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Human Molecular Genetics Advance Access published online on October 7, 2003
Human Molecular Genetics, doi:10.1093/hmg/ddg334
© 2003 by Oxford University Press
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©2003 Oxford University Press

Article

A frameshifting mutation in CHRNE unmasks skipping of the preceding exon

Kinji Ohno 1*, Margherita Milone 1, Xin-Ming Shen 1, and Andrew G. Engel 1

1 Department of Neurology and Neuromuscular Research Laboratory, Mayo Clinic, Rochester, MN

* To whom correspondence should be addressed. E-mail: ohnok{at}mayo.edu.


  Abstract

A frameshifting 7-bp deletion ({varepsilon}553del7) in exon 7 of CHRNE encoding the acetylcholine receptor {varepsilon} subunit, observed in seven congenital myasthenic syndrome patients, enhances expression of an aberrantly spliced transcript that skips the preceding 101-bp exon 6. To recapitulate the aberrant splicing, we cloned the entire CHRNE spanning 12 exons and 11 introns and expressed it in COS cells. Scanning mutagenesis revealed that {varepsilon}553del7 does not disrupt an exonic splicing enhancer. Inhibition of protein synthesis and of nonsense-mediated mRNA decay (NMD) by anisomycin shows that even wild-type CHRNE produces an exon 6-skipped transcript, and that even {varepsilon}553del7-CHRNE yields a normally spliced transcript. Both transcripts, however, are likely degraded by NMD due to a premature stop codon. In contrast, the normally spliced transcript from wild-type CHRNE and the exon 6-skipped transcript from {varepsilon}553del7-CHRNE carry no premature stop codon and hence are immune to NMD. Optimization of splicing signals for exon 6 prevents it being skipped even in the presence of anisomycin and/or {varepsilon}553del7, indicating that inherently weak splicing signals for exon 6 account for its skipping. We suggest that a similar mechanism likely operates in other genes in skipping of remote exons. Presence of weak splicing signals for exon 6 also prompted us to search for mutations in exon 6 that disrupt an exonic splicing enhancer. Indeed, we found that {varepsilon}EF157V and {varepsilon}E154X in exon 6, observed in two other patients, caused aberrant splicing of exon 6.


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