The major defect in Ashkenazi Jews with Tay-Sachs disease is an insertion in the gene for the alpha-chain of beta-hexosaminidase

The Ashkenazi Jewish population is enriched for carriers of a fatal form of Tay-Sachs disease, an inherited disorder caused by mutations in the alpha-chain of the lysosomal enzyme, beta-hexosaminidase A. Until recently it was presumed that Tay-Sachs patients from this ethnic isolate harbored the same alpha-chain mutation. This was disproved by identification of a splice junction defect in the alpha-chain of an Ashkenazi patient which could be found in only 20-30% of the Ashkenazi carriers tested. In this study we have isolated the alpha-chain gene from an Ashkenazi Jewish patient, GM515, with classic Tay-Sachs disease who was negative for the splice junction defect. Sequence analysis of the promoter region, exon and splice junctions regions, and polyadenylation signal area revealed a 4-base pair insertion in exon 11. This mutation introduces a premature termination signal in exon 11 which results in a deficiency of mRNA in Ashkenazi patients. A dot blot assay was developed to screen patients and heterozygote carriers for the insertion mutation. The lesion was found in approximately 70% of the carriers tested, thereby distinguishing it as the major defect underlying Tay-Sachs disease in the Ashkenazi Jewish population.     

A gel electrophoretic assay for detecting the insertion defect in Ashkenazi Jewish carriers of Tay-Sachs disease

A simple, rapid, nonradioactive assay for detecting the 4-bp insertion defect found in the beta-hexosaminidase alpha-chain gene of 70% of the Ashkenazi Jewish carriers of Tay-Sachs disease is described. In this assay, DNA derived from such carriers serves as a template for the polymerase chain reaction. Following amplification of a 159-bp fragment of exon 11 inclusive of the insertion, a portion of the product is subjected to electrophoresis in a 4% NuSieve agarose minigel. Visualization of the DNA with ethidium bromide demonstrates that heterozygote carriers for the defect display two distinct bands. In contrast, DNA from carriers of the splice junction defect, a mutation found in 30% of the Ashkenazi Jewish carriers of Tay-Sachs disease, displays only one band.     

Tay-Sachs disease in Moroccan Jews: deletion of a phenylalanine in the alpha-subunit of beta-hexosaminidase.

Tay-Sachs disease is an inherited lysosomal storage disorder caused by defects in the beta-hexosaminidase alpha-subunit gene. The carrier frequency for Tay-Sachs disease is significantly elevated in both the Ashkenazi Jewish and Moroccan Jewish populations but not in other Jewish groups. We have found that the mutations underlying Tay-Sachs disease in Ashkenazi and Moroccan Jews are different. Analysis of a Moroccan Jewish Tay-Sachs patient had revealed an in-frame deletion (delta F) of one of the two adjacent phenylalanine codons that are present at positions 304 and 305 in the alpha-subunit sequence. The mutation impairs the subunit assembly of beta-hexosaminidase A, resulting in an absence of enzyme activity. The Moroccan patient was found also to carry, in the other alpha-subunit allele, a different, and as yet unidentified, mutation which causes a deficit of mRNA. Analysis of obligate carriers from six unrelated Moroccan Jewish families showed that three harbor the delta F mutation, raising the possibility that this defect may be a prevalent mutation in this ethnic group.     

A splicing defect due to an exon-intron junctional mutation results in abnormal beta-hexosaminidase alpha chain mRNAs in Ashkenazi Jewish patients with Tay-Sachs disease

Abnormal beta-hexosaminidase alpha chain mRNAs from an Ashkenazi Jewish patient with the classical infantile Tay-Sachs disease contained intact or truncated intron 12 sequences. Sequence analysis showed a single nucleotide transversion at the 5' donor site of intron 12 from the normal G to C. This provides the first evidence that this junctional mutation, also found independently in two other laboratories by analysis of genomic clones, results in functional abnormality. Analysis with normal and mutant oligonucleotides as probes indicated that our patient was a compound heterozygote with only one allele having the transversion. The patient studied in the other two laboratories was also a compound heterozygote. Another Ashkenazi Jewish patient was normal in this region in both alleles. Thus, the splicing defect is the underlying genetic cause in some but not all Ashkenazi Jewish patients with Tay-Sachs disease.     

A deletion involving Alu sequences in the beta-hexosaminidase alpha-chain gene of French Canadians with Tay-Sachs disease

French Canadians living in eastern Quebec are carriers of a severe type of Tay-Sachs disease, known as the classic form, 10 times more often than the general population. The alpha-chain of beta-hexosaminidase A, a lysosomal enzyme composed of two chains (alpha, beta), bears the mutation in this inherited disorder. We previously reported that the 5' end of the alpha-chain gene was deleted in two such patients (Myerowitz, R., and Hogikyan, N.D. (1986) Science, 232, 1646-1648). The present study reports the size, precise location, and environment of the deletion. A clone encompassing the deletion was isolated from a genomic library constructed in lambda EMBL3 with DNA from a patient's fibroblasts. Comparison of the restriction maps of the clone with that of the normal gene (Proia, R.L., and Soravia, E. (1987) J. Biol. Chem. 262, 5677-5681) showed that the deletion was 7.6 kilobases long and included part of intron 1, all of exon 1 and extended 2000 base pairs upstream past the putative promotor region of the alpha-chain gene. These data are consistent with the inability to detect mRNA and immunoprecipitable alpha-chain protein in this mutant. Sequence analysis of the deletion junction in the mutant and corresponding regions of the normal gene demonstrated the presence of similarly oriented Alu sequences at the 5' and 3' deletion boundaries. The data are in accord with the possibility that the deletion may have arisen during homologous recombination from unequal crossing over between Alu sequences.     

Faulty association of alpha- and beta-subunits in some forms of beta-hexosaminidase A deficiency

We have previously described the kinetics of association of the alpha- and beta-subunits of beta-hexosaminidase A in intact cultured human fibroblasts, using biosynthetic labeling and immunoprecipitation with antisera that distinguish between monomeric and associated alpha-chains (Proia, R. L., d'Azzo, A., and Neufeld, E. F. (1984) J. Biol. Chem. 259, 3350-3354). We now show lack of alpha-beta association in fibroblasts of several individuals deficient in beta-hexosaminidase A (5 patients with nonclassic forms of Tay-Sachs disease and 2 asymptomatic siblings). Defective association was accompanied by markedly reduced (less than one-tenth of normal) conversion of the alpha-chain precursor of Mr = 67,000 to the mature lysosomal form of Mr = 54,000. Analysis by hybridization with fibroblasts lacking the alpha- or beta-chain showed that the association defect resided in the alpha-chain. Most of the cell strains studied also had decreased synthesis of the alpha-chain, suggesting compound heterozygosity with the Ashkenazi Tay-Sachs (no synthesis) allele. An unusual feature of the association defect is the variability in the resulting clinical manifestations, even within families, implying that other factors determine the adequacy of the residual associated beta-hexosaminidase A in vivo.     

Isolation of cDNA clones coding for the alpha-subunit of human beta-hexosaminidase. Extensive homology between the alpha- and beta-subunits and studies on Tay-Sachs disease

The lysosomal beta-hexosaminidases (N-acetyl-beta-glucosaminidase, EC 3.2.1.30) occur as two major isozymes, hexosaminidase A (alpha beta a beta b) and hexosaminidase B (2(beta a beta b)). To facilitate the investigations of the biosynthesis and structure of the enzymes and the nature of mutation in Tay-Sachs disease, we have isolated cDNA clones coding for the alpha-subunit. The polypeptide chains of hexosaminidase A (30 mg) were digested with trypsin, and peptides were isolated by reverse phase high pressure liquid chromatography and their amino acid sequences determined. One of alpha-chain peptides contained a string of seven amino acids from which two sets of oligonucleotides were specified. They were used to screen the SV40-transformed human fibroblast cDNA library of Okayama and Berg. Three cDNA clones, designated pHexA, identified from among 5 X 10(5) clones screened, contained the deduced amino-acid sequences of five alpha-chain peptides. Genomic DNA homologous to pHexA cDNA mapped to human chromosome 15 in somatic cell hybrids, as expected for the pre-alpha-polypeptide. Two of the clones contained identical polyadenylation sites, while the third was polyadenylated about 450 base pairs downstream. The two types of clones were found to correspond to a major 2.0-kilobase pair and a minor 2.3-kilobase pair mRNA species. Blot hybridizations of mRNA and DNA from Tay-Sachs variant fibroblasts revealed absence or reduction of levels of both mRNA species among infantile and juvenile variants, but no observable DNA alterations. Alignment of the pre-alpha- and pre-beta-polypeptides revealed 55% nucleotide and 57% amino acid homology. These data suggest a common origin of the HEXA and HEXB genes and account for the similar substrate specificities of the alpha-dimer subunit, hexosaminidase S, and hexosaminidase B.     

Alpha-locus hexosaminidase genetic compound with juvenile gangliosidosis phenotype: clinical,genetic, and biochemical studies.

A 3-year-old boy developed progressive neurological deterioration in his third year, characterized by dementia, ataxia, myoclonic jerks, and bilateral macular cherry-red spots. Hexosaminidase A (HEX A) was partially decreased in the patient''s serum, leukocytes, and cultured skin fibroblasts. Hexosaminidase was studied in serum and leukocytes from family members. Four members of the paternal branch appeared to be carriers of classical infantile Tay-Sachs allele, HEX alpha 2, probably receiving the gene from one great-grandparent of Ashkenazi origin. In the maternal branch, no one was a carrier of classical infantile Tay-Sachs disease, but five individuals were carriers of a milder alpha-locus defect. The patient, therefore, was a genetic compound of two different alpha-locus hexosaminidase mutations. At least 21 families with late-infantile or juvenile GM2 gangliosidosis have been reported, 18 of them with alpha-locus mutations, and three with beta-locus mutations. Genetic compounds of hexosaminidase have been reported in at least seven families, five with alpha-locus mutations and two with beta-locus mutations. The compound had the phenotype of infantile Tay-Sachs disease in one family, infantile Sandhoff disease in another, and the normal phenotype in the rest.     

Juvenile sandhoff disease: Complementation tests with Sandhoff and Tay-Sachs disease using polyethylene glycol-induced cell fusion

Summary Juvenile Sandhoff, Sandhoff, and Tay-Sachs fibroblasts were mixed in paired combinations and treated with polyethylene glycol (PEG) to promote cell fusion. The hexosaminidase (hex) isozymes of PEG-treated mixed-cell cultures were determined and compared with those of untreated control cultures. Fusions involving juvenile Sandhoff and Sandhoff fibroblasts did not show an increase in either total hexosaminidase or heat-stable hex B. Fusions of juvenile Sandhoff (or Sandhoff) and Tay-Sachs fibroblasts showed an increase of heat-labile hex A. Thus, juvenile Sandhoff cells show complementation with Tay-Sachs cells but not Sandhoff cells. Consequently, the genetic defect in juvenile Sandhoff disease probably represents an allelic mutation of the gene that is defective in Sandhoff disease.     

Impaired proteolytic processing of lysosomal N-acetyl-beta-hexosaminidase in cultured fibroblasts from patients with infantile generalized N-acetylneuraminic acid storage disease

Cultured skin fibroblasts from patients suffering with infantile generalized N-acetylneuraminic acid (NeuAc) storage disease accumulate free NeuAc in a population of lysosomes less dense than those observed in normal fibroblasts (1.035 vs. greater than 1.07 mean density), as assessed by the distribution of lysosomal enzyme activities and NeuAc on Percoll gradients after subcellular fractionation. In the present study, normal and affected fibroblasts were labeled with [35S]methionine, and cell homogenates or subcellular fractions from Percoll gradients were immunoprecipitated with polyclonal antibodies to lysosomal N-acetyl-beta-hexosaminidase (Hex); immunoprecipitated polypeptides were analyzed by SDS-polyacrylamide gel electrophoresis. The synthesis and initial processing of Hex polypeptides were comparable in normal and affected fibroblasts, but mature polypeptides were quantitatively localized in "buoyant" lysosomes of affected cells, along with Hex activity; moreover, mature alpha-chain of Hex was approximately 2 kDa larger than that observed in normal cells. The molecular weight difference was apparently due to impaired proteolytic processing of alpha-chain in affected fibroblasts, since treatment of immunoprecipitated alpha-chain from normal and affected cells with neuraminidase and endo-beta-N-acetylglucosaminidase H failed to resolve the molecular weight difference. The impaired processing was observed to be persistent (after a chase of up to 200 h), but had no apparent effect on the turnover or activity of Hex in affected fibroblasts. The observed proteolytic processing defect may be primary or secondary in infantile NeuAc storage disease.     

Multiple abnormal beta-hexosaminidase alpha chain mRNAs in a compound-heterozygous Ashkenazi Jewish patient with Tay-Sachs disease

Abnormal beta-hexosaminidase alpha chain cDNA clones were isolated from fibroblasts of an Ashkenazi Jewish patient with Tay-Sachs disease. Four abnormal cDNA clones were sequenced in their entirety. We showed previously that three of these mRNAs retained intron 12 with a mutation from G to C at the 5' donor site and that the patient was heterozygous with respect to this splicing defect (Ohno, K., and Suzuki, K., (1988) Biochem. Biophys. Res. Commun. 153, 463-469). One clone retained, in addition to intron 12, intron 13, which was truncated and polyadenylated due to a polyadenylation signal within intron 13. The fourth clone did not contain intron 12 and was missing exon 12. Some of these abnormal mRNAs were also missing one or more of upstream exons. The regions of exon 12-intron 12 and of upstream exons were evaluated in a total of 30 clones, including those completely sequenced, by restriction mapping and Southern analysis with appropriate probes. Of the 25 cDNA clones that included the exon 12-intron 12 region, 11 contained the exon 12-intron 12 sequence with the junctional transversion, and 11 were missing both exon 12 and intron 12. Among the 12 clones that included the region of exon 3-exon 9, 7 were missing one or more of upstream exons. Three clones gave results expected of normal cDNA in the region of exons 12 and 13. One of the three, furthermore, was 3.6-kilobases long and contained the completely normal beta-hexosaminidase alpha chain mRNA sequence on the 3' side and an abnormal 1.7-kilobase segment at the 5' end. These findings suggest that the splicing defect results in either retention of intron 12 or skipping of exon 12 in approximately equal proportions and that remote upstream exons are also frequently excised out. The three clones that were normal in the exon 12-intron 12 region could have derived from the other yet-to-be-characterized mutant allele. However, we were unable to obtain firm evidence that the abnormal upstream sequence is directly related to Tay-Sachs disease.     

A shortened beta-hexosaminidase alpha-chain in an Italian patient with infantile Tay-Sachs disease.

Fibroblasts derived from a beta-hexosaminidase A (HexA)-deficient infant with clinically classic Tay-Sachs disease synthesized a precursor alpha-chain that was smaller than its normal counterpart. Fibroblasts from the infant''s parents, who were consanguinous, produced both normal and mutant alpha-chains. The size difference, estimated to be 2-3 kilodaltons on the basis of sodium dodecyl sulfate-polyacrylamide-gel electrophoresis, persisted after removal of oligosaccharides with endo-H and is therefore attributable to a shortened polypeptide. The mutant alpha-chain did not undergo the further posttranslational modifications characteristic of its normal counterpart--i.e., synthesis of the mannose phosphate recognition marker, association with the beta-chain to give HexA, and proteolytic conversion to the mature form. Nor was it secreted, even in the presence of NH4Cl. Instead, it disappeared in the course of a 20-h chase. These results suggest that the mutant alpha-chain was trapped in an early biosynthetic compartment, either the endoplasmic reticulum or the cis-Golgi. The mutation appears to be different from all those previously described in patients with clinically classic Tay-Sachs disease.     

The intron 7 donor splice site transition: a second Tay-Sachs disease mutation in French Canada

Mutations at the hexosaminidase A (HEXA) gene which cause Tay-Sachs disease (TSD) have elevated frequency in the Ashkenazi Jewish and French-Canadian populations. We report a novel TSD allele in the French-Canadian population associated with the infantile form of the disease. The mutation, a GA transition at the +1 position of intron 7, abolishes the donor splice site. Cultured human fibroblasts from a compound heterozygote for this transition (and for a deletion mutation) produce no detectable HEXA mRNA. The intron 7+1 mutation occurs in the base adjacent to the site of the adult-onset TSD mutation (G805A). In both mutations a restriction site for the endonuclease EcoRII is abolished. Unambiguous diagnosis, therefore, requires allele-specific oligonucleotide hybridization to distinguish between these two mutant alleles. The intron 7+1 mutation has been detected in three unrelated families. Obligate heterozygotes for the intron 7+1 mutation were born in the Saguenay-Lac-St-Jean region of Quebec. The most recent ancestors common to obligate carriers of this mutation were from the Charlevoix region of the province of Quebec. This mutation thus has a different geographic centre of diffusion and is probably less common than the exon 1 deletion TSD mutation in French Canadians. Neither mutation has been detected in France, the ancestral homeland of French Canada.     

Distribution of three alpha-chain beta-hexosaminidase A mutations among Tay-Sachs carriers.

DNA from 176 carriers of the Tay-Sachs gene was tested for the presence of the three mutations most commonly found among Ashkenazi Jews: the so-called insertion, splice junction, and adult mutations. Among 148 Ashkenazi Jews tested, 108 had the insertion mutation, 26 had the splice junction mutation, five had the adult mutation, and nine had none of the three. Among 28 non-Jewish carriers tested, most of whom were obligate carriers, four had the insertion mutation, one had the adult mutation, and the remaining 23 had none of the three.     

Juvenile GM2 gangliosidosis caused by substitution of histidine for arginine at position 499 or 504 of the alpha-subunit of beta-hexosaminidase

Juvenile GM2 gangliosidosis is a rare neurodegenerative disorder closely related to Tay-Sachs disease but of later onset and more protracted course. The biochemical defect lies in the alpha-subunit of the lysosomal enzyme beta-hexosaminidase. Cultured fibroblasts derived from patient A synthesized an alpha-subunit which could acquire mannose 6-phosphate and be secreted, but which failed to associate with the beta-subunit to form the enzymatically active heterodimer. By contrast, fibroblasts from patient B synthesized an alpha-subunit that was retained in the endoplasmic reticulum. To identify the molecular basis of the disorder, RNA from fibroblasts of these two patients was reverse-transcribed, and the cDNA encoding the alpha-subunit of beta-hexosaminidase was amplified by the polymerase chain reaction (PCR) in four overlapping fragments. The PCR fragments were subcloned and shown by sequence analysis to contain a G to A transition corresponding to substitution of histidine for arginine at position 504 in the case of patient A and at position 499 in the case of patient B. The mutations were confirmed by hybridization of allele-specific oligonucleotides to PCR-amplified fragments of DNA corresponding to exon 13 of the alpha-subunit gene. The Arg504----His mutation was found on both alleles of patient A as well as of another unrelated patient; the homozygosity of this mutant allele is attributable to consanguinity in the two families. The Arg499----His mutation was found in patient B in compound heterozygosity with a common infantile Tay-Sachs allele. There is additional heterogeneity in juvenile GM2 gangliosidosis, as neither mutation was found in the DNA of a fourth patient. The Arg----His mutations at positions 499 and 504 are located at CpG dinucleotides, which are known to be mutagenic "hot spots."