Expression of the beta-hexosaminidase alpha subunit gene with the four-base insertion of infantile Jewish Tay-Sachs disease.

One of the two mutations responsible for the classical infantile Jewish form of Tay-Sachs disease is a four-base insertion in exon 11 of the beta-hexosaminidase alpha subunit gene. The gene is known to be transcribed normally, but the mRNA is essentially undetectable. It is not clear why such a relatively minor abnormality results in complete failure to generate stable mRNA. The four-base insertion was introduced into the normal beta-hexosaminidase cDNA by site-directed mutagenesis. When COS-1 cells were transfected with the resultant mutant cDNA, it generated stable mRNA and a truncated, relatively stable but catalytically inactive enzyme protein. The mutant enzyme protein was not processed nor released into the culture medium. The mutant cDNA also generated the truncated enzyme protein in an in vitro translation system with rabbit reticulocyte lysate. COS-1 cells transfected with a 3' end of the gene segment, from intron 8 through the 3' terminus, generated processed RNA of approximately 2 kilobases, the size expected from normal splicing, irrespective of presence or absence of the four-base insertion in exon 11. These results indicate that the four-base insertion does not destabilize properly spliced mRNA, nor does it interfere with normal splicing of the transcript, at least in the expression system utilized. If the four-base insertion is responsible for the undetectable mRNA in the mutant cells, it must interfere with some other steps in the processing/splicing/transport of the primary transcript yet to be examined. On the other hand, the possibility cannot be excluded definitively that another still unidentified abnormality in the same allele might be responsible for the nearly complete absence of mRNA.