Multimodal identification of skippable exons in CEP120and CC2D2A and mapping of reported truncating mutations
The CEP120 transcript ENST00000328236 contains 20 coding exons. Among them, the nucleotide length of 11 exons is a multiple of three and therefore amenable to exon skipping without change in reading frame (Figure 4A). Considering the location of protein domains of functional importance (coiled-coil and C2 domains), only exons 14 and 15 are predicted to be skippable without inducing loss of protein function. As codons are not overlapping between exons (phase 0), skipping of exons 14 and 15 will not lead to potential amino acid substitutions. Based on the GTEx alternative splicing data presented above, exon 2 (predicted to be skipped in kidney, see transcript ENST00000306481) might represent an additional target for tolerated exon skipping with an alternative start codon functional in exon 3. However, none of the reported CEP120mutations to date fall in either one of these identified exons, suggesting that CEP120 , at the current state of knowledge, is not a good candidate gene to apply exon skipping therapies.
The CC2D2A transcript ENST00000503292 contains 36 coding exons, 17 of which are amenable to exon skipping without change in reading frame (Figure 4B). Considering the location of protein domains of functional importance (coiled-coil and C2 domains) (Bachmann-Gagescu et al., 2012; Noor et al., 2008) exons 4, 7, 8, 9, 12, 13, 22, 23, 31, 32, 33, 34 and 35 might be skippable without inducing loss of protein function. These exons all contain full complements of codons (phase 0) and can therefore be skipped without introducing potential amino acid substitutions. Based on the data presented above, exon 30 appears as another promising candidate for exon skipping. Furthermore, exon 30 is predicted to encode only the last 3 amino acids of the C2 domain or to have no overlap with the C2 domain at all (Bachmann-Gagescu et al., 2012; Gorden et al., 2008; Noor et al., 2008; Srour et al., 2012). Prediction tools and available literature provide conflicting data with respect to C2 domain overlap with exon 25, indicating the need for functional studies confirming this potential exon skipping target (http://smart.embl-heidelberg.de/)(Bachmann-Gagescu et al., 2012; Noor et al., 2008; Srour et al., 2012). Because of their clear pathogenic implications, we focus on truncating variants as potential targets for exon skipping approaches. Table 2 lists the reported patients harbouring at least one truncating variants, in any of theCC2D2A skippable exons. Furthermore, all CC2D2A truncating variants falling into any of the predicted skippable exons are represented in Figure 4B. 14 different truncating variants represent potential targets for exon skipping and are located in exons 8, 13, 22, 23, 25, 30 and 31 (Figure 4B). Four of these truncating variants have been described in homozygosis: c.517C>T (p.Arg173Ter, exon 8), c.2848C>T (p.Arg950Ter, exon 23), c.2875del (p. Glu959AsnfsTer3, exon 23) and c.3084del (p. Lys1029ArgfsTer3, exon 25). Skipping of each of the 7 identified exons that harbour truncating variants and are potentially tolerant to skipping is leading to a predicted near-full length protein product (Figure S4).