Total
RNA was isolated from cultured fibroblasts from 12 unrelated patients with
Tay-Sachs disease, an autosomal recessive disorder due to
beta-hexosaminidase A deficiency.
beta-Hexosaminidase mRNA was amplified by
cDNA-PCR in four overlapping segments spanning the entire coding sequence. In two patients, abnormal size
cDNA-PCR fragments in which exons were removed resulted from splicing mutations that were characterized at the genomic
DNA level: both were G to A transitions, at the first position of intron 2 and at the fifth position of intron 4. Five other mutations have been identified by
cDNA-PCR chemical mismatch analysis and direct sequencing of an amplified fragment containing the mismatch site. One missense mutation alters the
codon for Ser210 to Phe in exon 6 and the other one alters the
codon for Arg504 to Cys in exon 13. A 3-bp deletion results in the deletion of a
phenylalanine residue in exon 8. Two
nonsense mutations in exon 3 (Arg137 to stop) and in exon 11 (Arg393 to stop) are associated with a marked decrease of
mRNA abundance, probably because they result in mRNA instability. Three of the six single base mutations involve the conversion of a CpG dinucleotide in the sense strand to TpG. These results demonstrate the extreme molecular heterogeneity of mutations causing
Tay-Sachs disease. The procedure described in this paper allows the rapid detection of any type of mutation, except those impairing the promoter function. Applicable even to patients with splicing or
nonsense mutations and very low
mRNA abundance, it has therefore a potentially broad application in human genetics, for both diagnostic and fundamental purposes.