Transcriptional Profile Analysis of RPGRORF15 Frameshift Mutation Identifies Novel Genes Associated with Retinal Degeneration

The term retinitis pigmentosa (RP) refers to a group of many different inherited retinal diseases characterized by progressive rod or rod–cone photoreceptor degeneration that causes subsequent visual impairment and blindness. Some of the causative genes have clear, well-identified roles (e.g., involvement in phototransduction, in maintaining photoreceptor structure, or in RPE retinoid metabolism; RetNet: http://www.sph.uth.tmc.edu/RetNet/ provided in the public domain by the University of Texas Houston Health Science Center, Houston, TX). However, there remain a large number of diseases caused by genes with poorly understood functions and for which the mechanism linking the genes and/or mutations with photoreceptor disease and degeneration is unknown. Among these is the RP3 form of X-linked RP (XLRP), a uniformly severe, early-onset retinal disease in humans that is caused by mutations in the RP GTPase regulator (RPGR) gene.1 Although estimates vary depending on the sample population and methods of testing, it is generally accepted that mutations in RPGR account for >70% of XLRP cases.2–4 Furthermore, the carboxyl-terminal exon open reading frame 15 (ORF15) of RPGR, a mutational hot spot, has been shown to be mutated in 22% to 60% of XLRP patients.2,5,6 RPGR is essential for the maintenance of photoreceptor viability.7 The protein, which has a series of six RCC1-like domains (RLDs) characteristic of the highly conserved guanine nucleotide exchange factors, is found in the rod and cone photoreceptor connecting cilia.8 RPGR has complex interactions with other proteins that have microtubular-based transport functions in the retina and that are presumed to function in the photoreceptor centrosome, inner and outer segments, and ciliary axoneme region.9,10 Among these, the genes coding for nephrocystin-4,11 -5,12 and -69; PDE6D13; RPGR interacting protein (RPGRIP1)11; and RPGRIP1L14 cause retinal disease when mutated, thus emphasizing the critical importance of this protein complex in maintaining photoreceptor structure, function, and viability. One approach to developing insights into the cell- or tissue-specific functions of genes or to examining the molecular mechanisms of disease is microarray-based global profiling of gene expression in combination with bioinformatic analysis. In several studies, the transcriptome of the mouse and human retinas has been analyzed by characterizing changes in expression profiles during development and aging.15–17 More recently, transcriptomic data of distinct retinal cells18–20 and a web-based platform containing numerous retinal gene expression studies have been made available (http://alnitak.u-strasbg.fr/RetinoBase/ provided in the public domain by University Louis Pasteur, Strasbourg, France). In addition, studies based on differential gene expression in mouse retinal disease models provide useful information to aid in discerning the role of disease-causing genes with respect to other genes and in evaluating their involvement in gene pathways and cascades.21–23 These approaches have specific limitations in terms of human retinal diseases, not the least being the lack of adequate sample sizes at the appropriate disease stages. However, the constraints can be overcome by using animal models of homologous diseases. These models provide a powerful tool for translational studies, provided that the human disease modeled and the corresponding animal disease are comparable. Natural mutations in RPGRORF15 occur in humans and dogs,2,24 and X-linked progressive retinal atrophy (XLPRA) is the dog homolog of human XLRP. In dogs, two different ORF15 microdeletions have been identified: XLPRA1 is a postdevelopmental, slowly progressive photoreceptor degeneration resulting from a 5-bp deletion in ORF15 that truncates the translated protein, whereas XLPRA2 is an early-onset, progressive rod and cone photoreceptor disease caused by a 2-bp deletion that creates a frameshift and premature stop in the translated protein. The deduced peptide sequence is changed by the inclusion of 34 additional basic residues that increase the isoelectric point of the truncated protein.25 Beltran et al.26,27 described in detail the course of retinal disease in canine XLPRA2, the phenotype of which replicates the salient features of RPGR-XLRP.28,29 The purpose of the present study was to identify the genes and molecular mechanisms associated with disease onset and progression in normal and XLPRA2 mutant canine retinas. We examined the global retinal gene expression profiles at 7 and 16 weeks, the most relevant disease-related ages. Kinetics of photoreceptor cell death show a burst of dying cells between 6 and 7 weeks, whereas at 16 weeks, when the retina has lost approximately 40% of its photoreceptors, there is a constant but decreased rate of cell death.26 For this, we used a validated custom retinal cDNA microarray30,31 and performed real-time quantitative reverse transcription-PCR (qRT-PCR), Western blot analysis, and immunohistochemistry, to confirm and expand the microarray results. We detected several genes that were differentially expressed (DE) at critical time points in the degenerating XLPRA2 retina and that are specific for the disease stages examined. The downregulation of rod-specific genes also suggests the differential and preferential damage of rods in the early stages of the disease.