Complex alleles of the acid beta-glucosidase gene in Gaucher disease.

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 seven point mutations in the acid beta-glucosidase (beta-Glc) gene have been identified. By means of sequence-specific oligonucleotides (SSO), mutation 6433C has been detected homozygously in neuronopathic type 2 (acute) and type 3 (subacute) patients, as well as in children with severe visceral involvement who are apparently free of neuronopathic disease. To investigate the molecular basis for this puzzling finding, amplified beta-Glc cDNAs from 6433C homozygous type 2 and type 3 Gaucher disease patients were cloned and sequenced. The Swedish type 3 Gaucher disease patient was truly homozygous for alleles only containing the 6433C mutation. In comparison, the type 2 patient contained a singly mutated 6433C allele and a "complex" allele with multiple discrete point mutations (6433C, 6468C, and 6482C). Each of the mutations in the complex allele also was present in the beta-Glc pseudogene. SSO hybridization of 6433C homozygotes revealed that both type 2 patients contained additional mutations in one allele, whereas the 6433C alone was detected in both type 3 and in young severe type 1 Gaucher disease patients. These results suggest that the presence of the complex allele influences the severity of neuronopathic disease in 6433C homozygotes and reveal the central role played by the pseudogene in the formation of mutant alleles of the beta-Glc gene. Analysis of additional cDNA clones also identified two new alleles in a type 3 patient, emphasizing the molecular heterogeneity of neuronopathic Gaucher 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)     

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.     

Gaucher disease: A G+1----A+1 IVS2 splice donor site mutation causing exon 2 skipping in the acid beta-glucosidase mRNA.

Gaucher disease is the most frequent lysosomal storage disease and the most prevalent Jewish genetic disease. About 30 identified missense mutations are causal to the defective activity of acid beta-glucosidase in this disease. cDNAs were characterized from a moderately affected 9-year-old Ashkenazi Jewish Gaucher disease type 1 patient whose 80-year-old, enzyme-deficient, 1226G (Asn370----Ser [N370S]) homozygous grandfather was nearly asymptomatic. Sequence analyses revealed four populations of cDNAs with either the 1226G mutation, an exact exon 2 (delta EX2) deletion, a deletion of exon 2 and the first 115 bp of exon 3 (delta EX2-3), or a completely normal sequence. About 50% of the cDNAs were the delta EX2, the delta EX2-3, and the normal cDNAs, in a ratio of 6:3:1. Specific amplification and characterization of exon 2 and 5' and 3' intronic flanking sequences from the structural gene demonstrated clones with either the normal sequence or with a G+1----A+1 transition at the exon 2/intron 2 boundary. This mutation destroyed the splice donor consensus site (U1 binding site) for mRNA processing. This transition also was present at the corresponding exon/intron boundary of the highly homologous pseudogene. This new mutation, termed "IVS2 G+1----A+1," is the first splicing mutation described in Gaucher disease and accounted for about 3.4% of the Gaucher disease alleles in the Ashkenazi Jewish population. The occurrence of this "pseudogene"-type mutation in the structural gene indicates the role of acid beta-glucosidase pseudogene and structural gene rearrangements in the pathogenesis of this disease.     

Seven novel mutations in the methylenetetrahydrofolate reductase gene and genotype/phenotype correlations in severe methylenetetrahydrofolate reductase deficiency.

5-Methyltetrahydrofolate, the major form of folate in plasma, is a carbon donor for the remethylation of homocysteine to methionine. This form of folate is generated from 5,10-methylenetetrahydrofolate through the action of 5,10-methylenetetrahydrofolate reductase (MTHFR), a cytosolic flavoprotein. Patients with an autosomal recessive severe deficiency of MTHFR have homocystinuria and a wide range of neurological and vascular disturbances. We have recently described the isolation of a cDNA for MTHFR and the identification of two mutations in patients with severe MTHFR deficiency. We report here the characterization of seven novel mutations in this gene: six missense mutations and a 5' splice-site defect that activates a cryptic splice site in the coding sequence. We also present a preliminary analysis of the relationship between genotype and phenotype for all nine mutations identified thus far in this gene. A nonsense mutation and two missense mutations (proline to leucine and threonine to methionine) in the homozygous state are associated with extremely low activity (0%-3%) and onset of symptoms within the 1st year of age. Other missense mutations (arginine to cysteine and arginine to glutamine) are associated with higher enzyme activity and later onset of symptoms.     

Human acid beta-glucosidase: inhibition studies using glucose analogues and pH variation to characterize the normal and Gaucher disease glycon binding sites

Comparative kinetic studies with glycon inhibitors were used to investigate the properties of the active site of human acid beta-glucosidase (EC 3.2.1.45) from normal placenta and spleens of type 1 Ashkenazi Jewish Gaucher disease (AJGD) patients. With the pure normal enzyme, the specificity of glycon binding was assessed with 35 glucose derivatives and epimers. Most glycons were mixed type inhibitors with a predominantly competitive nature (i.e., Kis much less than Kii) and had low apparent affinity for the enzyme (Kisapp = 20-500 mmol/l). beta-Glucose-1-phosphate was unusual, since it inhibited 4-methylumbelliferyl-beta-glucoside hydrolysis in an uncompetitive pattern (Kiapp = 0.55 mmol/l) but had no effect on glucosyl ceramide hydrolysis. C-1- (1-deoxy-1-amino-beta-D-glucose) and C-3- (3-deoxy-3-amino-D-glucose) amino and C-5-imino [1-deoxynojirimycin (dNM), nojirimycin and castanospermine] substituted sugars were highly potent inhibitors with Kisapp(beta-glucose)/Kisapp approximately equal to 10(3)-10(5); an amine at C-2 did not alter Kisapp compared to beta-glucose. The variation of Kisapp with pH for the 5-imino- and 1-deoxy-1-aminoglycosides conformed to a model for the unprotonated inhibitors binding to the protonated forms (EH and EH2) of the diprotic (Vmaxapp and Vmaxapp/Kmapp) normal enzyme (pK1 = 4.7; pK2 = 6.7) with pH-independent Kisapp values of 2.9-9.0 mumol/l and 0.22 mmol/l, respectively. Several of the amine-containing inhibitors competitively protected the enzyme from inactivation by conduritol B epoxide, a covalent active site-directed inhibitor, indicating interaction with residues at that site. With the partially purified AJGD splenic enzymes, the results were the same except that Kisapp(AJGD)/Kisapp(normal) = 4-17 for dNM and 1-deoxy-1-amino-beta-glucose; this ratio was approximately equal to 1 with most other glycons, and particularly, nojirimycin and castanospermine. The results of these studies indicated that the glycon binding site of the normal acid beta-glucosidase contains important residues for interaction with the C-2, C-3 and C-4 hydroxyl groups of beta-glucose and a residue with pKa = 6.7 which was critical to the binding of amine-containing inhibitors and the hydrolysis of substrates. The findings were consistent with a specific alteration in or near the glycon binding site which results in the functional abnormalities of the mutant AJGD acid beta-glucosidase.     

Gaucher disease: gene frequencies in the Ashkenazi Jewish population.

DNA from over 2,000 Ashkenazi Jewish subjects has been examined for the four most common Jewish Gaucher disease mutations, which collectively account for about 96% of the disease-producing alleles in Jewish patients. This population survey has made possible the estimation of gene frequencies for these alleles. Eighty-seven of 1,528 individuals were heterozygous for the 1226G (N370S) mutation, and four presumably well persons were homozygous for this mutation. The gene frequency for the 1226G allele was calculated to be .0311, and when these data were pooled with those obtained previously from another 593 Jewish subjects, a gene frequency of .032 with a standard error of .004 was found. Among 2,305 normal subjects, 10 were found to be heterozygous for the 84GG allele, giving a gene frequency of .00217 with a standard error of .00096. No examples of the IVS2(+1) mutation were found among 1,256 samples screened, and no 1448C (L444P) mutations were found among 1,528 samples examined. Examination of the distribution of Gaucher disease gene frequencies in the general population shows that the ratio of 1226G mutations to 84GG mutations is higher than that in the patient population. This is presumed to be due to the fact that homozygotes for the 1226G mutation often have late-onset disease or no significant clinical manifestations at all. To bring the gene frequency in the patient population into conformity with the gene frequency in the general population, nearly two-thirds of persons with a Gaucher disease genotype would be missing from the patient population, presumably because their clinical manifestations were very mild.     

Posttranslational processing of human lysosomal acid beta-glucosidase: a continuum of defects in Gaucher disease type 1 and type 2 fibroblasts.

The major processing steps in the maturation of the lysosomal hydrolase, acid beta-glucosidase, were examined in fibroblasts from normal individuals and from patients with types 1 and 2 Gaucher disease. In pulse-chase studies with normal fibroblasts, remodeling of N-linked oligosaccharides resulted in the temporal appearance of three molecular-weight forms of acid beta-glucosidase. An initial 64-kDa form, containing high mannose-type oligosaccharide side chains, was processed quantitatively, within 24 h, to a sialylated 69-kDa form. During the subsequent 96 h, some of the 69-kDa form is processed to 59 kDa. Glycosidase digestion studies revealed that the increase in the apparent molecular weight of the normal enzyme from 64 kDa to 69 kDa resulted primarily from the addition to sialic acid residues in the Golgi apparatus. The polypeptide backbone of both the 64-kDa and 69-kDa forms was 55.3 kDa. Processing of acid beta-glucosidase in fibroblasts from three of four type 1 (nonneuronopathic) Ashkenazi Jewish Gaucher disease patients was nearly normal. With fibroblasts from one Ashkenazi Jewish and three non-Jewish type 1 as well as from two type 2 (acute neuronopathic) Gaucher disease patients, only a 64-kDa form of acid beta-glucosidase was detected. Inefficient and incomplete processing to the 69-kDa form was found in one type 2 cell line (GM2627). These results indicate that no firm correlation exists between the type or degree of abnormal processing of acid beta-glucosidase in fibroblasts and the phenotype of Gaucher disease.     

Gaucher disease types 1, 2, and 3: differential mutations of the acid beta-glucosidase active site identified with conduritol B epoxide derivatives and sphingosine.

To elucidate the genetic heterogeneity in Gaucher disease, the residual beta-glucosidase in cultured fibroblasts from affected patients with each of the major phenotypes was investigated in vitro and/or in viable cells by inhibitor studies using the covalent catalytic site inhibitors, conduritol B epoxide or its bromo derivative, and the reversible cationic inhibitor, sphingosine. These studies delineated three distinct groups (designated A, B, and C) of residual activities with characteristic responses to these inhibitors. Group A residual enzymes had normal I50 values (i.e., the concentration of inhibitor that results in 50% inhibition) for the inhibitors and normal or nearly normal t1/2 values for conduritol B epoxide. All neuronopathic (types 2 and 3) and most non-Jewish nonneuronopathic (type 1) patients had group A residual activities and, thus, could not be distinguished by these inhibitor studies. Group B residual enzymes had about four- to fivefold increased I50 values for the inhibitors and similarly increased t1/2 values for conduritol B epoxide. All Ashkenazi Jewish type 1 and only two non-Jewish type 1 patients had group B residual activities. The differences in I50 values between groups A and B also were confirmed by determining the uninhibited enzyme activity after culturing the cells in the presence of bromo-conduritol B epoxide. Group C residual activity had intermediate I50 values for the inhibitors and represented a single Afrikaner type 1 patient: this patient was a genetic compound for the group A (type 2) and group B (type 1) mutations. These inhibition studies indicated that: Gaucher disease type 1 is biochemically heterogeneous, neuronopathic and non-Jewish nonneuronopathic phenotypes cannot be reliably distinguished by these inhibitor studies, and the Ashkenazi Jewish form of Gaucher disease type 1 results from a unique mutation in a specific active site domain of acid beta-glucosidase that leads to a defective enzyme with a decreased Vmax.     

Human acid beta-glucosidase: use of inhibitors, alternative substrates and amphiphiles to investigate the properties of the normal and Gaucher disease active sites

Comparative studies with lipoidal inhibitors and alternative substrates were conducted to investigate the properties of the active site of human acid beta-glucosidase (D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45) from normal placenta and spleens of Type 1 Ashkenazi Jewish Gaucher disease (AJGD) patients. With the normal enzyme, the inhibitory potencies of series of alkyl(Cn; n = 0-18)amines, alkyl beta-glucosides and alkyl-1-deoxynojirimycins were a biphasic function of increasing chain length: i.e., large decreases in Ki,app or IC50 were found only with n greater than 4 and limiting values were approached with n = 12-14. This biphasic function of alkyl chain length was observed in the presence or absence of detergents and/or negatively charged lipids. In the presence of Triton X-100 concentrations greater than the critical micellar concentration, the relative (to deoxynojirimycin) inhibitory potencies of the N-Cn-deoxynojirimycins (n greater than 4) were decreased about 3-5-fold, due to an energy requirement to extract the inhibitors from Triton X-100 micelles. The Ki,app or IC50 of N-hexylglucosylsphingosine was inversely related to the Triton X-100 concentration and was not affected by the presence of 'co-glucosidase'. The mutual exclusion of glucon, N-Cn-deoxynojirimycin and sphingosine derivatives from the normal enzyme suggested a shared region for binding in the active site. Increasing the fatty-acid acyl chain length of glucosyl ceramide from 1 to 24 carbons had minor effects on Km,app ( = Kis,app) (8-40 microM), but increased Vmax,app up to 13-fold. With the AJGD enzyme, the inhibitor and alternative substrate findings were similar to those with the normal enzyme, except that Kis,app(AJGD)/Kis,app(normal) = 4 to 11 for the Cn-glycons and sphingosine derivatives. These results indicated that (1) the Ki,app or Km,app values for amphiphilic inhibitors or substrates reflect a balance of binding energies for two hydrophobic subsites within the enzyme's active site and Triton X-100 micelles and (2) the abnormal properties of the AJGD enzyme result from an amino-acid alteration(s) within or near a hydrophilic region which is shared by the glycon-binding site and the two hydrophobic sites of the active site.     

Gaucher disease type III (Norrbottnian type) is caused by a single mutation in exon 10 of the glucocerebrosidase gene.

Three major forms (types I-III) of Gaucher disease (GD) have been identified. The largest group of patients with type III GD has been reported from the province of Norrbotten in Sweden. In the present study the genomes from two GD patients of Norrbottnian origin were examined for abnormalities in the glucocerebrosidase gene. In both individuals, a single nucleotide substitution was found in exon 10. This mutation, which results in the substitution of proline for leucine, is identical to the NciI mutation described by Tsuji and co-workers in GD patients of other ethnic origins. Nine additional patients with Norrbottnian GD were shown to be homozygous for the same mutation by restriction-enzyme digestion of DNA amplified by PCR.     

Genotype assignment in Gaucher disease by selective amplification of the active glucocerebrosidase gene.

Genomic DNA prepared from human cells in culture was amplified by the polymerase chain-reaction technique using two primers specific for the active human glucocerebrosidase gene. The 1,036-bp amplified fragment derived from the active gene was tested for the existence of three mutations--designated "370," "NciI," and "HhaI"--by allele-specific oligonucleotide hybridization. The results obtained from the cell lines examined permitted a clear distinction between homozygous affected, heterozygous, and normal genotypes. However, 28% of the possible affected loci were normal with respect to the three mutations, indicating the presence of additional mutations that remain to be elucidated. While the NciI mutation could be found in both Ashkenazi Jewish and non-Jewish type 1 patients, the only homozygotes with this mutation had the neurological (type 2 or type 3) form of the disease. The 370 mutation, on the other hand, was only present in type 1 patients and was not identified among any of the patients with neurologic forms of the disease.     

Crystal structures of complexes of N-butyl- and N-nonyl-deoxynojirimycin bound to acid beta-glucosidase: insights into the mechanism of chemical chaperone action in Gaucher disease

Gaucher disease is caused by mutations in the gene encoding acid beta-glucosidase (GlcCerase), resulting in glucosylceramide (GlcCer) accumulation. The only currently available orally administered treatment for Gaucher disease is N-butyl-deoxynojirimycin (Zavesca, NB-DNJ), which partially inhibits GlcCer synthesis, thus reducing levels of GlcCer accumulation. NB-DNJ also acts as a chemical chaperone for GlcCerase, although at a different concentration than that required to completely inhibit GlcCer synthesis. We now report the crystal structures, at 2A resolution, of complexes of NB-DNJ and N-nonyl-deoxynojirimycin (NN-DNJ) with recombinant human GlcCerase, expressed in cultured plant cells. Both inhibitors bind at the active site of GlcCerase, with the imino sugar moiety making hydrogen bonds to side chains of active site residues. The alkyl chains of NB-DNJ and NN-DNJ are oriented toward the entrance of the active site where they undergo hydrophobic interactions. Based on these structures, we make a number of predictions concerning (i) involvement of loops adjacent to the active site in the catalytic process, (ii) the nature of nucleophilic attack by Glu-340, and (iii) the role of a conserved water molecule located in a solvent cavity adjacent to the active site. Together, these results have significance for understanding the mechanism of action of GlcCerase and the mode of GlcCerase chaperoning by imino sugars.