Fig. 2. Types of pathogenic splice aberrations discovered in patients.

RNA-seq identified a range of aberrations caused by both coding and noncoding variants, such as (A) exon skipping caused by an essential splice site variant in patient D7, (B) exon extension caused by a donor +3 A>C extended splice site variant in nemaline myopathy patient C9 (where disruption of splicing at the canonical splice site results in splicing from intact GTA motifs from the intron), (C) exonic splice gain caused by a C>T donor splice site–creating variant in patient N22 with a donor +5-G sequence context, resulting in a stronger splice motif than the existing canonical splice site, and (D) intronic splice gain in patient N33 caused by a C>T donor splice site–creating deep intronic variant. Evidence for wild-type splicing in addition to the inclusion of the pseudoexon in the patient is in line with the milder Becker’s muscular dystrophy phenotype. Splice aberrations shown in (B) to (D) result in the introduction of a premature stop codon to the transcript.