title: A frameshifting mutation in CHRNE unmasks skipping of the preceding exon.
abstract: A frameshifting 7 bp deletion (epsilon553del7) in exon 7 of CHRNE encoding the acetylcholine receptor epsilon 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 epsilon553del7 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 epsilon553del7-CHRNE yields a normally spliced transcript. Both transcripts, however, are 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 epsilon553del7-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 epsilon553del7, indicating that inherently weak splicing signals for exon 6 account for its skipping. We suggest that a similar mechanism probably operates in other genes in skipping of remote exons. The 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 epsilonEF157V and epsilonE154X in exon 6, observed in two other patients, caused aberrant splicing of exon 6.
authors.affiliation: Department of Neurology, Mayo Clinic, Rochester, MN, USA. ohnok@mayo.edu
authors.forename: Kinji
authors.initials: K
authors.lastname: Ohno
authors.forename: Margherita
authors.initials: M
authors.lastname: Milone
authors.forename: Xin-Ming
authors.initials: XM
authors.lastname: Shen
authors.forename: Andrew G
authors.initials: AG
authors.lastname: Engel
mesh_terms: D000293:Adolescent; D000328:Adult; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002648:Child; D002522:Chlorocebus aethiops; D002886:Chromosomes, Human, Pair 17; D004252:DNA Mutational Analysis; D005091:Exons; D005260:Female; D016368:Frameshift Mutation; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D020294:Myasthenic Syndromes, Congenital / Q000235:genetics*; D010375:Pedigree; D012326:RNA Splicing; D011978:Receptors, Nicotinic / Q000235:genetics*; D017384:Sequence Deletion
chemical_list: D011978:Receptors, Nicotinic