MOLECULAR EPIDEMIOLOGY OF TAY-SACHS-DISEASE IN EUROPE

The abnormalities in the gene coding for the beta-hexosaminidase alpha subunit were analysed from fibroblast's RNAs of 42 Tay-Sachs patients (seven with adult or late onset of Tay-Sachs disease and 35 with infa ntile Tay-Sachs disease). After first strand synthesis by random primi ng, PCR was used to amplify in two overlapping fragments (868 and 949 bp) the entire coding region. These amplified products were first stud ied for changes in size by agarose gel electrophoresis to screen for s plicing mutations leading to exon skipping or cryptic splice site acti vation. For each patient, the two overlapping cDNA fragments were subj ected to chemical mismatch cleavage analysis using hydroxylamine to mo dify C-containing mismatches and osmium tetroxide to modify T-containi ng mismatches. DGGE was used to screen for mutations in the coding reg ion spanning exon 2 to exon 6, a region putatively encompassing the ac tive site and therefore a potential hot spot of mutations associated w ith Tay-Sachs disease. To increase the sensitivity of the technique, a 30 bp GC-clamp has been added at the 5' end of the sense oligonucleot ide to amplify a fragment of 629 bp. The computerized analysis found t hat single base changes in domain spanning from nt 313 to nt 693 can b e distinguished. Fragments displaying an altered melting behavior or a cleaved product were further analysed by direct sequencing of the amp lified material. These methods as a whole allowed us to identify 30/38 alleles studied (79%) with 15 point mutations and one 4 bp insertion detected. Thirteen alleles (34%) were known mutations with exon 11 ins ertion and Gly(269) --> Ser mutations accounting for four alleles each . Seventeen alleles (45%) carried 14 different novel mutations (two sp licing and 12 missense mutations). The region scanned by DGGE containe d seven out of the 30 identified alleles (238) of which six carried no vel mutations. The novel Tay-Sachs mutations detected in this study in crease the number of mutant alleles identified in our laboratory to 22 . These results provide further insight into the molecular heterogenei ty of Tay-Sachs disease.