title: A glycine250--> aspartate substitution in the alpha-subunit of hexosaminidase A causes juvenile-onset Tay-Sachs disease in a Lebanese-Canadian family.
abstract: The mutation causing juvenile Tay-Sachs disease (TSD) in two sibs of Lebanese-Maronite origin is described. An mRNA-containing extract of cultured fibroblasts obtained from one of the probands was used as a template to amplify the coding sequence of the hexosaminidase A (Hex A) alpha-subunit. Sequencing of amplified cDNA fragments revealed a single alteration, guanine to adenine at nt 749 creating a G250D mutation. The mutation introduces a new recognition site for the restriction enzyme Eco RV, permitting identification of heterozygotes for this allele following PCR amplification and Eco RV digestion of exon 7 sequences from genomic DNA templates. In order to test the effect of this substitution, an in vitro mutagenized cDNA construct was introduced into a mammalian expression vector and transfected into monkey Cos-1 cells separately or along with a beta-cDNA expression vector. When the mutant alpha-cDNA was the only gene introduced into COS cells no enzymatic activity above endogenous COS cell activity was detected. Cotransfection of normal alpha-cDNA and beta-cDNA followed by immunoprecipitation of human Hex A resulted in 20-fold increase in the ratio between positive and negative (mock transfection) control values. This allowed the detection of some residual activity (12% of the positive control) when the mutant alpha-cDNA replaced its wild-type counterpart. The predicted protein environment in which the mutation occurs is compared to that of the adult-onset Tay-Sachs disease mutation caused by a Gly269-->Ser substitution in exon 7.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: DeBelle Lab for Biochemical Genetics, McGill University Montreal Children's Hospital Research Institute, Quebec, Canada.
authors.forename: I
authors.initials: I
authors.lastname: Trop
authors.forename: F
authors.initials: F
authors.lastname: Kaplan
authors.forename: C
authors.initials: C
authors.lastname: Brown
authors.forename: D
authors.initials: D
authors.lastname: Mahuran
authors.forename: P
authors.initials: P
authors.lastname: Hechtman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid; D001483:Base Sequence; D002170:Canada; D002460:Cell Line; D002522:Chlorocebus aethiops; D003241:Consanguinity; D005260:Female; D005998:Glycine; D054818:Hexosaminidase A; D006801:Humans; D007668:Kidney; D007861:Lebanon / Q000208:ethnology; D046911:Macromolecular Substances; D008297:Male; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009838:Oligodeoxyribonucleotides; D010375:Pedigree; D016133:Polymerase Chain Reaction / Q000379:methods; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013661:Tay-Sachs Disease / Q000201:enzymology / Q000235:genetics*; D014162:Transfection; D001619:beta-N-Acetylhexosaminidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D001224:Aspartic Acid; D054818:Hexosaminidase A; D001619:beta-N-Acetylhexosaminidases; D005998:Glycine