Impact of premature stop codons on mRNA levels in infantile sandhoff disease

Sandhoff disease is an autosomal recessive lysosomal storage disease resulting from mutations of the HEXB gene encoding theβ subunit of β-hexosaminidase A. Fibroblast lines from four patients with the infantile form of the disease were investigated for mutations by single strand conformation polymorphism analysis and direct sequencing of PCR products. Two of the cell lines were homozygous for a common, 16 kb deletion of the 5' end of HEXB gene. The two other cell lines contained the 16 kb deletion along with a second mutant allele generating a stop codon: In one case a nonsense mutation, C₈₅₀-T, which generated a stop codon at codon 284; and in the other, a single base deletion, ΔT1344, which generated a stop codon at codon 451. One additional cell line investigated was a compound heterozygote for two frameshift mutations,ΔG₇₇₄in exton 7 and ΔAG_1305-1306 In exon 11 (McInnes et al. 1992, Biochim. BIophys. Acta 1138: 315-317). Stop codons were generated in this cell line at codons 274 and 454, respectively. We took advantage of these genotypes to investigate the steady-state level of mRNA produced by cells containing stop codons using a competitive polymerase chain reaction technique. The mRNA levels were, as percent of normal per single gene dose: for the stop codon at codon 451, 30% for those at codons 274 and 454, combined percentage of 1.7%; and at codon 284, 0.8%. These studies demonstrate a dramatic difference In the steady-state level of Hexβ mRNA In the cell lines with stop codons in close proximity to each other (codons 451 vs 454). The difference in mRNA level could not be accounted for by second site mutations of by alternative splicing of the sequence. It is possible that a specific sequence in the mRNA, normally present or created by the mutation, is particularly sensitive (or reslstant) to degradation when exposed through premature termination.