A glycine250--> aspartate substitution in the alpha-subunit of hexosaminidase A causes juvenile-onset Tay-Sachs disease in a Lebanese-Canadian family.

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). 
