It is now widely accepted that aberrant splicing of constitutive exons is often caused by mutations affecting cis-acting splicing regulatory elements, but there is a misconception that all exons have an equal dependency on SREs and thus a similar susceptibility to aberrant splicing.

We investigated exonic mutations in ACADM exon 5 to experimentally examine their effect on splicing and found that seven out of eleven tested mutations affected exon inclusion, demonstrating that this constitutive exon is particularly vulnerable to exonic splicing mutations. Employing ACADM exon 5 and 6 as models, we demonstrate that the balance between splicing enhancers and silencers, flanking intron length, and flanking splice site strength are important factors that determine exon definition and splicing efficiency of the exon in question.

Our study shows that two constitutive exons in ACADM have different inherent vulnerabilities to exonic splicing mutations. This suggests that in silico prediction of potential pathogenic effects on splicing from exonic mutations may be improved by also considering the inherent vulnerability of the exon. Moreover, we show that SNPs that affect either of two different exonic splicing silencers, located far apart in exon 5, all protect against both immediately flanking and more distant exonic splicing mutations.

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