Transcriptional profile analysis of RPGRORF15 frameshift mutation identifies novel genes associated with retinal degeneration.
OBJECTIVE
To identify genes and molecular mechanisms associated with photoreceptor degeneration in a canine model of XLRP caused by an RPGR exon ORF15 microdeletion. Methods. Expression profiles of mutant and normal retinas were compared by using canine retinal custom cDNA microarrays. qRT-PCR, Western blot analysis, and immunohistochemistry (IHC) were applied to selected genes, to confirm and expand the microarray results.


RESULTS
At 7 and 16 weeks, respectively, 56 and 18 transcripts were downregulated in the mutant retinas, but none were differentially expressed (DE) at both ages, suggesting the involvement of temporally distinct pathways. Downregulated genes included the known retina-relevant genes PAX6, CHML, and RDH11 at 7 weeks and CRX and SAG at 16 weeks. Genes directly or indirectly active in apoptotic processes were altered at 7 weeks (CAMK2G, NTRK2, PRKCB, RALA, RBBP6, RNF41, SMYD3, SPP1, and TUBB2C) and 16 weeks (SLC25A5 and NKAP). Furthermore, the DE genes at 7 weeks (ELOVL6, GLOD4, NDUFS4, and REEP1) and 16 weeks (SLC25A5 and TARS2) are related to mitochondrial functions. qRT-PCR of 18 genes confirmed the microarray results and showed DE of additional genes not on the array. Only GFAP was DE at 3 weeks of age. Western blot and IHC analyses also confirmed the high reliability of the transcriptomic data.


CONCLUSIONS
Several DE genes were identified in mutant retinas. At 7 weeks, a combination of nonclassic anti- and proapoptosis genes appear to be involved in photoreceptor degeneration, whereas at both 7 and 16 weeks, the expression of mitochondria-related genes indicates that they may play a relevant role in the disease process.
Genini
Sem
S
Section of Ophthalmology, Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Zangerl
Barbara
B
Slavik
Julianna
J
Acland
Gregory M
GM
Beltran
William A
WA
Aguirre
Gustavo D
GD
D000818:Animals; D015153:Blotting, Western; D004252:DNA Mutational Analysis; D004195:Disease Models, Animal; D004283:Dog Diseases / Q000235:genetics* / Q000473:pathology; D004285:Dogs; D005091:Exons / Q000235:genetics; D005136:Eye Proteins / Q000235:genetics*; D016368:Frameshift Mutation / Q000235:genetics*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000502:physiology*; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D016366:Open Reading Frames / Q000235:genetics*; D012174:Retinitis Pigmentosa / Q000235:genetics / Q000473:pathology / Q000662:veterinary*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D005136:Eye Proteins; C099929:RPGR protein, human