A "G" to "A" mutation at position -1 of a 5' splice site in a late infantile form of Tay-Sachs disease.

Tay-Sachs disease is an autosomal recessive genetic disease caused by a deficiency in beta-hexosaminidase A. We have characterized a new mutation in a Tunisian patient displaying a late infantile form of Tay-Sachs disease. Northern blot analysis of patient's fibroblast total RNAs shows a broad, fast migrating band in the region of the normal beta-hexosaminidase alpha transcripts. The mRNA coding for beta-hexosaminidase alpha subunit was first reverse transcribed and then amplified in four overlapping segments spanning the entire coding sequence by polymerase chain reaction. We found in the products of polymerase chain reaction (PCR) that amplify the segment spanning exons 2-7, in addition to a normal fragment, two smaller size fragments, one of which is also seen in normal control fibroblasts. The analysis of the patient's specific abnormal fragment by hybridization with exon-specific oligonucleotides and then sequencing allowed us to conclude that this fragment lacked exon 5. The other smaller species lacked exons 4 and 5 in both patient and normal control. The sequence of a genomic fragment containing exon 5 and of the patient's normal cDNA fragment spanning exons 2-7, revealed a point mutation G to A at the last nucleotide of exon 5. This mutation doesn't change the sense of the affected codon. Northern blot of patient's fibroblast poly(A+) RNAs allowed us to quantify two of the forms of transcripts seen by PCR. In the patient, the normal size transcript and the exon 5-deleted transcript represent, respectively, 3 and 7% of the normal control beta-hexosaminidase alpha mRNA. We propose that this point mutation is responsible for an inefficient and abnormal processing of the mutant transcript resulting in the appearance of two low abundance spliced mRNAs. One is lacking exon 5 and most likely codes for an inactive protein; the other is similar to normal beta-hexosaminidase alpha mRNA, except for the presence of the silent G to A mutation and codes therefore for a normal enzyme accounting for the 2.5% residual beta-hexosaminidase A activity measured in patient's fibroblasts by a fluorometric assay. The third form, without exons 4 and 5, is also evidenced in normal fibroblasts by PCR so that we think that it is not related to Tay-Sachs disease.