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 hexoaminidase A (Hex A) a-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 P-cDNA expression vector.
When the mutant a-cDNA was the only gene introduced into COS cells no enzymatic activity above endogenous COS cell activity was detected. Cotransfection of normal a-cDNA and P-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 a-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 GIyza9 -+ Ser substitution in exon 7. We suggest that the more severe clinical phenotype linked to the Gly,,, + Asp mutation may be explained by the less conservative substitution of the large, charged aspartate residue as compared to the small neutral serine, and the placement of the Glyzso in a hydrophobic section of an a-helix.