Classical Tay-Sachs Disease Research Articles

Catabolism of GM2 in man and mouse

Tay–Sachs disease is caused by the deficiency of either the enzyme or the activator protein that is responsible for the catabolism of GM2. The biochemical basis of this disease remained elusive until the discovery of two β-hexosaminidase (Hex) isozymes, Hex A and Hex B, in human spleen using 4-methylumbelliferyl-β-GlcNAc as substrate. This finding led to the revelation of the deficiency of Hex A in classical Tay–Sachs disease. In the late 1960s, based on the accumulation of GM2 and the deficiency of Hex A in patients with classical Tay–Sachs disease, it was concluded that Hex A was solely responsible for the hydrolysis of the GalNAc from GM2. Upon investigation of the degradation of GM2, we found in the early 1970s that Hex A required a protein cofactor, GM2 activator (GM2-Act), to assist the hydrolysis of GM2. During the last two decades, GM2-Act has been isolated and characterized from various tissues. The gene encoding this activator has also been cloned, and the recombinant GM2-Act has been expressed in Escherichia coli and other expression systems. Although human GM2-Act was very effective in stimulating the hydrolysis of GM2 by Hex A, it was not effective in stimulating the hydrolysis of asialo-GM2 (GA2) by the same enzyme. To explain the possible function of GM2-Act, we have synthesized an analog of GM2, called 6′GM2 [GalNAcβ1,6(NeuAcα2,3)-Galβ1,4GlcCer]. We have shown that the GalNAc and the NeuAc in 6′GM2 were readily hydrolyzed by Hex A and sialidase, respectively, without the assistance of GM2-Act. Thus, the resistance of GM2 to Hex A is caused by the rigid conformation of the terminal trisaccharide in GM2, and GM2-Act may be able to modify this conformation. The recombinant mouse GM2-Act shares 74.1% amino acid identity with the human GM2-Act. While the mouse GM2-Act can efficiently stimulate the hydrolysis of both GM2 and GA2 by Hex A, and to a lesser extent can also stimulate Hex B to hydrolyze GA2, the human GM2-Act can only effectively stimulate the hydrolysis of GM2 by Hex A. We have subsequently identified a narrow region (Asn 106–Tyr 114) in the mouse GM2-Act sequence that is responsible for stimulating the hydrolysis of GA2. Our results provide clear evidence of the existence of an additional pathway for GM2 catabolism in mouse by converting GM2 to GA2 and subsequently to LacCer. These results provide an explanation for the lack of excessive GM2 accumulation in the Hexa gene-disrupted mice.

12. The search for the genetic lesion in Ashkenazi Jews with classic Tay-Sachs disease

Publisher Summary This chapter gives an overview on the search for the genetic lesion in Askenazi Jews with Classic Tay-Sachs diseases (TSD). Elucidation of the mutation underlying classic TSD in the Ashkenazi Jewish population posed more of a challenge. Restriction enzyme digestion patterns of the mutant α-chain gene were identical to those of the normal gene, indicating that the mutation involved a small deletion or single base change. Amplification of the region encompassing the mutation by polymerase chain reaction followed by digestion of the amplified product with Dde1, then Southern analysis of the sample, reveals a 120-base-pair DNA (deoxyribonucleic acid) fragment from the normal allele in contrast to an 85-base pair piece from the mutant. It has generally been assumed that only one mutation gives rise to the classic form of TSD in the Ashkenazi Jewish population. PCR-based assay for the insertion mutation which involved amplification of a segment of exon 11 inclusive of the region of insertion and detection of the lesion by probing duplicate samples of the amplified product with oligonucleotides specific for either the normal or mutant sequence was developed.

15. Recognition and delineation of β-hexosaminidase α-chain variants: A historical and personal perspective

Publisher Summary In this chapter, recognition and delineation of β- Hexosaminidase α-chain variants: a historical and personal perspective, is discussed. Three major classes were recognized among G M2 -gangliosidoses, N-acetyl-β-hexosaminidase (Hex) A deficiency, B deficiency, and the form in which both isozymes appeared normal. As more is found out about the enzymology of β-hexosaminidases and about the metabolism of G M2 -ganglioside and related compounds, it became clear that the above three categories of G M2 -gangliosidosis are not only enzymologically distinct but also are caused by abnormalities in three different genes—Hex α, Hex β, and the G M2 activator protein. The classical Tay-Sachs disease in the Ashkenazi Jewish population was found to be a cross-reacting material (CRM)-negative. 1,830 different genotypes are theoretically possible among patients even within the already known mutations, and this number is constantly increasing. In autosomal recessive disorders, two abnormal alleles contribute to the phenotypic expression.

Molecular basis of adult-onset and chronic GM2 gangliosidoses in patients of Ashkenazi Jewish origin: substitution of serine for glycine at position 269 of the alpha-subunit of beta-hexosaminidase.

Chronic and adult-onset GM2 gangliosidoses are neurological disorders caused by marked deficiency of the A isoenzyme of beta-hexosaminidase; they occur in the Ashkenazi Jewish population, though less frequently than classic (infantile) Tay-Sachs disease. Earlier biosynthetic studies had identified a defective alpha-subunit that failed to associate with the beta-subunit. We have now found a guanosine to adenosine transition at the 3' end of exon 7, which causes substitution of serine for glycine at position 269 of the alpha-subunit [designated 269 (Gly----Ser) substitution]. An RNase protection assay was used to localize the mutation to a segment of mRNA from fibroblasts of a patient with the adult-onset disorder. That segment of mRNA (after reverse transcription) and a corresponding segment of genomic DNA were amplified by the polymerase chain reaction and sequenced by the dideoxy method. The sequence analysis, together with an assay based on the loss of a ScrFI restriction site, showed that the patient was a compound heterozygote who had inherited the 269 (Gly----Ser) mutation from his father and an allelic null mutation from his mother. The 269 (Gly----Ser) mutation, in compound heterozygosity with a presumed null allele, was also found in fetal fibroblasts with an association-defective phenotype and in cells from five patients with chronic GM2 gangliosidosis. It was not found in beta-hexosaminidase A-deficient cells obtained from patients with infantile Tay-Sachs disease nor in cells from individuals who do not have beta-hexosaminidase A deficiency. However, there must be additional mutations with similar consequences, since the 269 (Gly----Ser) substitution was not present in fibroblasts from two patients with juvenile GM2 gangliosidosis even though these had an association-defective alpha-subunit.

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.

Splice junction mutation in some Ashkenazi Jews with Tay-Sachs disease: evidence against a single defect within this ethnic group.

Tay-Sachs disease is an inherited disorder in which the alpha chain of the lysosomal enzyme beta-N-acetylhexosaminidase A bears the mutation. Ashkenazi Jews are found to be carriers for a severe type of Tay-Sachs disease, the classic form, 10 times more frequently than the general population. Ashkenazi Jewish patients with classic Tay-Sachs disease have appeared to be clinically and biochemically identical, and the usual assumption has been that they harbor the same alpha-chain mutation. In this study I have isolated the alpha-chain gene from an Ashkenazi Jewish patient, GM2968, with classic Tay-Sachs disease and compared its nucleotide sequences with that of the normal alpha-chain gene in the promoter region, exon and splice junction regions, and polyadenylylation signal area. Only one difference was observed between these sequences: at the 5' boundary of intron 12, a guanosine in the conserved splice junction dinucleotide sequence G-T had been altered to a cytidine. The alteration is presumed to be functionally significant and to result in aberrant mRNA splicing. Utilizing the polymerase chain reaction to amplify the region encompassing the mutation, I developed an assay to screen patients and heterozygote carriers for this mutation. Surprisingly, in each of two Ashkenazi patients, only one alpha-chain allele harbored the splice junction mutation. Only one parent of each of these patients was positive for the defect. Another Ashkenazi patient did not bear this mutation at all nor did either of the subject's parents. In addition, 30% of obligate heterozygotes tested carried the splice junction mutation, whereas 20 Ashkenazi Jews designated noncarriers by enzymatic assay were negative for this alteration. The data are consistent with the presence of more than one mutation underlying the classic form of Tay-Sachs disease in the Ashkenazi Jewish population.

Presence of beta-hexosaminidase A alpha-chain mRNA in two different variants of GM2-gangliosidosis.

Tay-Sachs disease displays a variety of forms on the clinical and biochemical level. On the molecular level it has been shown, that poly (A)+ RNA preparations from fibroblasts of patients with classical Tay-Sachs disease lack detectable alpha-chain message when analyzed by Northern blotting with complementary DNA encoding the alpha-chain of human beta-hexosaminidase A. In this report the p beta H alpha-5 clone was used to investigate whether patients with two different variants of Tay-Sachs disease also lack the alpha-chain message. On the basis of RNA hybridization analyses, we could show that our patients which synthesize an altered alpha-chain, as judged by testing enzyme activity and substrate specificity, have the 2.1 kb mRNA which is also seen in healthy control patients.

Composition of gangliosides and neutral glycosphingolipids of brain in classical Tay-Sachs and Sandhoff disease: more lyso-GM2 in Sandhoff disease?

The ganglioside composition of the brain from an individual with classical Tay-Sachs disease and from an individual with Sandhoff disease was examined using our new quantitative methods for ganglioside content determination and compared with that of age-matched control brains. The concentration of GM2 was found to be 12.2 and 13.0 mumol/g of fresh tissue in Tay-Sachs disease and in Sandhoff disease cerebral gray matter, respectively. GM2 was 86 and 87% respectively, of total gangliosides. The concentration of GM1 and, in particular, GM3 ganglioside was also found to be increased, whereas the concentration of the major di- and trisialogangliosides (GD1a, GD1b, and GT1b) had diminished markedly. There was no significant increase in level of any other ganglioside than lyso-GM2. Its concentration was 12 and 16 nmol/g in cerebral gray matter of two Tay-Sachs disease brains and 43 nmol/g in Sandhoff disease brain. The Sandhoff disease brain also differed from the classical Tay-Sachs disease brain by having a much higher concentration of gangliotriaosylceramide and globotetraosylceramide. The structures of relevant gangliosides and neutral glycolipids were established by fast atom bombardment-mass spectrometry and permethylation studies.

Accumulation of Ganglioside GM2 in Cerebrospinal Fluid of a Patient with the Variant AB of Infantile GM2 Gangliosidosis

Brain biopsy has been used for the diagnosis of the variant AB of infantile GM2 gangliosidosis. Accumulation of ganglioside GM2 (300 ng of neuraminic acid per milliliter) was observed in the CSF of a patient with this disorder. GM2 was found also in the CSF of a patient with classic Tay-Sachs disease. Normal CSF did not contain any measurable amounts of GM2. In addition, a glycolipid with a mobility, by thin-layer chromatography, similar to that of paragloboside was observed in the CSF of the patient with the variant AB of GM2 gangliosidosis. These findings indicate that the variant AB can be diagnosed by demonstrating accumulation of GM2 in the CSF of patients with normal hexosaminidase activity.

HISTOLOGICAL OBSERVATION OF THE BRAIN OF TAY‐SACHS DISEASE WITH SEIZURE AND CHRONIC DPH INTOXICATION

The patients was a 3-year-old boy with psychomotor retardation and attacked by seizures since 8 months of age. On funduscopy, the maculla presented a cherry-red spot. Serum hexosaminidase A activities were as low as 8.2 percent. Both parents were carriers. The patient was diagnosed as classical Tay-Sachs disease by neurological examination. Diphenylhydantoin was continuously given for 2 years and 2 months till his death. Autopsy revealed swelling of the cerebrum, atrophy and sclerosis of the cerebellum, hepatomegaly and mild enlargement of the lymph nodes. Histologically, the cerebrum showed ballooned swelling of nerve cells, slight gliosis and demyelination, while cerebellar Purkinje's cells and granular cells were degenerated and disappeared. The cerebellar cortex showed small focal spongy degeneration. By electron microscopy, membranous cytoplasmic bodies were found in the nerve cells. The change of brain observed in this case were interpreted as a combined result of (i) essential change to classical Tay-Sachs disease, (ii) ischemic change due to frequently repeated seizures, (iii) chronic toxicity by long-term anticonvulsant administration.

Distortion of neuronal geometry and formation of aberrant synapses in neuronal storage disease

Golgi and electron microscope studies of cortical neurons in several lysosomal storage diseases were carried out to elucidate structural features of the large neural processes (meganeurites) that develop as storage sites for accumulated undigestible substrates. Meganeurites occur preferentially in pyramidal neurons wherein they develop between the base of the perikaryon and the initial portion of the axon. They frequently give rise to secondary neurites which bear filopodium-like processes. Meganeurites may possess spines some of which are contacted by presynaptic processes containing synaptic vesicles. The extent of meganeurite development is related to the onset, severity and clinical course of neuronal storage disease. Extensive development of bizarre and pleomorphic meganeurites occurs in classical Tay-Sachs disease (infantile GM2-gangliosidosis, B variant), whereas a smaller proportion of neurons exhibits meganeurites in juvenile GM2-hangliosidosis and Hurler's disease. Meganeurites with spines and spine synapses were prominent in GM2-gangliosidosis, AB variant. It is proposed that meganeurites and meganeurite synapses contribute to the onset and progression of neuronal dysfunction in storage diseases by altering electrical properties of the neuron and modifying integrative operations of somadendritic synaptic inputs.

GM2-Gangliosidosis, AB variant

Clinical and neuropathological studies of a case of AB variant GM2-gangliosidosis have been presented. The patient was a 14 months old black female infant who had "black cherry spot" in the retinas. The total activities of beta-galactosidase and N-acetyl-beta-hexosaminidase, as well as the proportion of hexosaminidase A and B components in her serum and leukocytes were normal when the assays were carried out with artificial fluorogenic substrate. Diagnosis of GM2-gangliosidosis AB variant was established by an abnormal increase of GM2-ganglioside in the biopsied brain tissue, similar to classical Tay-Sachs disease. Her clinical manifestation appeared to be similar but somewhat milder than those of classical Tay-Sachs disease. Light microscopic features of the cerebral biopsy were also closely similar to Tay-Sachs disease and Sandhoff disease but gliosis and neuronal loss were less pronounced. Electron microscopic study revealed numerous membranous cytoplasmic bodies (MCB) and zebra bodies in neurons. In addition, varieties of large intracytoplasmic inclusions in astrocytes, a feature distinctly different from classical Tay-Sachs disease, were observed. Numerous cytoplasmic inclusions were also present in oligodendroglia, pericytes and microglial cells.

Generalized accumulation of neutral glycosphingolipids with GM2 ganglioside accumulation in the brain

Analyses have been made of glycosphingolipids from visceral organs and brain of a patient with an unusual lipid storage disorder diagnosed initially as classical Tay-Sachs disease. Levels of the lipids from fresh-frozen sections of gray and white matter, kidney, spleen, liver, and heart from this patient were compared with those of normal juvenile controls, and the fatty acid composition of accumulated glycosphingolipids was compared with reference compounds. This patient was found to have abnormally high concentrations of a globoside in liver, kidney, and spleen, asialo GM2 ganglioside in brain and liver, and GM2 ganglioside in the brain. On the basis of these findings along with the clinical manifestations of Tay-Sachs disease with visceral involvement (hepatosplenomegaly) and demonstration of total deficiency of both A and B components of β-N-acetylhexosaminidase activity, this glycosphingolipidosis is the same as two previously reported cases of GM2 gangliosidosis with globoside accumulation and total β-N-acetylhexosaminidase deficiency.

Generalized gangliosidosis: Another inborn error of ganglioside metabolism?

Introduction IN 1959 Norman et al1described a patient with a specific form of amaurotic idiocy which they called Disease With Visceral Involvement. In this patient the clinical and pathological picture resembled Tay-Sachs disease, plus accumulation of lipid-laden histiocytes in liver, spleen, thymus, and other organs. Their patient's case differed chemically from Niemann-Pick disease, in which there is also combined neural and visceral involvement, in that the stored material in the brain was a ganglioside. In Niemann-Pick disease the characteristic lipid, which accumulates to a moderate degree in the brain and to a marked degree in the viscera, is sphingomyelin. Their patient's case also differed from classical Tay-Sachs disease since visceral involvement is absent in this disease. Also in 1959, Craig et al2described an infant with foam-cell histiocytosis of the viscera, involvement of renal glomerular epithelium, and clinical and radiological features suggestive of Hurler's disease. Since

Generalized Gangliosidosis

Introduction IN 1959 Norman et al1described a patient with a specific form of amaurotic idiocy which they called "Tay-Sachs Disease With Visceral Involvement." In this patient the clinical and pathological picture resembled Tay-Sachs disease, plus accumulation of lipid-laden histiocytes in liver, spleen, thymus, and other organs. Their patient's case differed chemically from Niemann-Pick disease, in which there is also combined neural and visceral involvement, in that the stored material in the brain was a ganglioside. In Niemann-Pick disease the characteristic lipid, which accumulates to a moderate degree in the brain and to a marked degree in the viscera, is sphingomyelin. Their patient's case also differed from classical Tay-Sachs disease since visceral involvement is absent in this disease. Also in 1959, Craig et al2described an infant with "foam-cell" histiocytosis of the viscera, involvement of renal glomerular epithelium, and clinical and radiological features suggestive of Hurler's disease. Since