Peter Hechtman

Synthesis of 4-methylumbelliferyl-β--N-acetylglucos-amine-6-sulfate and its use in classification of GM2 gangliosidosis genotypes☆

Measurement of hexosaminidase A (Hex A) is an important clinical chemical procedure in the classification of GM2 gangliosidosis genotypes. We have synthesized a new substrate which may be useful in both the biochemical diagnosis of GM2 gangliosidosis and the detection of heterozygotes for the Tay-Sachs disease (TSD) allele. 4-Methylumbelliferyl-beta-D-N-acetylglucosamine-6-sulfate (4MUGS) was synthesized by sulfation of 4MU-beta-D-N-acetylglucosamine (4MUG) with chlorosulfonic acid and purified through gel filtration and ion-exchange chromatography. The structure of 4MUGS was verified by elemental analysis and NMR. Hex A is approximately 100 times more active toward 4MUGS than Hex B. The advantage of this increased specificity is that Hex A can be determined in a one-step procedure which allows separation of normal control serum values from those of obligate heterozygotes. Alternatively, assay values obtained using both substrates can be transformed by application of an empirical equation that allows the calculation of both Hex A and Hex B without the requirement of thermal fractionation. Lower values for % Hex A in serum have been obtained for Tay-Sachs homozygotes using the 4MUGS assay procedure. The results of Hex A assays on fibroblast cell strains obtained from Tay-Sachs homozygotes, variant forms of GM2 gangliosidosis and normal controls are also discussed.

The French Canadian Tay-Sachs disease deletion mutation: identification of probable founders

SummaryTay-Sachs disease (TSD) is an inherited neurodegenerative ganglioside storage disorder caused by deficiency of the hexosaminidase A enzyme. A deletion allele (FCD) at the HEXA locus has attained high frequency in the French Canadian population. The distribution of affected probands shows a likely center of diffusion for this mutation located in the Bas-St.-Laurent and Gaspésie regions of the province of Quebec. We have reconstructed the genealogies of 15 obligate carriers of the FCD allele to an average depth of 12 generations identifying 60 ancestors and 80 European founders common to all of them. The ancestral origins of the European founders show a significantly greater number of individuals born in the French provinces of Normandy and Perche than expected based on information regarding the origins of the 8,500 immigrants who settled the colony of New France during the French regime. We have identified common ancestors among the 10 who were born in Quebec who appear to be likely candidates for the origin of the FCD mutation. One such couple had 11 children, 5 of whom settled in regions of Quebec or New Brunswick that today have elevated heterozygote frequencies for the FCD. The five offspring are ancestors of all known carriers. By contrast, the absence of FCD alleles among TSD probands in France suggests that the mutation did not occur in a European founder.

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 G→A 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.

Human Genetics of Membrane Transport with Emphasis on Amino Acids

This chapter is concerned with membrane transport and its genetic control in man. A discussion in this context would be unmanageable if one were to consider all the known physical and chemical aspects of cell membranes, their relevant functions, and the inherited disorders thereof. On the other hand, since no one can yet define what the critical physical and chemical features of biological membranes are, nor define precisely how the process of membrane transport is accomplished,163 it is still necessary to consider transport as a rather black-box phenomenon. We do know of several rare inherited disorders of membrane transport in man and in other species which are viewed with great interest because of the insight they bring concerning the organization and function of membrane transport processes.

Tay-Sachs disease: B1 variant

This first child of non-Jewish parents had nystagmus at 4 months of age, bilateral cherry-red macular spots at 7 months of age, and hyperacusis at 8 months of age; the patient has deteriorated progressively following a clinical course typical of Tay-Sachs disease B variant. Total beta-N-acetylhexosaminidase assayed with 4-methylumbelliferyl-beta-glucosamine (4 MU GlcNAc) as substrate was within the normal range in plasma and cultured dermal fibroblasts and 2/3 the normal mean in leukocytes. The hexosaminidase A activity, assayed with the same substrate in plasma and cultured fibroblasts, approximated Tay-Sachs disease heterozygote levels; however, the activity of hexosaminidase A assayed with 4 MU Glc NAc-6-sulfate in the plasma, leukocytes, and cultured fibroblasts was less than 8, 2, and 1%, respectively of the control mean. This female infant with the B1 variant of Tay-Sachs disease demonstrated an earlier onset and more rapidly progressive course than was observed in 4 of the 5 previously reported patients with this Tay-Sachs disease variant.

Gamma-aminobutyric acid metabolism in brain homogenates of the spastic mouse.

Mice that are homozygous for the spa gene (Chai, 1961) exhibit a number of neurological defects of which the most striking is impaired ability to right themselves when turned over on their backs. Chai et al. (1962) tound that spasticity could be alleviated by administration of the vitamin B 6 antagonist aminooxyacetic acid (AOA), suggesting that the primary defect in the spastic mouse is in the metabolism of a neurotransmitter. Six neurotransmitters are metabolized by pyridoxal phosphate (PLP) dependent enzymes (Clark, 1963). Furthermore, the anticonvulsant activity of AOA has been described in rats (DaVanzo et al., 1961), mice (Bushnel and Lehman, 1963), and dogs (Essig, 1963). In vivo, AOA preferentially inhibits 7-aminobutyric acid transaminase (GABA-T) (Baxter and Roberts, 1961), causing up to fourto fivefold elevation of brain GABA (Wood and Peesker, 1975). An attractive hypothesis concerning the nature of the metabolic defect in spastic mice is that GABA concentration is lowered because of either a decrease in its rate of formation or an increase in the rate of breakdown. Chai et al. (1962), however, were not able to detect differences in the total brain concentration of GABA between spastic mice and normal littermates.

The isolation and properties of a β-alanine permeaseless mutant of Pseudomonas fluorescens

A mutant of Pseudomonas fluorescens has been isolated which is markedly deficient in the active transport of l-alanine, l-proline, and β-alanine. This mutant, which is also deficient in β-alanine transaminase, is capable of catalyzing facilitated diffusion and exchange transport of β-alanine at low temperatures but can maintain little or no uptake of this amino acid against a concentration gradient. In the transport mutant, competitive stimulation of β-alanine by l-proline can be observed, whereas in the parent strain, l-proline inhibits β-alanine uptake. The rate of efflux of β-alanine from preformed pools is 4-fold greater in the transport mutant than in the parent strain. It thus appears that the functional block in this transport mutant resides not in the membrane carrier but in the ability of the cell to couple energy to the membrane carrier.

Allele-specific amplification of genomic DNA for detection of deletion mutations: identification of a French-Canadian Tay-Sachs mutation.

SummaryA rapid and efficient method for the detection of a 7.6-kb deletion in theβ-hexosaminidase Aα-subunit gene, a mutant allele causing Tay-Sachs disease in French Canadians, is described. The protocol involves PCR (polymerase chain reaction) amplification of target sequences on normal and mutant chromosomes. Three amplification primers, a single 5′ primer complementary to normal and mutant DNA templates and two 3′ primers specific for normal and mutant DNA templates are required. The primers direct amplification of two unique fragments (normal and mutant) that are easily separated by gel electrophoresis. Allele-specific oligonucleotide hybridization using normal and mutant probes to genomic DNA samples from normal, heterozygous and homozygous individuals confirms these results and is consistent with results of genotypic classification of individuals using Southern analysis. The method is applicable to detection of deletion mutations in cases where some deletion-flanking sequence is known.

High-capacity method for purification of human liver hexosaminidase B using hydrophobic chromatography

Hydrophobic chromatography was investigated as a purification procedure for human liver hexosaminidases. Both phenyl-Sepharose and valine-Sepharose have a high binding capacity of hexosaminidases. A degree of resolution between the A and B ioszymes is achieved with phenyl-Sepharose. Both hydrophobic supports must be used close to their capacity in order to recover the applied enzyme. Two purification procedures for human liver hexosaminidase B were employed, which resulted in recoveries of approximately 48 and 24% with final specific activities of 33,400 and 4840 nmole/min.mg, respectively.