Infantile Sandhoff Disease Research Articles

A Novel Missense Mutation (C522Y) Is Present in the β-Hexosaminidase β-Subunit Gene of a Japanese Patient with Infantile Sandhoff Disease

A novel missense mutation (1565G→A) was identified in the cDNA and genomic DNA coding for the β-hexosaminidase β-subunit of a Japanese patient with infantile Sandhoff disease. The patient was homozygous for this mutation, which should result in a cysteine-to-tyrosine substitution at codon 522. Computer-assisted analysis of this amino acid substitution predicted alteration in the secondary structure in the region of a highly conserved sequence. An immunofluorescence study revealed the accumulation of GM2 ganglioside in cultured fibroblasts from the patient with this mutation.

A second, large deletion in the HEXB gene in a patient with infantile Sandhoff disease.

A second, large deletion in the HEXB gene in a patient with infantile Sandhoff disease.

Sandhoff disease in Argentina: high frequency of a splice site mutation in the HEXB gene and correlation between enzyme and DNA-based tests for heterozygote detection.

The level of beta-hexosaminidase activity in plasma and leukocytes and the frequency of three known HEXB mutations were studied in an Argentinean deme with high incidence of infantile Sandhoff disease. Two mutations were previously identified in one of two Sandhoff patients from the region, a splice mutation, IVS-2 + 1 G-->A, and a 4-bp deletion, delta CTTT782-785. These mutations, and a 16-kb deletion from the 5' end of the HEXB gene common in non-Argentineans, were screened in 9 Sandhoff patients (all unrelated), 24 obligate heterozygotes, 33 additional individuals belonging to families with affected members, and 64 randomly ascertained individuals from the high risk region. Of 31 independent alleles examined, including those of the two patients previously reported, 30 had the IVS-2 splice mutation and only the originally reported patient had the delta CTTT deletion. The 16-kb deletion was not observed. Further, among the 57 unaffected members of families with a previous history of Sandhoff disease, and absolute correlation was found between carrier diagnosis by enzyme assay of leukocytes and the DNA-based tests for mutation. One of the 64 controls was classified as a carrier by enzyme assay but did not have one of the three mutations screened. We conclude that a single mutation predominates in this Argentinean population and that the DNA-based test can be an effective supplement or alternative to enzyme-based testing.

Impact of premature stop codons on mRNA levels in infantile Sandhoff disease.

Sandhoff disease is an autosomal recessive lysosomal storage disease resulting from mutations of the HEXB gene encoding the beta subunit of beta-hexosaminidase A. Fibroblast lines from four patients with the infantile form of the disease were investigated for mutations by single strand conformation polymorphism analysis and direct sequencing of PCR products. Two of the cell lines were homozygous for a common, 16 kb deletion of the 5' end of HEXB gene. The two other cell lines contained the 16 kb deletion along with a second mutant allele generating a stop codon: in one case a nonsense mutation, C850-->T, which generated a stop codon at codon 284; and in the other, a single base deletion, delta T1344, which generated a stop codon at codon 451. One additional cell line investigated was a compound heterozygote for two frameshift mutations, delta G774 in exon 7 and delta AG1305-1306 in exon 11 (McInnes et al. 1992, Biochim. Biophys. Acta 1138: 315-317). Stop codons were generated in this cell line at codons 274 and 454, respectively. We took advantage of these genotypes to investigate the steady-state level of mRNA produced by cells containing stop codons using a competitive polymerase chain reaction technique. The mRNA levels were, as percent of normal per single gene dose: for the stop codon at codon 451, 30%; for those at codons 274 and 454, combined percentage of 1.7%; and at codon 284, 0.8%. These studies demonstrate a dramatic difference in the steady-state level of Hex beta mRNA in the cell lines with stop codons in close proximity to each other (codons 451 vs 454).(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular basis of an adult form of Sandhoff disease: Substitution of glutamine for arginine at position 505 of the β-chain of β-hexosaminidase results in a labile enzyme

Sandhoff disease is a lysosomal storage disorder characterized by accumulation of G M2 ganglioside due to mutations in the β-chain of β-hexosaminidase. Hexosaminidase activity is negligible in infantile Sandhoff disease whereas residual activity is present in juvenile and adult forms. Here we report the molecular basis of the first described adult form of Sandhoff disease. Southern analysis of chromosomal DNA indicated the absence of chromosomal deletions in the gene encoding the β-chain. Northern analysis of RNA from cultured fibroblasts demonstrated that at least one of the β-chain alleles was transcribed into normal-length mRNA. Sequence analysis of the entire cDNA prepared from poly-adenylated RNA showed that only one point mutation was present, consisting of a G→A transition at nucleotide position 1514. This mutation changes the electric charge at amino acid position 505 by substitution of glutamine for arginine in a highly conserved part of the β-chain, present even in the slime mold Dictyostelium discoideum. The nucleotide transition generated a new restriction site for Dde I, which was present in only one of the alleles of the patient. Reverse transcription of mRNA followed by restriction with Dde I resulted in complete digestion at the mutation site, demonstrating that the secone allel was of an mRNA-negative type. Transfection of COS cells with a cDNA construct containing the mutation but otherwise the normal sequence resulted in the expression of a labile form of β-hexosaminidase. These results show that the patient is a genetic compound, and that the lability of β-hexosaminidase found in this form of Sandhoff disease is based on a single nucleotide transition.

Two small deletion mutations of the HEXB gene are present in DNA from a patient with infantile Sandhoff disease

Lysosomal β-hexosaminidase (EC 3.2.1.52) occurs as two major isozymes hexosaminidase A (αβ) and B (ββ). The α subunit is encoded by the HEXA gene and the β subunit by HEXB gene. Defects in the α or β subunits lead to Tay-Sachs or Sandhoff disease, respectively. While many HEXA gene mutations have been reported only three HEXB gene mutations are known. We report the characterization of two rare HEXB mutations present in genomic DNA from a single fibroblast cell line, GM203, taken from a patient with the infantile form of Sandhoff disease. The first is a single base pair deletion in exon 7 changing the codon for Gly-258, GGA, to GA and the second, a two base pair deletion in exon 11 changes the codons for Arg-435/Val-436, AGA/GTC, to AGTC. Each mutation produces a frame shift in the affected allele that results in a premature stop codon 17 or 20 codons downstream, respectively. These mutations also result in the inability to detect β-mRNA by Northern blot analysis of total mRNA. These data are consistent with the idea that the severe infantile form of Tay-Sachs or Sandhoff disease is associated with a total lack of residual hexosaminidase A activity.

Juvenile Sandhoff disease: a Japanese patient carrying a mutation identical to that found earlier in a Canadian patient

A 35-year-old Japanese man with juvenile Sandhoff disease is described. He showed progressive neurogenic muscular atrophy, cerebellar ataxia and mental deterioration, beginning at age 10 years. The accumulation of GM 2 ganglioside in the submucosal nerve cell was confirmed by positive immunostaining using anti-GM 2 ganglioside antibody. Biochemical evaluation revealed nearly absent β-hexosaminidase A and B activities in leukocytes and cultured fibroblasts. Hydrolysis of [ 3H]globoside I in the intact fibroblasts was apparently disturbed but the rate of hydrolysis was higher than those seen in cells from patients with infantile Sandhoff disease. Analysis of the β-hexosaminidase β-subunit gene of the patient disclosed a point mutation (a G-to-A transition) within intron 12. The mutation generates a new splice junction resulting in a 24-base insertion between exons 12 and 13 in the processed mRNA and consequently an 8-amino acid insertion in the translation product. This mutation is identical to that originally found in a Canadian patient with juvenile Sandhoff disease. A possible relationship with the clinical phenotype and the gene abnormality is discussed.

Distribution of saposin proteins (sphingolipid activator proteins) in lysosomal storage and other diseases.

Saposins (A, B, C, and D) are small glycoproteins required for the hydrolysis of sphingolipids by specific lysosomal hydrolases. Concentrations of these saposins in brain, liver, and spleen from normal humans as well as patients with lysosomal storage disease were determined. A quantitative HPLC method was used for saposin A, C, and D and a stimulation assay was used for saposin B. In normal tissues, saposin D was the most abundant of the four saposins. Massive accumulations of saposins, especially saposin A (about 80-fold increase over normal), were found in brain of patients with Tay-Sachs disease or infantile Sandhoff disease. In spleen of adult patients with Gaucher disease, saposin A and D accumulations (60- and 17-fold, respectively, over normal) were higher than that of saposin C (about 16-fold over normal). Similar massive accumulations of saposins A and D were found in liver of patients with fucosidosis (about 70- and 20-fold, respectively, over normal). Saposin D was the primary saposin stored in the liver of a patient with Niemann-Pick disease (about 30-fold over normal). Moderate increases of saposins B and D were found in a patient with GM1 gangliosidosis. Normal or near normal levels of all saposins were found in patients with Krabbe disease, metachromatic leukodystrophy, Fabry disease, adrenoleukodystrophy, I-cell disease, mucopolysaccharidosis types 2 and 3B, or Jansky-Bielschowsky disease. The implications of the storage of saposins in these diseases are discussed.