A Large Animal Model for <i>CNGB1</i> Autosomal Recessive Retinitis Pigmentosa

<div><p>Retinal dystrophies in dogs are invaluable models of human disease. Progressive retinal atrophy (PRA) is the canine equivalent of retinitis pigmentosa (RP). Similar to RP, PRA is a genetically heterogenous condition. We investigated PRA in the Papillon breed of dog using homozygosity mapping and haplotype construction of single nucleotide polymorphisms within a small family group to identify potential positional candidate genes. Based on the phenotypic similarities between the PRA-affected Papillons, mouse models and human patients, <i>CNGB1</i> was selected as the most promising positional candidate gene. <i>CNGB1</i> was sequenced and a complex mutation consisting of the combination of a one basepair deletion and a 6 basepair insertion was identified in exon 26 (c.2387delA;2389_2390insAGCTAC) leading to a frameshift and premature stop codon. Immunohistochemistry (IHC) of pre-degenerate retinal sections from a young affected dog showed absence of labeling using a C-terminal CNGB1 antibody. Whereas an antibody directed against the N-terminus of the protein, which also recognizes the glutamic acid rich proteins arising from alternative splicing of the CNGB1 transcript (upstream of the premature stop codon), labeled rod outer segments. CNGB1 combines with CNGA1 to form the rod cyclic nucleotide gated channel and previous studies have shown the requirement of CNGB1 for normal targeting of CNGA1 to the rod outer segment. In keeping with these previous observations, IHC showed a lack of detectable CNGA1 protein in the rod outer segments of the affected dog. A population study did not identify the <i>CNGB1</i> mutation in PRA-affected dogs in other breeds and documented that the <i>CNGB1</i> mutation accounts for ∼70% of cases of Papillon PRA in our PRA-affected canine DNA bank. <i>CNGB1</i> mutations are one cause of autosomal recessive RP making the <i>CNGB1</i> mutant dog a valuable large animal model of the condition.</p></div

Papillon mutation in <i>CNGB1</i> exon 26.

<p>Sanger dideoxy-sequencing traces for part of <i>CNGB1</i> exon 26 are shown for an unaffected (A) and an affected (B) Papillon. Panel C shows the codon and amino acid alignment inferred from the traces in panels A and B, for the unaffected sequence (top) and affected Papillon mutation sequence (bottom). The complex Papillon mutation includes a 6 bp AGCTAC insertion between reference bases chr2:61,502,599–61,502,600 (yellow highlight in panel C and within yellow box in panel B) and an adenine deletion at chr2: 61,502,597 (red highlight in panel C and red triangle in panel B). The deletion causes a frameshift and premature stop codon within seven residues, including the two new, inserted codons.</p

Intron and exon boundries for canine CNGB1 gene.

<p>1. Locations are all in respect to UCSC Genome Browser CanFam2.0 (<a href="http://genome.ucsc.edu" target="_blank">http://genome.ucsc.edu</a>).</p><p>2. Capital letters are exonic DNA sequences and lower case bases are intronic regions.</p><p>End of coding region marked in Exon 33 row by underlined TGA.</p

Representative ERG tracings from a normal control Papillon and a PRA-affected Papillon, both 10 weeks of age.

<p>A. Dark-adapted ERG recordings at −2.4, −1.2 and 0.4 log cdS/m². B. Light-adapted flash and flicker (33 Hz) ERG tracings. Background white light of 30 cd/m² and flash intensity of 0.4 log cdS/m². The vertical bars on the flicker ERG indicates the flash timing.</p

Immunohistochemistry on frozen retinal sections from age (8 wk) and sex matched (female) control and affected dogs.

<p>Images on the left are from the control dog and images on the right are from the affected dog. Panels A and B are stained with GARP-CNGB1 N-terminal antibody (green) and DAPI (blue). GARP proteins are present in both the control and affected samples. Panels C and D were stained with CNGB1 C-terminal antibody (green) and DAPI. CNGB1 full length protein is not detected in the affected sample. Panels E and F are stained with CNGA1 antibody (green) and DAPI. CNGA1 is not detected in the affected sample, presumably due to necessity for CNGB1 to form viable channels and normal trafficking. Size bar: 20 µm. OS- photoreceptor outer segment, IS – photoreceptor inner segment, ONL – outer nuclear layer, IPL – inner plexiform layer, INL – inner nuclear layer.</p

SD-OCT cross sectional images of the central retina from <i>CNGB1</i> mutant Papillons and wild-type controls.

<p>Note the progressive thinning of the outer nuclear layer of the retina in the affected animals. NFL/GCL – nerve fiber layer/ganglion cell layer; IPL – inner plexiform layer; INL – inner nuclear layer; OPL – outer plexiform layer; ONL – outer nuclear layer; PR – photoreceptor inner and outer segments; C – choroid. (Note the retinal pigment epithelium is not labeled).</p

PRA type 1 genotypes and clinical status for 139 Papillons.

<p>1. Genotyping results: (+/+) means wild-type <i>CNGB1</i> sequence. M = mutant (c.2387delA;2389_2390insAGCTAC) genotype.</p><p>2. Not including colony dogs to avoid inflation of mutation presence in the general population of Papillon dogs.</p