July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
A point mutation in the novel lncRNA, ROBO2-AS, correlates with phenotype severity in canine RPGR-XLPRA1
Author Affiliations & Notes
  • Tatyana Appelbaum
    Clinical Studies, U of Penn School Vet Med, Philadelphia, Pennsylvania, United States
  • Leonardo Murgiano
    Clinical Studies, U of Penn School Vet Med, Philadelphia, Pennsylvania, United States
  • Doreen Becker
    Clinical Studies, U of Penn School Vet Med, Philadelphia, Pennsylvania, United States
  • Evelyn Santana
    Clinical Studies, U of Penn School Vet Med, Philadelphia, Pennsylvania, United States
  • Gustavo D Aguirre
    Clinical Studies, U of Penn School Vet Med, Philadelphia, Pennsylvania, United States
  • Footnotes
    Commercial Relationships   Tatyana Appelbaum, None; Leonardo Murgiano, None; Doreen Becker, None; Evelyn Santana, None; Gustavo Aguirre, None
  • Footnotes
    Support  EY-06855, -17549, the Foundation Fighting Blindness, the Van Sloun Fund for Canine Genetic Research, and Hope for Vision
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 2347. doi:
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      Tatyana Appelbaum, Leonardo Murgiano, Doreen Becker, Evelyn Santana, Gustavo D Aguirre; A point mutation in the novel lncRNA, ROBO2-AS, correlates with phenotype severity in canine RPGR-XLPRA1. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2347.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Canine X-linked progressive retinal atrophy 1 (XLPRA1) is caused by a 5nt deletion in RPGR 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 incomplete penetrance of XLPRA1.

Methods : Phenotypes from a pedigree derived from one affected male dog were scored as mild, moderate and severe. SNPs were genotyped using SNaPshot Multiplex Kit. Genome wide association study (GWAS) was performed using CanineHD Genotyping BeadChip. PHASE software was utilized for haplotype reconstruction. Fisher exact test was used for statistical analysis. Protein expression was evaluated by Western blot and IHC. Gene expression was determined by qRT-PCR.

Results : GWAS identified a single locus on chromosome 31 with notable trend toward association with XLPRA1 phenotype. SNP data from GWAS were re-analyzed as haplotypes and two XLPRA1 phenotypes (severe and moderate) were tested for association with each haplotype identified. We found significant association (p=0.02) of the major haplotype with the moderate phenotype. Two genes from the identified 5 MB interval, ROBO1 and ROBO2, known to be important for normal retinal functioning were selected as candidate XLPRA1 genetic modifiers. Noticeably, gene and protein expression of ROBO1 and ROBO2 were downregulated in affected retinas. In this study, we determined the complete structure of ROBO1 and ROBO2 previously uncharacterized in dogs. ROBO1 encompasses of 31 exons and encodes 1651aa protein. Two isoforms of ROBO2 [ROBO2a (1405aa) and ROBO2b (1382aa)] driven by alternative promoters were found expressed in the canine retina. These two isoforms composed of 27 coding exons differ only in their first exon. Here we also report two novel genes, lncRNAs ROBO1-AS and ROBO2-AS found in the antisense direction of the canine ROBO1 and ROBO2, respectively. All exons as well as 5’- and 3’-regulatory regions of ROBO genes were screened for mutations. Of total 11 SNPs identified, one, located in the ROBO2-AS exon, showed significant association with the moderate XLPRA1 phenotype (p=0.02).

Conclusions : The correlation between the SNP in ROBO2-AS with the moderate disease phenotype suggests a potential protective role of lncRNA ROBO2-AS in the pathogenesis of the canine disease.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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