Abstract
Purpose :
Canine X-linked progressive retinal atrophy 1 (XLPRA1) is caused by a 5nt deletion in RPGR exon ORF15. Affected dogs within a closed research colony show extensive phenotypic heterogeneity in disease severity and progression. We hypothesized that an unlinked modifier gene(s) contributes to expressivity of phenotype severity.
Methods :
Phenotypes from a pedigree derived from one affected male dog were scored as moderate and severe. Genome wide association study (GWAS) was performed using CanineHD Genotyping BeadChip, PHASE software for haplotype reconstruction, and Fisher exact test was used for statistical analysis. Assessment of RNA secondary structures was done with the Vienna RNA package, and gene expression was determined by qRT-PCR.
Results :
GWAS in an XLPRA1 phenotype informative pedigree and the subsequent haplotype phasing revealed two novel long-noncoding RNAs (lncRNAs) ROBO1-AS and ROBO2-AS at the XLPRA1 modifier locus on cfa31 as prospective phenotype modifier genes. The ROBO1-AS and ROBO2-AS transcripts are 5’-capped, subject to alternative splicing, and display overlapping gene organization with ROBO1 and ROBO2, respectively. We found that expression level of sense-antisense ROBO genes is retained at the same level in retinas of different ages (pre-mature: 3wks, 5wks, 7wks and fully developed: 16wks), suggesting an important role of these genes in retinal function. Next, we found that ROBO1-AS and ROBO2-AS act in cis to form lncRNA/mRNA duplexes with ROBO1 and ROBO2, possibly regulating their transcriptional and/or translational output. Moreover, ROBO2-AS acts also in trans to target RPGR and ENOPH1 mRNAs. The study further showed that the specific sequence variants of ROBO1-AS and ROBO2-AS strongly correlate with moderate XLPRA1 phenotype, pointing to potential advantageous pleiotropic effects of the ROBO1-AS and ROBO2-AS alleles on disease phenotype. Finally, using computational modeling we demonstrated that the ROBO1-AS and ROBO2-AS sequencing variants significantly alter the RNA structural ensemble of ROBO1-AS and ROBO2-AS, possibly interfering with their molecular function.
Conclusions :
The results provide important insight into the role of novel genes ROBO1-AS and ROBO2-AS in retinal function and point towards potential clinical interventions capable to modulate time course of retinal degeneration.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.