| First Author | Bacchi N | Year | 2015 |
| Journal | Invest Ophthalmol Vis Sci | Volume | 56 |
| Issue | 8 | Pages | 4846-56 |
| PubMed ID | 26218913 | Mgi Jnum | J:230951 |
| Mgi Id | MGI:5766582 | Doi | 10.1167/iovs.15-16410 |
| Citation | Bacchi N, et al. (2015) A New Splicing Isoform of Cacna2d4 Mimicking the Effects of c.2451insC Mutation in the Retina: Novel Molecular and Electrophysiological Insights. Invest Ophthalmol Vis Sci 56(8):4846-56 |
| abstractText | PURPOSE: Mutations in CACNA2D4 exon 25 cause photoreceptor dysfunction in humans (c.2406C-->A mutation) and mice (c.2451insC mutation). We investigated the feasibility of an exon-skipping therapeutic approach by evaluating the splicing patterns and functional role of targeted exons. METHODS: Splicing of the targeted alpha2delta4 (CACNA2D4) exons in presence and absence of the mutation was assessed by RT-PCR in vivo on mouse retinae and in vitro in HEK293T cells using splicing-reporter minigenes. Whole-cell patch-clamp recordings were performed to evaluate the impact of different Cacna2d4 variants on the biophysical properties of Cav1.4 L-type calcium channels (CACNA1F). RESULTS: Splicing analysis revealed the presence of a previously unknown splicing isoform of alpha2delta4 in the retina that truncates the gene open reading frame (ORF) in a similar way as the c.2451insC mutation. This isoform originates from alternative splicing of exon 25 (E25) with a new exon (E25b). Moreover, the c.2451insC mutation has an effect on splicing and increases the proportion of transcripts including E25b. Our electrophysiological analyses showed that only full-length alpha2delta4 was able to increase Cav1.4/beta3-mediated currents while all other alpha2delta4 variants did not mediate such effect. CONCLUSIONS: The designed exon-skipping strategy is not applicable because the resulting skipped alpha2delta4 are nonfunctional. alpha2delta4 E25b splicing variant is normally present in mouse retina and mimics the effect of c.2451insC mutation. Since this variant does not promote significant Cav1.4-mediated calcium current, it could possibly mediate a different function, unrelated to modulation of calcium channel properties at the photoreceptor terminals. |
