possible therapeutic effects of myxobacterial metabolites on type I Gaucher disease

Gaucher disease (GD) is the most prevalent lysosomal storage disorder caused by an inherited deficiency of glucocerebrosidase. In the present study, we aimed to determine whether myxobacterial metabolites exhibit a potential therapeutic effect in the cells from a patient with type I GD. We screened 288 bioactive compounds of myxobacteria in the skin fibroblasts from a patient with type I GD. MTT assays were performed to determine their effects on cell viability. The expression levels of Bcl-2-associated X protein (Bax), ATP-citrate synthase (ATP-CS), E3-binding protein (E3BP), and acetyl-coenzyme A acetyltransferase 1 (ACAT1) were determined by western blotting to understand the molecular mechanisms of myxobacterial metabolites in cells. Thin-layer chromatography (TLC) was carried out to measure changes in glucosylceramide levels in the cultured fibroblasts. This screening process identified 4 compounds that increased cell viability more than 1.45 times. After exposure to these compounds, the expression level of Bax decreased, whereas those of ATP-CS, E3BP, and ACAT1 increased. TLC revealed reduced amounts of intracellular glucosylceramides in patient cells. Here we suggest that myxobacterial metabolites can relieve the stress due to glucosylceramide accumulation, and that it may be utilized as a new therapeutic approach.     

Phenotype/genotype correlations in Gaucher disease type I: clinical and therapeutic implications.

Gaucher disease is the most frequent lysosomal storage disease and the most prevalent genetic disease among Ashkenazi Jews. Gaucher disease type 1 is characterized by marked variability of the phenotype and by the absence of neuronopathic involvement. To test the hypothesis that this phenotypic variability was due to genetic compounds of several different mutant alleles, 161 symptomatic patients with Gaucher disease type 1 (> 90% Ashkenazi Jewish) were analyzed for clinical involvement, and their genotypes were determined. Qualitative and quantitative measures of disease involvement included age at onset of the disease manifestations, hepatic and splenic volumes, age at splenectomy, and severity of bony disease. Highly statistically significant differences (P < .005) were found in each clinical parameter in patients with the N370S/N370S genotype compared with those patients with the N370S/84GG, N370S/L444P, and N370S/? genotypes. The symptomatic N370S homozygotes had onset of their disease two to three decades later than patients with the other genotypes. In addition, patients with the latter genotypes have much more severely involved livers, spleens, and bones and had a higher incidence of splenectomy at an earlier age. These predictive genotype analyses provide the basis for genetic care delivery and therapeutic recommendations in patients affected with Gaucher disease type 1.     

Biochemical and ultrastructural findings in Epstein-Barr virus-transformed lymphoid cell lines from type 1 Gaucher disease

Lymphoid cell lines established by Epstein-Barr Virus (EBV)-transformation of peripheral blood B-lymphocytes from patients affected with type 1 Gaucher disease showed a severe deficiency of glucosylceramide-beta-glucosidase activity (residual activity around 15%-30% of control activity). Ultrastructural investigations showed, in these lymphoid cell lines from type 1 Gaucher disease, the presence of numerous membrane-bound inclusion bodies characteristic of Gaucher cells.     

Glucocerebrosidase mutations in Gaucher disease.

BACKGROUND: Thirty-six mutations that cause Gaucher disease, the most common glycolipid storage disorder, are known. Although both alleles of most patients with the disease contain one of these mutations, in a few patients one or both disease-producing alleles have remained unidentified. Identification of mutations in these patients is useful for genetic counseling. MATERIALS AND METHODS: The DNA from 23 Gaucher disease patients in whom at least one glucocerebrosidase allele did not contain any of the 36 previously described mutations has been examined by single strand conformation polymorphism (SSCP) analysis, followed by sequencing of regions in which abnormalities were detected. RESULTS: Eight previously undescribed mutations were detected. In exon 3, a deletion of a cytosine at cDNA nt 203 was found. In exon 6, three missense mutations were identified: a C-->A transversion at cDNA nt 644 (Ala176-->Asp), a C-->A transversion at cDNA nt 661 that resulted in a (Pro182-->Thr), and a G-->A transition at cDNA nt 721 (Gly202-->Arg). Two missense mutations were found in exon 7: a G-->A transition at cDNA nt 887 (Arg257-->Gln) and a C-->T at cDNA nt 970 (Arg285-->Cys). Two missense mutations were found in exon 9: a T-->G at cDNA nt 1249 (Trp378-->Gly) and a G-->A at cDNA nt 1255 (Asp380-->Asn). In addition to these disease-producing mutations, a silent C-->G transversion at cDNA nt 1431, occurring in a gene that already contained the 1226G mutation, was found in one family. CONCLUSIONS: The mutations described here and previously known can be classified as mild, severe, or lethal, on the basis of their effect on enzyme production and on clinical phenotype, and as polymorphic or sporadic, on the basis of the haplotype in which they are found. Rare mutations such as the new ones described here are sporadic in nature.     

Impaired autophagosomes and lysosomes in neuronopathic Gaucher disease

Gaucher disease is an inherited autosomal recessive disease caused by mutations of acid β-glucosidase, a lysosomal hydrolase specific for degradation of glucosylceramide and glucosylsphingosine in the glycosphingolipid metabolic pathway. Clinically, Gaucher disease is classified into three types: type 1 is a visceral disease, whereas types 2 and 3 are acute and chronic neuronopathic variants, respectively. In types 2 and 3, the CNS pathology displays neuronal inclusions and neuron death. The underlying mechanism(s) by which the glycosphingolipid storage leads to this pathology is not fully understood. A mouse model whose phenotype mimicked that of the human neuronopathic variants was generated in our lab. In the brain of this model, abnormal autophagosomes and lysosomes implicate autophagy in the neuronal degeneration of Gaucher disease.     

Plasma glucosylceramide and ceramide in type 1 Gaucher disease patients: correlations with disease severity and response to therapeutic intervention

The concentrations of plasma glucosylceramide (GlcCer) and ceramide (Cer) were determined in a cohort of type 1 Gaucher disease patients. In plasma of untreated patients, GlcCer concentrations were on average 3-fold increased (median Gaucher: 17.5 nmol/ml, range: 6.5-45.5 (n=27); median control: 5.9 nmol/ml, range 4.0-8.6 (n=15)). Although plasma Cer concentrations were not significantly different between the two groups (median Gaucher: 7.2 nmol/ml, range: 4.2-10.9 (n=27); median control: 7.8 nmol/ml, range 5.7-11.9 (n=15)) in individual patients plasma GlcCer/Cer ratio yields slightly better discrimination between Gaucher disease patients and normal individuals than the GlcCer levels. Positive correlations were detected between plasma GlcCer concentration and GlcCer/Cer ratio and severity of disease, plasma chitotriosidase and CCL18, surrogate markers of storage cells. Gaucher disease is treated by enzyme replacement and substrate reduction therapy. Both therapies were found to result in decreases in plasma GlcCer already within 6 months, without causing abnormal plasma GlcCer or Cer concentrations. The corrections in plasma GlcCer were most robust in patients with a pronounced clinical response. In conclusion, plasma GlcCer concentration and GlcCer/Cer ratio is of value to monitor Gaucher disease manifestation and response to therapeutic intervention.     

Protective effect of catechin in type I Gaucher disease cells by reducing endoplasmic reticulum stress

Gaucher disease (GD) is the most common lysosomal storage disorder (LSD) and is divided into three phenotypes, I, II, and III. Type I is the most prevalent form and has its onset in adulthood. The degree of endoplasmic reticulum (ER) stress is one of the factors that determine GD severity. It has recently been reported that antioxidants reduce ER stress and apoptosis by scavenging the oxidants that cause oxidative stress. For this report, we investigated the possibility that catechin can act on type I GD patient cells to alleviate the pathogenic conditions of GD. We treated GD cells with catechin and examined the expression level of GRP78/BiP (an ER stress marker) by western blots and fluorescence microscopy, the proliferation rate of GD cells, and scratch-induced wound healing activity. Our results show that catechin reduces the expression level of GRP78/BiP, leads to cell proliferation rates of GD cells similar levels to normal cells, and improves wound healing activity. We conclude that catechin protects against ER stress in GD cells and catechin-mediated reductions in ER stress may be associated with enhanced cell survival.     

Leukocyte beta-glucosidase in homozygotes and heterozygotes for Gaucher disease

Human leukocytes contain at least two isozymes of 4-methylumbelliferyl-β-glucosidase acting optimally at pH 4.0 and 4.8; in Gaucher disease, only the former is deficient. Brief exposure of the leukocyte homogenate to pH 4.0 at room temperature results in irreversible inactivation of the pH 4.8 activity, while the activity at pH 4.0 remains unaffected. The more acidic isozyme is stimulated four- to fivefold by 0.2% sodium taurodeoxycholate (TDC) with a shift in the pH optimum to 5.0. The less acidic isozyme is completely suppressed in the presence of this detergent. Both leukocyte isozymes appear to be membrane-bound since gel filtration of Sephadex G-200 produces only one peak of activity located at the void volume, unlike in liver and kidney where a second peak also can be demonstrated. Heat inactivation analysis indicated that in controls, assayed in the absence of detergent, pH 4.0 activity is more thermostable than pH 4.8 activity. However, in Gaucher disease, the residual β-glucosidase at pH 4.0 is just as thermolabile as the unaffected pH 4.8 activity. Heat inactivation of the enzyme in the presence of TDC resulted in rapid loss of activity, suggesting a direct effect of the bile salt on the configuration of the enzyme decreasing its thermal stability. In the absence of detergent, acid β-glucosidase shows two K_m's, one at 3.2 mM and another at 0.9 mM. In the presence of detergent, only the higher K_m at 3.3 mM is obtained. In patients with Gaucher disease and in obligate carriers, the K_m remains essentially unaffected while the V_max shows the expected deficiency.

A reliable and reproducible selective assay technique has been developed for the diagnosis of Gaucher disease homozygotes and obligate heterozygotes and for the carrier screening of individuals at risk for this inherited disorder. The efficacy of this technique has been demonstrated by studying the activity in 42 controls, 26 patients, 32 obligate heterozygotes, and 23 healthy relatives of patients with Gaucher disease.

     

Neurofilaments and neurological disease

Neurofilaments are one of the major components of the neuronal cytoskeleton and are responsible for maintaining the calibre of axons. They are modified by post-translational changes that are regulated in complex fashions including by the interaction with neighbouring glial cells. Neurofilament accumulations are seen in several neurological diseases and neurofilament mutations have now been associated with Charcot-Marie-Tooth disease, Parkinson's disease and amyotrophic lateral sclerosis. In this review, we discuss the structure, normal function and molecular pathology of neurofilaments.     

Acid lability of the mutated glucosylceramide-beta-glucosidase in a lymphoid cell line from type 2 Gaucher disease

Lymphoid cell lines from patients with infantile (type-2) and juvenile (type 3) Gaucher disease have been established by Epstein-Barr virus transformation and investigated and compared with the adult phenotype (type 1) with the view to enzymology. The enzymatic defect in glucosylceramide(GlcCer)-beta-glucosidase activity was more severe in type 2 and 3 than in type 1 cells. The mutant GlcCer-beta-glucosidase from our studied type 2 lymphoid cells was profoundly labile at pH 4.0 and 37 degrees C, whereas the residual GlcCer-beta-glucosidase from type 1 and type 3 were stable similar to the normal enzyme. In contrast to the distinct stability of the GlcCer-beta-glucosidases from the three phenotypes, the acid lability of the nonspecific membrane-bound beta-glucosidases from type 1, 2 and 3 were quite similar.