Spectrum of splicing errors caused by CHRNE mutations affecting introns and intron/exon boundaries

Abstract

Background: Mutations in CHRNE, the gene encoding the muscle nicotinic acetylcholine receptor e 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 GRT substitution at the 39 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 39 splice sites (g.IVS10-9_c.1167dup16), results in silencing of the downstream 39 splice site. This conforms to the scanning model of recognition of the 39 splice site, which predicts that the first "ag" occurring after the branch point is selected for splicing.

Molecular characterisation of congenital myasthenic syndromes in Southern Brazil

“…Other previously published CHRNE mutations detected in our patients are: CHRNE c.1293insG,5 found in four patients from two families and CHRNE p.R286M6 detected homozygously in two patients from two kinships.…”

Natural Variation in the Splice Site Strength of a Clock Gene and Species-Specific Thermal Adaptation

“…Importantly, a suboptimal base at position +6 can be offset by an optimal base at position -1, and vice-versa (Carmel et al, 2004). Indeed, analysis of human splicing mutations in position -1, support the idea that a mismatch at this position can be compensated for by matches at positions +3 to +6, especially at position +6 (Ohno et al, 2005). Thus, although both dmpi8 and dyp3’ introns are flanked by an A at position -1, the T at position +6 of dyp3’ likely compensates, contributing to the more efficient and temperature independent splicing of dyp3’ in D. yakuba .…”

Identification of previously unreported mutations in CHRNA1, CHRNE and RAPSN genes in three unrelated Italian patients with congenital myasthenic syndromes

“…3). Both mutations are located on the TPR region of rapsyn which is important for self-association [4,5]; and while the N88K mutation affects AChR clustering [9], the splice mutation is likely to affect exon splicing, resulting in skipping of exon 1 or retention of intron 1, and hence leading to truncated rapsyn polypeptide and AChR deficiency [11]. A muscle biopsy of patient 3 was not available to determine how this mutation effects rapsyn RNA splicing.…”