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.     

Analyses of variant acid beta-glucosidases: effects of Gaucher disease mutations

Acid beta-glucosidase (GCase) is a 497-amino acid, membrane-associated lysosomal exo-beta-glucosidase whose defective activity leads to the Gaucher disease phenotypes. To move toward a structure/function map for disease mutations, 52 selected single amino acid substitutions were introduced into GCase, expressed in an insect cell system, purified, and characterized for basic kinetic, stability, and activator response properties. The variant GCases from Gaucher disease patients and selected variant GCases from the mouse had decreased relative k(cat) and differential effects on active site binding and/or attachment of mechanism-based covalent (conduritol B epoxide) or reversible (deoxynojirimycin derivatives) inhibitors. A defect in negatively charged phospholipid activation was present in the majority of variant GCases but was increased in two, N370S and V394L. Deficits in saposin C enhancement of k(cat) were present in variant GCases involving residues 48-122, whereas approximately 2-fold increases were obtained with the L264I GCase. About 50% of variant GCases each had wild-type or increased sensitivity to in vitro cathepsin D digestion. Mapping of these properties onto the crystal structures of GCase indicated wide dispersion of functional properties that can affect catalytic function and stability. Site-directed mutagenesis of cysteine residues showed that the disulfide bonds, Cys(4)-Cys(16) and Cys(18)-Cys(23), and a free Cys(342) were essential for activity; the free Cys(126) and Cys(248) were not. Relative k(cat) was highly sensitive to a His substitution at Arg(496) but not at Arg(495). These studies and high phylogenetic conservation indicate localized and general structural effects of Gaucher disease mutations that were not obvious from the nature of the amino acid substitution, including those predicted to be nondisruptive (e.g. Val --> Leu). These results provide initial studies for the engineering of variant GCases and, potentially, molecular chaperones for therapeutic use.     

Gaucher disease

Although Gaucher disease is a rare disorder, recent developments in novel means for therapeutic intervention have invigorated both academic research and pharmaceutical industry discovery programmes. The common mutations found in the lysosomal enzyme deficient in Gaucher disease, beta-glucocerebrosidase, earmark these proteins for destruction by the endoplasmic reticulum-localised protein folding machinery, resulting in enzyme insufficiency, lysosomal glycolipid storage and subsequent pathology. However, many of these mutants can be rescued from global misfolding to preserve glycolipid substrate binding and eventual catalysis in the lysosome, by the addition of subinhibitory concentrations of pharmacologically active small molecules. This novel, chaperon-mediated approach has benefited from insights into the molecular understanding of beta-glucocerebrosidase structure, drug design and development in cellular models for disease.     

Heterogeneity of mutations in the acid beta-glucosidase gene of Gaucher disease patients

Gaucher disease is inherited in an autosomal recessive manner and is the most prevalent lysosomal storage disease. Gaucher disease has marked phenotypic variation and molecular heterogeneity, and several simple and complex alleles of the acid beta-glucosidase gene have been identified as causal to this disease. Certain combinations of alleles have been shown to correlate well with the severity of the disease, but many Gaucher disease patients exist whose disease is not explained by any of the published mutations. This study was undertaken to identify mutant alleles in such incompletely characterized Gaucher disease, in an attempt to find further correlations between clinical phenotype and the presence of acid beta-glucosidase alleles. RNA was isolated from Gaucher cell lines and converted to cDNA, the cDNA was amplified by PCR and cloned, and several clones for each allele were sequenced. Several new singly mutated and multiply mutated alleles were identified, and sequence-specific oligonucleotide hybridization was used to verify the presence of these mutations in the genome of these patients. All newly identified mutations occurred only rarely in the Gaucher disease population, making it difficult to determine whether inheritance of a particular combination of alleles always correlates with the clinical manifestations seen in the test patients. Three of the newly described alleles were single missense mutations in exon 8, one was a single missense mutation in exon 5, and the fifth was a complex allele, comprising a series of different point mutations scattered throughout exons 5 and 6.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prevalence of nine mutations among Jewish and non-Jewish Gaucher disease patients.

The frequency of nine different mutated alleles known to occur in the glucocerebrosidase gene was determined in 247 Gaucher patients, of whom 176 were of Jewish extraction, 2 were Jewish with one converted parent, and 69 were of non-Jewish origin. DNA was prepared from peripheral blood, active glucocerebrosidase sequences were amplified by using the PCR technique, and the mutations were identified by using the allele-specific oligonucleotide hybridization method. The N37OS mutation appeared in 69.77% of the mutated alleles in Jewish patients and in 22.86% of the mutated alleles in non-Jews. The 84GG mutation, which has not been found so far among non-Jewish patients, existed in 10.17% of the disease alleles among Jewish patients. The IVS + 1 mutation constituted 2.26% of the disease alleles among Jewish patients and 1.43% among the non-Jewish patients. RecTL, a complex allele containing four single-base-pair changes, occurred in 2.26% of the alleles in Jewish patients and was found in two (1.43%) of the patients of non-Jewish extraction. Another complex allele, designated "RecNciI" and containing three single-point mutations, appeared in 7.8% of alleles of non-Jewish patients and in only two (0.56%) of the Jewish families. The prevalence of the L444P mutation among non-Jewish Gaucher patients was 31.43%, while its prevalence among Jewish patients was only 4.24%. The prevalence of two other point mutations--D409H and R463C--was 5.00% and 3.57%, respectively, among non-Jewish patients and was not found among the Jewish Gaucher patient population. The prevalence of the R496H mutation, found so far only among Jewish patients, was 1.13%. The results presented demonstrate that seven mutations identify 90.40% of the mutations among Jewish patients and that these seven mutations allow diagnosis of only 73.52% of the non-Jewish patients. Identification of additional mutant alleles will enhance the accuracy of carrier detection.     

A new brain-penetrant glucosylceramide synthase inhibitor as potential Therapeutics for Gaucher disease

Gaucher disease (GD), the most common lysosomal storage disorders, is caused by GBA gene mutations resulting in glycosphingolipids accumulations in various tissues, such as the brain. While suppressing glycosphingolipid accumulation is the central strategy for treating peripheral symptoms of GD, there is no effective treatment for the central nervous system symptoms. As glycosphingolipid biosynthesis starts from ceramide glycosylation by glucosylceramide synthase (GCS), inhibiting GCS in the brain is a promising strategy for neurological GD. Herein, we discovered T-036, a potent and brain-penetrant GCS inhibitor with a unique chemical structure and binding property. T-036 does not harbor an aliphatic amine moiety and has a noncompetitive inhibition mode to the substrates, unlike other known inhibitors. T-036 exhibited sufficient exposure and a significant reduction of glucosylsphingolipids in the plasma and brain of the GD mouse model. Therefore, T-036 could be a promising lead molecule for treating central nervous system symptoms of GD.

     

Molecular characteristics in Japanese patients with lipidosis: Novel mutations in metachromatic leukodystrophy and Gaucher disease

The characterization of mutations in Japanese patients with lipidosis, particularly in metachromatic leukodystrophy (MLD) and Gaucher disease has been studied in detail. Metachromatic leukodystrophy is characterized by an accumulation of sulfatide in nervous tissues and kidney due to a deficiency of arylsulfatase A (ASA). We analyzed the presence of three known mutant arylsulfatase A alleles in Japanese patients with MLD. Among 10 patients of Japanese patients with MLD, we found that allele 445A mutation has moderately high incidence and also homozygosity of this mutation results in the late infantile form. Allele 2381T was not found in Japanese patients. Furthermore, we found novel mutation which is G- to A mutation at the 1070 nucleotide of the ASA gene (designated 1070 A) in Japanese patients with juvenile onset. This mutation results in a amino acid substitution of Gly245 by Arg and found in heterozygote form. Our studies of molecular analysis in 10 Japanese patients with MLD indicate that Japanese MLD patients have unique characteristics of ASA mutations compared with those of Caucasian patients. On the other hand, Gaucher disease is the most prevalent sphingolipidosis, characterized by an accumulation of glucocerebroside in macrophage derived cells due to a deficiency of lysosomal hydrolase glucocerebrosidase. To study the molecular basis of Gaucher disease in Japanese patients, we analyzed the presence of the two known mutations (6433C and 3548A) in the glucocerebrosidase gene of 15 patients with Gaucher disease. We found that the 6433C and 3548A mutations occur in all subtypes of Japanese patients with Gaucher disease. Most frequent mutations among them was the 6433C mutation, 40% of 30 chromosomes, whereas the novel mutation of the 3548A found in Japanese patients with neuronopathic Gaucher disease was found in 20% (6 out of 30 chromosomes). The characteristics of these mutations in Japanese patients with Gaucher disease is different from those of Caucasian populations reported previously.     

Detection of the frequencies of GBA gene major mutations in patients with Gaucher disease in Ukraine

The molecular diagnostics of 27 from 26 Ukrainian families has been performed. The common mutations in GBA gene (N370S, L444P and 84GG) accounted for up to 58% of all cases: mutation N370S was detected in 42.3% alleles, mutation L444P was observed in 15.4% alleles and mutation 84GG was not found at all. The other mutations were: P178S, W184R and Rec Nci I (in compounds with N370S) in the patients with nonneuronopathic form of Gaucher disease, and the genotypes G377S/c 999G --> A and D409H/R120W/G202R were detected in patients with chronic neuronopathic form of Gaucher disease. The data analysis of the genotype and disease progression in the patients allows confirming the known genotype-phenotype correlation.     

Two new mutations and a linkage map of Anopheles stephensi

Genetic analyses of two new mutations in Anopheles stephensi are presented. Spotless wings (sl) and 2nd-3rd costal spots fused (2-3f) have been mapped on chromosome 2, approximately 79.5 map units apart. A preliminary linkage map for this species also is presented.     

Determining the frequency of common mutations in the GBA gene in patients with Gaucher disease in Ukraine

A molecular-genetics investigation is conducted on 27 patients from 26 families. Common mutations in the GBA gene (N370S, L444P, and 84GG) are studied. The overall frequency of the common mutations is nearly 58%, with the percentage of alleles that carry the N370S mutation close to 42.3% and the proportion that carry the L444P mutation, 15.4%. No allele containing the 84GG mutation was found. Besides other mutations, the rare mutations P178S, W184R, and Rec Nci I (together with N370S) were also found in the GBA gene in patients with the nonneuronopathic form of the disease, along with the genotypes G377S/c 999GA and D409H/R 120W/G202R in patients with the chronic neuronopathic form. An analysis of the correlation between the genotype and the course of the disease in the patients showed that the genotype-phenotype correlations were close to that described for European populations.     

Animal models for Gaucher disease research

Gaucher disease (GD), the most common lysosomal storage disorder (LSD), is caused by the defective activity of the lysosomal hydrolase glucocerebrosidase, which is encoded by the GBA gene. Generation of animal models that faithfully recapitulate the three clinical subtypes of GD has proved to be more of a challenge than first anticipated. The first mouse to be produced died within hours after birth owing to skin permeability problems, and mice with point mutations in Gba did not display symptoms correlating with human disease and also died soon after birth. Recently, conditional knockout mice that mimic some features of the human disease have become available. Here, we review the contribution of all currently available animal models to examining pathological pathways underlying GD and to testing the efficacy of new treatment modalities, and propose a number of criteria for the generation of more appropriate animal models of GD.     

Characterization of mutations in Gaucher patients by cDNA cloning.

Mutated cDNA clones containing the entire coding sequence of human glucocerebrosidase were isolated from libraries originated from Gaucher patients. Sequence analysis of a mutated cDNA derived from a type II Gaucher patient revealed a C-to-G transversion causing a substitution of an arginine for a proline at residue 415. This change creates a new cleavage site for the enzyme HhaI in the mutated cDNA. Allele-specific oligonucleotide hybridization made it possible to show that this mutation exists in the genomic DNA of the patient. From a cDNA library originated from a type I Gaucher patient, a mutated allele was cloned that contains a T-to-C transition causing a substitution of proline for leucine at residue 444 and creating a new NciI site. This mutation is identical to that described by S. Tsuji and colleagues in genomic DNA from type I, type II, and type III patients. Since the new NciI site generates RFLP, it was used to test the existence of this mutated allele in several Gaucher patients by Southern blot analysis. This allele was found in type I (Jewish and non-Jewish), type II, and type III Gaucher patients. These findings led us to conclude that the patient suffering from type II disease (denoted GM1260) carried both mutations described above. Any one of the amino acid changes described reduces the glucocerebrosidase activity as tested by transfection of COS cells with expression vectors harboring the mutated cDNAs. The base changes in the two mutated cDNAs do not affect the electrophoretic mobility of the corresponding polypeptides on an SDS polyacrylamide gel.     

Gaucher disease mouse models: point mutations at the acid beta-glucosidase locus combined with low-level prosaposin expression lead to disease variants

Gaucher disease is a common lysosomal storage disease caused by a defect of acid beta-glucosidase (GCase). The optimal in vitro hydrolase activity of GCase requires saposin C, an activator protein that derives from a precursor, prosaposin. To develop additional models of Gaucher disease and to test in vivo effects of saposin deficiencies, mice expressing low levels (4--45% of wild type) of prosaposin and saposins (PS-NA) were backcrossed into mice with specific point mutations (V394L/V394L or D409H/D409H) of GCase. The resultant mice were designated 4L/PS-NA and 9H/PS-NA, respectively. In contrast to PS-NA mice, the 4L/PS-NA and 9H/PS-NA mice displayed large numbers of engorged macrophages and nearly exclusive glucosylceramide (GC) accumulation in the liver, lung, spleen, thymus, and brain. Electron microscopy of the storage cells showed the characteristic tubular storage material of Gaucher cells. Compared with V394L/V394L mice, 4L/PS-NA mice that expressed 4--6% of wild-type prosaposin levels had approximately 25--75% decreases in GCase activity and protein in liver, spleen, and fibroblasts. These results imply that reduced saposin levels increased the instability of V394L or D409H GCases and that these additional decreases led to large accumulations of GC in all tissues. These models mimic a more severe Gaucher disease phenotype and could be useful for therapeutic intervention studies.     

A new glucocerebrosidase-gene missense mutation responsible for neuronopathic Gaucher disease in Japanese patients.

We have identified a new T-to-A single-base substitution at nucleotide 3548 (in the genomic sequence) in exon 6 in the glucocerebrosidase gene from a patient with Gaucher disease type 3. This mutation caused a substitution of isoleucine for phenylalanine at amino acid residue 213 (of 497 residues in the mature protein). By in vitro expression study in cultured mammalian cells, this mutation resulted in deficient activity of glucocerebrosidase. By allele-specific oligonucleotide hybridization of selectively PCR-amplified DNA from eight unrelated Japanese Gaucher disease patients, this mutant allele was observed in other neuronopathic Japanese Gaucher disease patients, in moderately frequent occurrence (three of six neuronopathic patients). This observation suggests that this allele was one of severe [corrected] alleles which were related to the development of neurological manifestations of Gaucher disease.     

Rapid identification of mutations in the glucocerebrosidase gene of Gaucher disease patients by analysis of single-strand conformation polymorphisms

To detect mutations in the glucocerebrosidase gene in Gaucher disease patients, we used the recently described technique of single-strand conformation polymorphism (SSCP) analysis in combination with selective amplification. We analyzed exon 8, 9, 10 and 11 of the glucocerebrosidase gene; these exons were sequentially amplified using the selectively amplified products as templates. We found variant SSCP patterns corresponding to the presence or absence of the 6433C mutation, which was detected by NciI digestion analysis, in exon 10. Furthermore, we detected four variant SSCP patterns in exon 8, 10 and 11. Sequencing analysis consistently revealed four single-base substitutions in the corresponding exons, three novel missense mutations (5409A, 6375G and 6682T) and one silent polymorphism (6594A). These mutations were found only in one patient; therefore, these findings have confirmed the marked genetic heterogeneity of Gaucher disease. SSCP analysis in combination with selective amplification is a rapid and sensitive procedure for the screening of the mutations in the glucocerebrosidase gene of patients with Gaucher disease.     

Gaucher disease: the origins of the Ashkenazi Jewish N370S and 84GG acid beta-glucosidase mutations

Type 1 Gaucher disease (GD), a non-neuronopathic lysosomal storage disorder, results from the deficient activity of acid beta-glucosidase (GBA). Type 1 disease is panethnic but is more prevalent in individuals of Ashkenazi Jewish (AJ) descent. Of the causative GBA mutations, N370S is particularly frequent in the AJ population, (q approximately .03), whereas the 84GG insertion (q approximately .003) occurs exclusively in the Ashkenazim. To investigate the genetic history of these mutations in the AJ population, short tandem repeat (STR) markers were used to map a 9.3-cM region containing the GBA locus and to genotype 261 AJ N370S chromosomes, 60 European non-Jewish N370S chromosomes, and 62 AJ 84GG chromosomes. A highly conserved haplotype at four markers flanking GBA (PKLR, D1S1595, D1S2721, and D1S2777) was observed on both the AJ chromosomes and the non-Jewish N370S chromosomes, suggesting the occurrence of a founder common to both populations. Of note, the presence of different divergent haplotypes suggested the occurrence of de novo, recurrent N370S mutations. In contrast, a different conserved haplotype at these markers was identified on the 84GG chromosomes, which was unique to the AJ population. On the basis of the linkage disequilibrium (LD) delta values, the non-Jewish European N370S chromosomes had greater haplotype diversity and less LD at the markers flanking the conserved haplotype than did the AJ N370S chromosomes. This finding is consistent with the presence of the N370S mutation in the non-Jewish European population prior to the founding of the AJ population. Coalescence analyses for the N370S and 84GG mutations estimated similar coalescence times, of 48 and 55.5 generations ago, respectively. The results of these studies are consistent with a significant bottleneck occurring in the AJ population during the first millennium, when the population became established in Europe.