Molecular epidemiology of Tay-Sachs disease in Europe

The abnormalities in the gene coding for the β-hexosaminidase a 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 infantile Tay-Sachs disease). After first strand synthesis by random priming, PCR was used to amplify in two overlapping fragments (868 and 949 bp) the entire coding region. These amplified products were first studied for changes in size by agarose gel electrophoresis to screen for splicing mutations leading to exon skipping or cryptic splice site activation. For each patient, the two overlapping cDNA fragments were subjected to chemical mismatch cleavage analysis using hydroxylamine to modify C-containing mismatches and osmium tetroxide to modify T-containing mismatches. DGGE was used to screen for mutations in the coding region spanning exon 2 to exon 6, a region putatively encompassing the active site and therefore a potential hot spot of mutations associated with Tay-Sachs disease. To increase the sensitivity of the technique, a 30 by GC-clamp has been added at the 5′ end of the sense oligonucleotide to amplify a fragment of 629 bp. The computerized analysis found that single base changes in domain spanning from nt 313 to nt 693 can be distinguished. Fragments displaying an altered melting behavior or a cleaved product were further analysed by direct sequencing of the amplified material. These methods as a whole allowed us to identify 30/38 alleles studied (79%) with 15 point mutations and one 4 by insertion detected. Thirteen alleles (34%) were known mutations with exon 1I insertion and Gly²⁶⁹ → Ser mutations accounting for four alleles each. Seventeen alleles (45%) carried 14 different novel mutations (two splicing and 12 missense mutations). The region scanned by DGGE contained seven out of the 30 identified alleles (23%) of which six carried novel mutations. The novel Tay-Sachs mutations detected in this study increase the number of mutant alleles identified in our laboratory to 22. These results provide further insight into the molecular heterogeneity of Tay-Sachs disease.