title: Rapid nonradioactive tracer method for detecting carriers of the major Ashkenazi Jewish Tay-Sachs disease mutations.
abstract: Tay-Sachs disease (TSD, GM2 gangliosidosis, Type I) is an autosomal recessive lysosomal storage disease caused by deficiency of beta-hexosaminidase A (Hex A) resulting from mutations in the gene (HEXA) encoding the alpha-subunit of the enzyme. Three mutations, in exons 7 and 11 and at the exon 12-intron 12 junction, account for > 90% of alleles identified in obligate Ashkenazi Jewish carriers. Mutation analysis requires amplification of available DNA by separate polymerase chain reactions (PCRs) and either restriction digestion and gel electrophoresis or 32P-labeled allele-specific oligonucleotide (ASO) probes. We developed a simple, nonradioisotopic method for rapidly identifying TSD carriers by a triplex PCR reaction followed by dot-blot analysis, using three wild-type and three mutant ASOs end-labeled with digoxigenin-dUTP (dig-ASO). Hybridization was demonstrated immunologically by reaction with an anti-digoxigenin-alkaline phosphatase conjugate followed by colorimetric demonstration of phosphatase activity. The results of analyses by the dig-ASO method of 65 carriers identified by serum enzyme activity and of 6 high-risk fetuses in prenatal testing were the same as those obtained by more conventional restriction analysis. Dig-ASO testing correctly reclassified 10 individuals who had tested inconclusively on analysis for leukocyte beta-hexosaminidase A activity; 3 were identified as carriers and 7 as noncarriers. The simplicity of the assay and the avoidance of the radioisotopes make this a potentially useful method for TSD carrier detection by mutation analysis in Ashkenazi Jews from populations in whom the identity and frequencies of the common TSD mutations are known.
authors.affiliation: Department of Pediatrics, Hospital for Sick Children, Toronto, Ontario, Canada.
authors.forename: P M
authors.initials: PM
authors.lastname: Strasberg
authors.forename: J T
authors.initials: JT
authors.lastname: Clarke
mesh_terms: D003124:Colorimetry; D004247:DNA / Q000235:genetics; D003856:Deoxyuracil Nucleotides; D004076:Digoxigenin / Q000031:analogs & derivatives; D005091:Exons; D005260:Female; D006580:Genetic Carrier Screening / Q000379:methods*; D005820:Genetic Testing / Q000379:methods*; D054818:Hexosaminidase A; D006801:Humans; D007124:Immunoenzyme Techniques; D007202:Indicators and Reagents; D007438:Introns; D007585:Jews; D009154:Mutation; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011296:Prenatal Diagnosis; D013661:Tay-Sachs Disease / Q000175:diagnosis / Q000235:genetics*; D001619:beta-N-Acetylhexosaminidases / Q000172:deficiency / Q000235:genetics
chemical_list: D003856:Deoxyuracil Nucleotides; D007202:Indicators and Reagents; D015345:Oligonucleotide Probes; C070202:digoxigenin-11-deoxyuridine triphosphate; D004247:DNA; D054818:Hexosaminidase A; D001619:beta-N-Acetylhexosaminidases; D004076:Digoxigenin