Two mutations remote from an exon/intron junction in the beta-hexosaminidase beta-subunit gene affect 3 '-splice site selection and cause Sandhoff disease

M. Fujimaru et al., Two mutations remote from an exon/intron junction in the beta-hexosaminidase beta-subunit gene affect 3 '-splice site selection and cause Sandhoff disease, HUM GENET, 103(4), 1998, pp. 462-469
Citations number
Categorie Soggetti
Molecular Biology & Genetics
Journal title
ISSN journal
0340-6717 → ACNP
Year of publication
462 - 469
SICI code
Four unrelated Japanese patients with infantile Sandhoff disease (beta-hexo saminidase beta-subunit deficiency) have been studied for the molecular bas is of their severe phenotype. Two patients had complex base substitutions; one patient was homoallelic for a triple mutation (P417L, K121R, and S255R) and the other was a compound heterozygote of a double (P417L and K121R) mu tation and the triple mutation. K121R is known to be a functional polymorph ism, while P417L (exon Il, +8 C-->T)generates predominantly an abnormally s pliced mRNA at base +112 of exon II and has been described in two patients with a juvenile form of the disease. The mild phenotype is attributed to th e presence of a small amount of normally spliced mRNA. S255R is a novel mut ation without prior description in the literature. An expression study of t he normally spliced cDNA with the double and the triple mutations gave abou t 70% and 30% of normal activity, respectively. This finding suggests that S255R further reduces the catalytic activity of the already below-threshold amount of normally spliced mRNA and accounts for the more severe phenotype in our patients. In the other two patients, a novel disease-causing base t ransition was found within intron 10, away from the intron/exon junction (- 17 a-->g). This mutation caused abnormal 3' splicing at position -37 of int ron 10, and no normally spliced product was detectable upon RT-P(SR analysi s. We noted an unusually low splice site score (61.8) for the exon 10/intro n Il junction and suspected that this might be partially responsible for th e aberrant splicing in these mutations. To test this hypothesis, we constru cted four chimeric cDNAs all with an additional intron 10 inserted and eval uated their splicing efficiency. They, respectively, had the normal sequenc e, P417L (exon 11, +8 C-->T), the intronic mutation (-17 a-->g), and the in tronic mutation with an artificially engineered intron 10/exon 11 junction of a higher splice site score (85.1). Of the total transcripts, 67% and 32% were correctly spliced in the normal chimeric construct and P417L, respect ively, while no normally spliced product was generated either in the chimer ic construct with -17 a-->g or in that with a high splice site score. The s equence around the adenosine -17 residue upstream of the normal acceptor sp lice site in this report, UGCAAU (-21 to -16), matches the consensus branch point sequence YNYRAY (Y, pyrimidine; R, purine; N, any base) reported in t he literature. The mutation in this study is most likely to abolish lariat formation because the artificial site of the high splice site score did not improve splicing efficiency.