Analysis of six candidate genes as potential modifiers of disease expression in canine XLPRA1, a model for human X-linked retinitis pigmentosa 3

PURPOSE: Canine X-linked progressive retinal atrophy (XLPRA) is caused by mutations in RPGR exon ORF15, which is also a mutation hotspot in human X-linked retinitis pigmentosa 3 (RP3). The XLPRA1 form of disease has shown extensive phenotypic variability in a colony of dogs that all inherited the same mutant X-chromosome. This variability in onset and severity makes XLPRA1 a valuable model to use to identify genes influencing photoreceptors degeneration in dog and to elucidate molecular mechanisms underlying RP in its human homolog. In this study, RPGRIP1, RANBP2, NPM1, PDE6D, NPHP5, and ABCA4 genes were selected on the basis of interaction with RPGR or RPGRIP1 or their implication in related retinal diseases, and were investigated as candidate genetic modifiers of XLPRA1. METHODS: A pedigree derived from an affected male dog outcrossed to unrelated normal mix bred or purebred females was used. Morphologic examination revealed phenotypic variability in the affected dogs characterized as mild, moderate, or severe. Single nucleotide polymorphisms (SNPs) and indel-containing markers spanning the entire genes were designed, based on the canine sequence and the Broad Institute SNP library, and genotyped on the pedigree. For each candidate gene, haplotypes were identified and their frequencies in severely and moderately affected dogs were compared to detect a putative correlation between a gene-specific haplotype(s), and severity level of the disease. Primers were derived from expressed sequence tags (ESTs) and predicted transcripts to assess the relative retinal expression of the six genes of interest in normal and affected retinas of different ages. RESULTS: Four to seven haplotypes per gene were identified. None of the haplotypes of RPGRIP1, NPM1, PDE6D, NPHP5, RANBP2, and ABCA4 were found to co-segregate with the moderate or severe phenotype. No significant difference in the retinal expression levels of the candidate genes was observed between normal and affected dogs. CONCLUSIONS: The haplotype distribution of RPGRIP1, NPM1, PDE6D, NPHP5, RANBP2, and ABCA4 suggests these genes are not modifiers of the disease phenotype observed in the XLPRA1 pedigree. The RPGRORF15 stop mutation does not affect the retinal expression of these genes at the mRNA level in the pre-degenerate stage of disease, but no conclusions can be made at this time about changes that may occur at the protein level.