Segregation of Tay-Sachs and Sandhoff alleles in a non-Jewish family.

A non-Jewish family is presented in which the genes for Tay-Sachs disease and Sandhoff disease are segregating. Individuals heterozygous for both alleles have low serum and white cell total hexosaminidase levels together with a proportion of heat-labile hexosaminidase A (HEX A) which falls in the normal range. The individuals would not be detected as carriers of Tay-Sachs disease or Sandhoff disease in a population screening program.     

Characterization of unusual hexosaminidase A (HEX A) deficient human mutants.

Two families with unusual hexosaminidase A (HEX A) mutations are described. In one, the proband had the Tay-Sachs disease phenotype with considerable HEX A activity. In the second, the proband was phenotypically normal with absent HEX A activity. Activities using ganglioside GM2 as substrate demonstrate markedly reduced activities in the first case and half-normal activities in the second. Pedigree analyses indicate the presence of two different mutations. In the first, the proband appears to be an allelic compound HEX A 2-4 where mutation HEX A 4 leads to a diminution of HEX A activity against GM2 but not for the synthetic substrate, 4MU-beta-D-N-acetyl-glucosaminide, with HEX A 2 being the Tay-Sachs disease (or similar) mutation. In the second family, the proband is an allelic compound HEX A 2-5 where mutation HEX A 5 leads to a diminution of HEX A activity against the synthetic substrate, 4MU-beta-D-N-acetyl-glucosaminide, but not for GM2. The presence of either mutation will lead to false-negative (HEX A 4) or false-positive (HEX A 5) assignments of heterozygosity or homozygosity for GM2 gangliosidosis when synthetic substrates are employed. In both families, DM2 N-acetyl-beta-D-galactosaminidase activity in fibroblasts was an accurate determinant of phenotype.     

Hereditary heat-labile hexosaminidase B: a variant whose homozygotes synthesize a functional HEX A.

Homozygosity for a mutant allele at the beta-chain locus of hexosaminidase (HEX), resulting in a variant of heat-labile HEX B, is reported for the first time in two healthy children. HEX activity in their sera, leukocytes, and cultured skin fibroblasts is severely deficient when measured on the synthetic substrate 4-MU-GLcNAc. However, their cultured skin fibroblasts synthesize and process both alpha and beta chains of HEX, and their lymphoid cells hydrolyze normally the natural ganglioside GM2. This mutation is, therefore, different from at least one of the beta-chain mutations found in previously published families with heat-labile HEX B.     

Complementation of genetic disease: a velocity sedimentation procedure for the enrichment of heterokaryons

Methodology is described to enrich for heterokaryons after mammalian cell fusion. A heterogeneous cell mixture can be separated on a Sta-Put apparatus into fractions of uniform size cells by sedimentation through a 1% bovine serum albumin-5% Ficoll gradient. Unfused RAG and LM/TK- cells, differing by 10% in diameter, have been sorted by size; following fusion, larger and faster sedimenting cells were shown to be hybrids. This methodology can be utilized in genetic complementation studies of human genetic diseases where selection procedures for proliferating hybrids do not exist. When fibroblasts from individuals with Tay-Sachs disease [deficient in hexosaminidase A (HEX A-)] and Sandhoff-Jatzkewitz disease (HEX A- and HEX B-) are fused, HEX A is generated, demonstrating complementation of two different mutations. After Sta-Put fractionation, the HEX A complementation product was associated with the faster sedimenting multinuclear cells and not with the mononuclear parental cells. This methodology will facilitate detection of genetic differences in fibroblasts from related inherited disorders.     

Inheritance of the enzyme deficiency in three neurolipidoses: variant 0 of Tay-Sachs disease (Sandhoff's disease), classic tay-sachs disease,and metachromatic leukodystrophy. Identification of the heterozygous carriers

Summary The recessive inheritance of the enzyme deficiencies (total hexosaminidase, hexosaminidase A, arylsulfatase A) in nine families with neurolipidoses (variant 0 of Tay-Sachs disease=Sandhoffs disease, classic Tay-Sachs disease, metachromatic leukodystrophy) has been studied. 62 family members were identified as: genotypically normals, phenotypically normal heterozygous carriers, or patients, according to the enzyme levels (normal, about half normal or low) in the leukocytes. The activities of the lysosomal enzymes involved in the diseases are related to the mean relative activity of three or four lysosomal reference enzymes thereby identifying the heterozygous carrier state by the enzyme level about half the normal enzyme. It cannot be completely excluded that the heterozygous state or total enzyme deficiency occur more frequently than would be expected according to Mendel's laws. This problem is discussed.

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Zusammenfassung Bei 9 Familien (62 untersuchten Personen) mit Neurolipidosen (Variante 0 der Tay-Sachsschen Erkrankung, klassische Tay-Sachssche Erkrankung, metachromatische Leukodystrophie) wird, der recessive Erbgang der Enzymdefekte (Total-Hexosaminidase, Hexosaminidase A, Arylsulfatase A) aufgezeigt. Die einzelnen Personen werden auf Grund der Enzymspiegel in den Leukocyten als genotypisch normal, heterozygot (ca. 50%ige Erniedrigung bei phänotypisch Normalen) oder krank (Enzymdefekt) identifiziert. —Werden die Werte der betroffenen lysosomalen Enzyme auf die durchschnittliche relative Aktivität von drei oder vier lysosomalen Referenzenzymen bezogen, so wird die Erniedrigung des Enzymspiegels bei Heterozygoten in fast jedem Fall erfaßt.—Es kann nicht völlig ausgeschlossen werden daß die Enzymdefekte etwas häufiger, als nach den Mendelschen Regeln anzunehmen ist, weitervererbt werden. Eine Hypothese dafür wird aufgestellt.

     

The biochemical genetics of the hexosaminidase system in man.

Tay-Sachs disease and related GM2 ganglioside storage disorders result from the absence of one form of hexosaminidase, HEX A. The persistence of a second major hexosaminidase isozyme, HEX B, does not protect against the lethal accumulation of GM2 ganglioside in the central nervous system. Using immunologic and biochemical techniques, it has been demonstrated that the two major isozymes of hexosaminidase, HEX A and HEX B, share a common subunit, the structure of HEX A being designated (alpha beta)n and the structure of HEX B being designated as (beta2)n. The minor isozyme, HEX S, is an alpha chain homopolymer designated (alpha2)n, and HEX C seems unrelated to the HEX A, B, S system. The structures of other minor isozymes have not been totally resolved, but HEX I1, I2, and P (which may be identical to I2) appear to represent forms of HEX B.     

Prenatal Diagnosis of Tay-Sachs Genotypes

Hexosaminidase activity was determined in cultured and uncultured amniotic fluid cells taken from seven pregnant women who had previously given birth to infants with Tay-Sachs disease. Complete deficiency of hexosaminidase A was found in one case, indicating a Tay-Sachs fetus. The diagnosis was confirmed on examination of various tissues after therapeutic abortion. Of the other six cases three were considered heterozygous and three homozygous normal. These diagnoses were confirmed postnatally on examination of cord blood leucocytes, peripheral leucocytes, and urine. The activity of hexosaminidase A is appreciably decreased in dead cells and hence in uncultured amniotic fluid cells. Hence reliable identification in utero of the three genotypes may be achieved only by examining the cultured living amniotic cells.     

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

Lysosomal beta-hexosaminidase (EC 3.2.1.52) occurs as two major isozymes hexosaminidase A (alpha beta) and B (beta beta). The alpha subunit is encoded by the HEXA gene and the beta subunit by HEXB gene. Defects in the alpha or beta 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 beta-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.     

Molecular analysis of HEXA gene in Argentinean patients affected with Tay-Sachs disease: possible common origin of the prevalent c.459+5A>G mutation

Tay-Sachs disease (TSD) is a recessively inherited disorder caused by the deficient activity of hexosaminidase A due to mutations in the HEXA gene. Up to date there is no information regarding the molecular genetics of TSD in Argentinean patients. In the present study we have studied 17 Argentinean families affected by TSD, including 20 patients with the acute infantile form and 3 with the sub-acute form. Overall, we identified 14 different mutations accounting for 100% of the studied alleles. Eight mutations were novel: 5 were single base changes leading to drastic residue changes or truncated proteins, 2 were small deletions and one was an intronic mutation that may cause a splicing defect. Although the spectrum of mutations was highly heterogeneous, a high frequency of the c.459+5G>A mutation, previously described in different populations was found among the studied cohort. Haplotype analysis suggested that in these families the c.459+5G>A mutation might have arisen by a single mutational event.     

Hereditary heat-labile hexosaminidase B: its implication for recognizing Tay-Sachs genotypes

Two pairs of alleles, at the two loci of hexosaminidase (HEX), were found to segregate in an Arab inbred family: the normal and the mutant Tay-Sachs (TSD) alleles of HEX A, and the normal and a mutant allele of HEX B. Since the mutant HEX B is heat labile, no reliable identification of TSD genotypes can be obtained in its presence, as long as the proportions of HEX A and B are estimated by the routinely used heat-inactivation method. The genotypes may be correctly identified in such cases by separation of the two isoenzymes on ion-exchange chromatography, estimating their individual activities, and calculating the ratio between them. Of the nine genotype combinations possible with these two pairs of alleles, five have been identified in the reported family by this procedure.     

Sandhoff disease: a prevalent form of infantile GM2 gangliosidosis in Lebanon.

All cases clinically diagnosed as Tay-Sachs disease at the American University Hospital, Beirut, during a period of 22 years (1957--1979) were reviewed. Of a total of 15 cases, seven had serum hexosaminidase tested and proved to have Sandhoff disease. In two other cases, parents were tested and found to be Sandhoff carriers. These results indicate that Sandhoff disease is relatively prevalent in Lebanon and that it may represent the more common form of infantile GM2 gangliosidosis in this country.     

Diagnosis and carrier detection of Tay-Sachs disease: direct determination of hexosaminidase A using 4-methylumbelliferyl derivatives of beta-N-acetylglucosamine-6-sulfate and beta-N-acetylgalactosamine-6-sulfate.

4-Methylumbelliferyl-6-sulfo-2-acetamido-2-deoxy derivatives of beta-D glucopyranoside and beta-D-galactopyranoside were prepared by direct sulfation of the commonly used unsulfated derivatives. Both sulfated substrates were highly specific for hexosaminidase A, and in fractionated serum, cells, and tissue preparations, less than 2.5% of these activities were associated with hexosaminidase B and the intermediate isozyme fractions. Serum and leukocytes from patients with infantile Tay-Sachs disease, including a patient with thermolabile hexosaminidase B, had less than 2% of noncarrier activities. Carrier values were clearly separated from those of noncarriers, and no problems were encountered in utilizing sera from pregnant women. The % hexosaminidase A values as derived from the ratio between the activities toward the sulfated and unsulfated substrates in the same specimen were comparable to those obtained by the heat-inactivation method (except for subjects with thermolabile hexosaminidase B) and may be helpful in genotype determination in borderline cases.     

Carrier detection in Sandhoff disease.

Three new cases of Sandhoff disease are reported. One infant was the second affected child in a large family. The parents, who were cousins, were part of a large kindred from an isolated community in northern Saskatchewan. We assayed total and heat-stable hexosaminidases in 38 other members of the kindred and found two distinct cohorts. Sixteen individuals had low total and low heat-stable hexosaminidase and were diagnosed as carriers of Sandhoff disease. The values for the remainder were within normal limits. In a retrospective study of data from more than 14,000 Ashkenazi Jews, who were screened for Tay-Sachs disease, six were identified as Sandhoff carriers. Our data indicate that carrier detection requires measurement of both total and heat-stable enzyme activity.     

The presence of two different infantile Tay-Sachs disease mutations in a Cajun population.

A study was undertaken to characterize the mutation(s) responsible for Tay-Sachs disease (TSD) in a Cajun population in southwest Louisiana and to identify the origins of these mutations. Eleven of 12 infantile TSD alleles examined in six families had the beta-hexosaminidase A (Hex A) alpha-subunit exon 11 insertion mutation that is present in approximately 70% of Ashkenazi Jewish TSD heterozygotes. The mutation in the remaining allele was a single-base transition in the donor splice site of the alpha-subunit intron 9. To determine the origins of these two mutations in the Cajun population, the TSD carrier status was enzymatically determined for 90 members of four of the six families, and extensive pedigrees were constructed for all carriers. A single ancestral couple from France was found to be common to most of the carriers of the exon 11 insertion. Pedigree data suggest that this mutation has been in the Cajun population since its founding over 2 centuries ago and that it may be widely distributed within the population. In contrast, the intron 9 mutation apparently was introduced within the last century and probably is limited to a few Louisiana families.     

Estimation of the frequency of hexosaminidase a variant alleles in the American Jewish population.

There appear to be several alleles of the hexosaminidase A (HEX A) gene that lead to different clinical syndromes. In addition to the infantile-onset Tay-Sachs disease (TSD), there is a juvenile-onset and an adult-onset form, which are also characterized by low HEX A levels. There are also apparently healthy adults with low HEX A activity. Based primarily on data from population screening for TSD carrier status, we estimate the allele frequency of the combined variant alleles for which data are available to be about 4.5 x 10(-4) and the frequency of adults showing zero HEX A levels (when tested using artificial substrate) to be about 1:67,000. The implications for population screening and prenatal diagnosis are discussed.     

Serum hexosaminidase activity in I-cell disease carriers

Summary Serum heat stable hexosaminidase activities are used to identify 47 I-cell disease heterozygotes in a large kindred. Serum beta-hexosaminidase isozyme patterns in normal individuals, Tay-Sachs disease carriers, I-cell disease carriers and in cord blood samples are compared.     

Studies on the substrate specificity of hexosaminidase A and B from liver

β-N-Acetylhexosaminidase A and B were partially purified from normal human liver using DEAE-cellulose column chromatography. Hexosaminidase B was also purified from the livers of patients who had died of Tay-Sachs disease. The hexosaminidase fractions were tested for their ability to hydrolyze the amino sugar moiety of synthetic substrates and of three amino sugar-containing glycolipids, GA₂, globoside, and GM₂.     

A novel mutation in the invariant AG of the acceptor splice site of intron 4 of the beta-hexosaminidase alpha-subunit gene in two unrelated American black GM2-gangliosidosis (Tay-Sachs disease) patients.

Samples of genomic DNA from three unrelated American black infants having both biochemical and clinical features of classical infantile Tay-Sachs disease were sequenced following PCR amplification. A G----T transversion was observed in the AG acceptor splice site preceding exon 5 of the beta-hexosaminidase alpha-subunit gene in the first black family. This transversion changed the acceptor splice site from the consensus sequence, AG, to AT, thereby interfering with splicing at this intron 4/exon 5 junction. The proband was homozygous for this mutation; his mother and a brother are heterozygous. The same mutation was found in a second, apparently unrelated, black GM2-gangliosidosis patient. The second patient was a compound heterozygote, as only one allele carried this mutation. The mother and a brother in this second family are carriers for this mutation, while the father and a noncarrier sister are normal for this region of the gene. The third proband did not have this mutation; nor did the mother of a fourth black proband. Eight other independently ascertained non-black, non-Jewish, GM2-gangliosidosis families did not have this mutation. The observation of the same novel mutation in two unrelated black GM2-gangliosidosis patients indicates that the American black population has segregating within it at least one GM2-gangliosidosis mutation which may be specific to this population and not a result of migration.     

Two abnormalities of hexosaminidase A in clinically normal individuals

Two abnormalities of beta-hexosaminidase A (HEX A) activity are described. One, found in two unrelated Jewish children, was characterized by the complete absence of HEX A activity in serum, but low levels of activity in leukocytes and fibroblasts using artificial substrate. The other, found in a non-Jewish man, was characterized by uniformly low levels of HEX A activity in leukocytes, fibroblasts, and serum against artificial substrate. In all cases, the pH optimum of HEX A was normal, there was no increased lability at 37 degrees C, and no inhibitor was detected to account for the deficiency of activity. Cultured fibroblasts of these individuals were capable of synthesizing and processing alpha- and beta-subunits of HEX A and capable of cleaving GM2 ganglioside. The patients, ranging in age from 6 to 30 years, are clinically normal. They are probably genetic compounds carrying the classical Tay-Sachs gene and a differently mutated allele that imparts the anomalous phenotypic features observed.     

Apparent hexosaminidase B deficiency in two healthy members of a pedigree.

A family is described in which all members have decreased serum and leukocyte hexosaminidase activity. Two individuals, the mother and the younger daughter, have a normal ratio of hexosaminidase B (HEX B) to total hexosaminidase, but their serum enzymes display respectively partial or complete lability to heat. It is proposed that the proband is a double heterozygote for the Sandhoff allele and for an allele producing thermolabile beta subunits.