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Two mutations produce intron insertion in mRNA and elongated beta-subunit of human beta-hexosaminidase
1Department of Biological Chemistry, School of Medicine, University of California, Los Angeles 90024.
Abstract:
An elongated beta-subunit of the lysosomal enzyme beta-hexosaminidase was found in fibroblast strains derived from two patients with juvenile Sandhoff disease and two asymptomatic individuals sharing an unusual isoenzyme pattern: a low level of residual A (alpha beta) isoenzyme activity (3-6% of normal for the juvenile Sandhoff and 9-10% for the asymptomatic strains) without B (beta beta) isoenzyme activity. The elongated beta-subunit was abnormal in other ways: It reacted with antiserum against the unfolded polypeptide, it was not phosphorylated on mannose residues, it was not processed to the mature form, and it was degraded rapidly. The increased length of the beta-subunit was caused by two different mutations. Cells from two juvenile Sandhoff and one asymptomatic individuals had the previously described G----A transition in intron 12 that creates a splice site, causing an in-frame insertion of 24 intronic nucleotides into mRNA (Nakano, T., and Suzuki, K. (1989) J. Biol. Chem. 264, 5155-5158). The second mutation was found in cells from the asymptomatic girl whose A+B- isoenzyme pattern had been designated "Hexosaminidase Paris" (Dreyfus, J. C., Poenaru, L., Vibert, M., Ravise, N., and Boue, J. (1977) Am. J. Hum. Genet. 29, 287-293); duplication of a region straddling the junction of intron 13 and exon 14 generates an alternate splice site, causing an in-frame insertion of 18 nucleotides into mRNA. Although the two new splice sites are used preferentially, the normal sites may be used to some extent, accounting for the residual A isoenzyme activity.
Insights
Two mutations cause an elongated beta-hexosaminidase subunit, leading to Sandhoff disease or unusual isoenzyme patterns in asymptomatic individuals. These mutations affect enzyme processing and stability, explaining residual activity.
Area of Science:
- Biochemistry
- Genetics
- Molecular Biology
Background:
- Juvenile Sandhoff disease is a lysosomal storage disorder.
- Beta-hexosaminidase (Hex) has A (αβ) and B (ββ) isoenzymes.
- A+B- isoenzyme pattern indicates specific genetic defects.
Purpose of the Study:
- Investigate the molecular basis of an unusual beta-hexosaminidase subunit.
- Identify mutations causing juvenile Sandhoff disease and asymptomatic isoenzyme patterns.
- Characterize the functional consequences of identified mutations.
Main Methods:
- Fibroblast culture from patients and asymptomatic individuals.
- Analysis of beta-hexosaminidase subunit structure and processing.
- Mutation identification using DNA sequencing and splice site analysis.
Main Results:
- Identified an elongated beta-hexosaminidase subunit in affected and asymptomatic individuals.
- Two distinct mutations altering mRNA splicing were found: a G-to-A transition in intron 12 and a duplication at intron 13/exon 14.
- Mutations resulted in abnormal subunit processing, phosphorylation, and rapid degradation, with some residual A isoenzyme activity.
Conclusions:
- Novel splice site mutations in the beta-hexosaminidase gene cause disease and asymptomatic phenotypes.
- Altered splicing leads to functional defects in the beta-hexosaminidase subunit.
- Understanding these mutations aids in diagnosing and characterizing Hexosaminidase deficiencies.