Abstract
Mutations in RPGRIP1 are associated with early onset retinal degenerations in humans and dogs. Dogs homozygous for a 44 bp insertion including a polyA29 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.ObjectiveWe 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.MethodsWe analysed alternate start sites and splicing of RPGRIP1 transcripts; variability of polyAn length in the insertion and slippage at polyAn during transcription/translation.Results and SignificanceWe 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 polyAn length was confirmed in DNA from different RPGRIP1 −/− dogs, yet polyAn variability did not correspond with the clinical phenotypes and no individual was found that carried a polyAn 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.
Highlights
Leber congenital amaurosis type 6 (LCA6) is a retinal dystrophy causing profound vision loss, often from birth, nystagmus and sometimes unrecordable electroretinogram (ERG)
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 Retinitis Pigmentosa GTPase Regulator Interacting Protein 1 (RPGRIP1) protein isoforms
Variable polyAn length was confirmed in DNA from different RPGRIP12/2 dogs, yet polyAn variability did not correspond with the clinical phenotypes and no individual was found that carried a polyAn tract capable of encoding an in-frame variant
Summary
Leber congenital amaurosis type 6 (LCA6) is a retinal dystrophy causing profound vision loss, often from birth, nystagmus and sometimes unrecordable electroretinogram (ERG) It is associated with homozygous (or compound heterozygous) nonsense or missense mutations in the Retinitis Pigmentosa GTPase Regulator Interacting Protein 1 (RPGRIP1) [1], [2], [3]. Patients with homozygous null mutations show profound loss of retinal function from early childhood, for some patients with missense mutations, vision is retained at birth but progressively lost in the first two decades (human cone-rod dystrophy type 13, CORD13) [4]. The disease in the colony segregated as autosomal recessive, showing association with an exonic insertion mutation in RPGRIP1 [8] This mutation is a polyA tract insertion of 29 nucleotides flanked by a 15 bp duplication. This was expected to lead to a change of reading frame and a stop codon early in the following exon of the RPGRIP1 gene, and truncation of the encoded protein
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