Multiple mechanisms contribute to leakiness of a frameshift mutation in canine cone-rod dystrophy

Mutations in RPGRIP1 are associated with early onset retinal degenerations in humans and dogs. Dogs homozygous for a 44 bp insertion including a polyA(29) tract potentially leading to premature truncation of the protein, show cone rod degeneration. This is rapid and blinding in a colony of dogs in which the mutation was characterised but in dogs with the same mutation in the pet population there is very variable disease severity and rate of progression.

We hypothesized that this variability must be associated with leakiness of the RPGRIP1 mutation, allowing continued RPGRIP1 production. The study was designed to discover mechanisms that might allow such leakiness.

We analysed alternate start sites and splicing of RPGRIP1 transcripts; variability of polyA(n) length in the insertion and slippage at polyA(n) during transcription/translation.

We observed a low rate of use of alternative start codons having potential to allow forms of transcript not including the insertion, with the possibility of encoding truncated functional RPGRIP1 protein isoforms. Complex alternative splicing was observed, but did not increase this potential. Variable polyA(n) length was confirmed in DNA from different RPGRIP1(-/-) dogs, yet polyA(n) variability did not correspond with the clinical phenotypes and no individual was found that carried a polyA(n) tract capable of encoding an in-frame variant. Remarkably though, in luciferase reporter gene assays, out-of-frame inserts still allowed downstream reporter gene expression at some 40% of the efficiency of in-frame controls. This indicates a major role of transcriptional or translational frameshifting in RPGRIP1 expression. The known slippage of reverse transcriptases as well as RNA polymerases and thermostable DNA polymerases on oligoA homopolymers meant that we could not distinguish whether the majority of slippage was transcriptional or translational. This leakiness at the mutation site may allow escape from severe effects of the mutation for some dogs.