Abstract

Background: Mutations in CHRNE, the gene encoding the muscle nicotinic acetylcholine receptor ε subunit, cause congenital myasthenic syndromes. Only three of the eight intronic splice site mutations of CHRNE reported to date have had their splicing consequences characterised.

Methods: We analysed four previously reported and five novel splicing mutations in CHRNE by introducing the entire normal and mutant genomic CHRNEs into COS cells.

Results and conclusions: We found that short introns (82–109 nucleotides) favour intron retention, whereas medium to long introns (306–1210 nucleotides) flanking either or both sides of an exon favour exon skipping. Two mutations are of particular interest. Firstly, a G→T substitution at the 3′ end of exon 8 predicts an R286M missense mutation, but instead results in skipping of exon 8. In human genes, a mismatch of the last exonic nucleotide to U1 snRNP is frequently compensated by a matching nucleotide at intron position +6. CHRNE intron 8 has a mismatch at position +6, and accordingly fails to compensate for the exonic mutation at position –1. Secondly, a 16 bp duplication, giving rise to two 3′ splice sites (g.IVS10-9_c.1167dup16), results in silencing of the downstream 3′ splice site. This conforms to the scanning model of recognition of the 3′ splice site, which predicts that the first "ag" occurring after the branch point is selected for splicing.