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Tay-Sachs disease is an autosomal recessive disorder affecting the central nervous system. The disorder results from mutations in the gene encoding the alpha-subunit of beta-hexosaminidase A, a lysosomal enzyme composed of alpha and beta polypeptides. Seventy-eight mutations in the Hex A gene have been described and include 65 single base substitutions, one large and 10 small deletions, and two small insertions. Because these mutations cripple the catalytic activity of beta-hexosaminidase to varying degrees, Tay-Sachs disease displays clinical heterogeneity. Forty-five of the single base substitutions cause missense mutations; 39 of these are disease causing, three are benign but cause a change in phenotype, and three are neutral polymorphisms. Six nonsense mutations and 14 splice site lesions result from single base substitutions, and all but one of the splice site lesions cause a severe form of Tay-Sachs disease. Eight frameshift mutations arise from six deletion- and two insertion-type lesions. One of these insertions, consisting of four bases within exon 11, is found in 80% of the carriers of Tay-Sachs disease from the Ashkenazi Jewish population, an ethnic group that has a 10-fold higher gene frequency for a severe form of the disorder than the general population. A very large deletion, 7.5 kilobases, including all of exon 1 and portions of DNA upstream and downstream from that exon, is the major mutation found in Tay-Sachs disease carriers from the French Canadian population, a geographic isolate displaying an elevated carrier frequency. Most of the other mutations are confined to single pedigrees. Identification of these mutations has permitted more accurate carrier information, prenatal diagnosis, and disease prognosis. In conjunction with a precise tertiary structure of the enzyme, these mutations could be used to gain insight into the structure-function relationships of the lysosomal enzyme.

We describe a partially automated DNA mutation assay for detecting the most frequent mutations in the alpha-subunit of beta-hexosaminidase A, the acid beta-glucosidase and the cystic fibrosis transmembrane conductance regulator genes for the Ashkenazi Jewish population. The assay detects carriers for Tay-Sachs disease, Gaucher disease, and cystic fibrosis with sensitivities of at least 92%, 96%, and 97%, respectively. Among 1,364 young adults of Ashkenazic ancestry in the Dor Yeshurim community who were tested, 52 were Tay-Sachs carriers, 110 were Gaucher carriers, and 62 were cystic fibrosis carriers. Ten individuals were carriers for two diseases, and three unsuspected cases were diagnosed with Gaucher disease based on mutation test results. In addition to Tay-Sachs mutation data, results for hexosaminidase A activity were also available. All of 1,254 samples normal by enzyme quantitation were also negative for the three alpha-subunit mutations tested, and all of 43 samples with 'inconclusive' enzyme results were negative by DNA. Only 52 of 67 samples positive by enzyme assay were also positive for one of the three mutations tested for Tay-Sachs disease. The data suggest a high degree of false positivity inherent in enzyme identification of carriers. There are no correlative methods to assess the sensitivity of Gaucher and CF carrier testing. The results show that population screening can be carried out efficiently by DNA analysis, with the accrual of carrier information for three separate diseases conducted as a single test. Furthermore, the DNA method for Tay-Sachs screening appears to exceed the specificity of hexosaminidase A enzyme testing.

Tay-Sachs disease (TSD) is an autosomal recessive genetic disorder resulting from mutation of the HEXA gene encoding the alpha-subunit of the lysosomal enzyme, beta-N-acetylhexosaminidase A (Hex A). We have discovered that a Tay-Sachs mutation, IVS-9 + 1 G-->A, first detected by Akli et al. (Genomics 11:124-134, 1991), is a common disease allele in non-Jewish Caucasians (10/58 alleles examined). A PCR-based diagnostic test, which detects an NlaIII site generated by the mutation, revealed a frequency among enzyme-defined carriers of 9/64 (14%). Most of those carrying the allele trace their origins to the United Kingdom, Ireland, or Western Europe. It was not identified among 12 Black American TSD alleles or in any of 18 Ashkenazi Jewish, enzyme-defined carriers who did not carry any of the mutations common to this population. No normally spliced RNA was detected in PCR products generated from reverse transcription of RNA carrying the IVS-9 mutation. Instead, the low levels of mRNA from this allele were comprised of aberrant species resulting from the use of either of two cryptic donor sites, one truncating exon 9 and the other within IVS-9, spliced to exon 10. Numerous additional splice products were detected, most involving skipping of one or more surrounding exons. Together with a recently identified allele responsible for Hex A pseudodeficiency (Triggs-Raine et al. Am J Hum Genet, 1992), these two alleles accounted for almost 50% (29/64) of TSD or carrier alleles ascertained by enzyme screening tests in non-Jewish Caucasians.

Following the birth of two infants with Tay-Sachs disease (TSD), a non-Jewish, Pennsylvania Dutch kindred was screened for TSD carriers using the biochemical assay. A high frequency of individuals who appeared to be TSD heterozygotes was detected (Kelly et al., 1975). Clinical and biochemical evidence suggested that the increased carrier frequency was due to at least two altered alleles for the hexosaminidase A alpha-subunit. We now report two mutant alleles in this Pennsylvania Dutch kindred, and one polymorphism. One allele, reported originally in a French TSD patient (Akli et al., 1991), is a GT-->AT transition at the donor splice-site of intron 9. The second, a C-->T transition at nucleotide 739 (Arg247Trp), has been shown by Triggs-Raine et al. (1992) to be a clinically benign "pseudodeficient" allele associated with reduced enzyme activity against artificial substrate. Finally, a polymorphism [G-->A (759)], which leaves valine at codon 253 unchanged, is described.

Although reduced acid beta-glucosidase activity appears to be the primary enzyme defect in type I Gaucher disease, patients with this disorder also have marked elevation of serum acid phosphatase and beta-hexosaminidase activities but with a normal level of lactic dehydrogenase activity. Moreover, there is a characteristic alteration in the hexosaminidase isozyme distribution with a striking increase in hexosaminidase B. Since these changes appear to be consistent and unlike those associated with other disorders or the hormonally induced alterations associated with pregnancy, routine serum testing for the Tay-Sachs carrier state may offer a useful approach for the presumptive diagnosis and screening for Gaucher disease. Unlike the changes in affected homozygotes, there are no characteristic alterations of acid phosphatase or hexosaminidase in heterozygotes for Gaucher disease.

Carriers of an inborn error of lysosomal catabolism can be recognized, as they have enzyme levels approximately half those of normal individuals. Of the various tissues readily available for assay, plasma and leukocytes and, in some situations, tears are preferred. Although mixed leukocytes have proved satisfactory in Tay-Sachs screening programs, purified preparations of granulocytes or lymphocytes will allow better discrimination in most situations. Enzymes are assayed relative to some other reference parameter which must be a constant or highly correlated with test enzyme activity. In the two mass screening programs in operation, beta-hexosaminidase A and alpha-mannosidase have both been assayed relative to total beta-hexosaminidase activity. Carrier detection is particularly important in X-linked diseases. The techniques used mostly involve hair roots or fibroblasts and depend on random inactivation of the X chromosome. In the mucolipidoses II and III, in which there are a number of deficient enzymes in cells, carriers may be identified on the basis of the ratio of beta-hexosaminidase I1 and I2 to total hexosaminidase.

The diagnostic usefulness of sulfated fluorogenic substrates in carrier detection of Tay-Sachs disease in serum during pregnancy was assessed by testing coded samples. Gradual increase in serum hexosaminidase activities toward these substrates was observed throughout pregnancy in both carrier and non-carriers of the Tay-Sachs gene, but absolute discrimination between the 2 genotypes could not be achieved even when values were compared within the same gestational age. Examination of isolated isozyme fractions with the sulfated substrates showed that the increased activities during pregnancy were due to a genuine increase in hexosaminidase A and not associated with the elevation of hexosaminidase I (or P), which was evident only with unsulfated substrates. The extent of the increase was influenced by the genotype of the fetus as indicated by higher values in pregnant carriers who carried non-carrier fetuses. We conclude that determination of serum hexosaminidase A during pregnancy by sulfated fluorogenic substrates may have a prenatal diagnostic value when used in obligate heterozygotes for Tay-Sachs disease, but is unreliable for screening purposes.