Project description:Purpose: To identify genes and molecular mechanisms associated with disease progression during (7 weeks of age) and after (16 weeks) the peak of photoreceptor death in dogs affected with XLPRA2, a canine model of early-onset XLRP caused by a microdeletion in RPGR exon ORF15. Methods: Expression profiles of diseased and normal dog retinas at both ages were compared using a canine retinal custom cDNA microarray. Data normalization and filtering were performed with GeneSpring, statistical analysis with Significance Analysis of Microarrays. Real-time PCR using Taqman probes, western blot and immunohistochemistry (IHC) were applied on selected genes to confirm and expand the microarray results. Results: At 7 and 16 weeks, respectively, 56 and 18 transcripts were down-regulated in mutant retinas compared to normals. None of the transcripts was differentially expressed (DE) at both ages, suggesting the involvement of temporally distinct molecular pathways. Down-regulated genes included PAX6, CHML, and RDH11 at 7 weeks and CRX and SAG at 16 weeks, which are necessary for visual system development and function. Genes directly or indirectly active in apoptotic processes were altered at 7 weeks (CAMK2G, NTRK2, PRKCB, RALA, RBBP6, RNF41, SEPT5, SMYD3, SPP1, and TUBB2C) and 16 weeks (SLC25A5 and NKAP). Furthermore, DE genes at 7 weeks (ELOVL6, GLOD4, NDUFS4, and REEP1) and 16 weeks (SLC25A5 and TARS2) are related to mitochondrial functions. Real-time PCR of 11 genes confirmed the microarray results and showed differential expression for additional genes not on the array, such as GFAP, RHO, OPN1SW, CNGB3 and the mutated RPGR. Western blotting and IHC analysis also confirmed the high reliability of the presented transcriptomic data. Conclusions: A list of mutated genes in RPGRORF15 diseased retinas, which are likely candidates to further study their role in age-related photoreceptor degeneration diseases, is reported at different crucial ages. The results indicate that at 7 weeks a combination of non-classical anti- and pro-apoptotic genes appears to be involved in photoreceptor degeneration, whereas at both 7 and 16 weeks expression of mitochondria related genes indicates they may play a relevant role in the disease process.

Project description:Purpose: To identify genes and molecular mechanisms associated with disease progression during (7 weeks of age) and after (16 weeks) the peak of photoreceptor death in dogs affected with XLPRA2, a canine model of early-onset XLRP caused by a microdeletion in RPGR exon ORF15. Methods: Expression profiles of diseased and normal dog retinas at both ages were compared using a canine retinal custom cDNA microarray. Data normalization and filtering were performed with GeneSpring, statistical analysis with Significance Analysis of Microarrays. Real-time PCR using Taqman probes, western blot and immunohistochemistry (IHC) were applied on selected genes to confirm and expand the microarray results. Results: At 7 and 16 weeks, respectively, 56 and 18 transcripts were down-regulated in mutant retinas compared to normals. None of the transcripts was differentially expressed (DE) at both ages, suggesting the involvement of temporally distinct molecular pathways. Down-regulated genes included PAX6, CHML, and RDH11 at 7 weeks and CRX and SAG at 16 weeks, which are necessary for visual system development and function. Genes directly or indirectly active in apoptotic processes were altered at 7 weeks (CAMK2G, NTRK2, PRKCB, RALA, RBBP6, RNF41, SEPT5, SMYD3, SPP1, and TUBB2C) and 16 weeks (SLC25A5 and NKAP). Furthermore, DE genes at 7 weeks (ELOVL6, GLOD4, NDUFS4, and REEP1) and 16 weeks (SLC25A5 and TARS2) are related to mitochondrial functions. Real-time PCR of 11 genes confirmed the microarray results and showed differential expression for additional genes not on the array, such as GFAP, RHO, OPN1SW, CNGB3 and the mutated RPGR. Western blotting and IHC analysis also confirmed the high reliability of the presented transcriptomic data. Conclusions: A list of mutated genes in RPGRORF15 diseased retinas, which are likely candidates to further study their role in age-related photoreceptor degeneration diseases, is reported at different crucial ages. The results indicate that at 7 weeks a combination of non-classical anti- and pro-apoptotic genes appears to be involved in photoreceptor degeneration, whereas at both 7 and 16 weeks expression of mitochondria related genes indicates they may play a relevant role in the disease process. 3 biological replicates each for normal and XLPRA2 affected retinas were analyzed at 7 and 16 weeks of age. Each individual sample was hybridized in a reference design using a custom-made retinal cDNA microarray against brain pool to enable cross comparison between groups. Retina was labeled with Cy5 in all samples but 5615 (GSM474350).

Project description:Background:Accurate clinical diagnosis and prognosis of retinal degeneration can be aided by the identification of the disease-causing genetic variant. It can confirm the clinical diagnosis as well as inform the clinician of the risk for potential involvement of other organs such as kidneys. It also aids in genetic counseling for affected individuals who want to have a child. Finally, knowledge of disease-causing variants informs laboratory investigators involved in translational research. With the advent of next-generation sequencing, identifying pathogenic mutations is becoming easier, especially the identification of novel pathogenic variants. Methods:We used whole exome sequencing on a cohort of 69 patients with various forms of retinal degeneration and in whom screens for previously identified disease-causing variants had been inconclusive. All potential pathogenic variants were verified by Sanger sequencing and, when possible, segregation analysis of immediate relatives. Potential variants were identified by using a semi-masked approach in which rare variants in candidate genes were identified without knowledge of the clinical diagnosis (beyond "retinal degeneration") or inheritance pattern. After the initial list of genes was prioritized, genetic diagnosis and inheritance pattern were taken into account. Results:We identified the likely pathogenic variants in 64% of the subjects. Seven percent had a single heterozygous mutation identified that would cause recessive disease and 13% had no obviously pathogenic variants and no family members available to perform segregation analysis. Eleven subjects are good candidates for novel gene discovery. Two de novo mutations were identified that resulted in dominant retinal degeneration. Conclusion:Whole exome sequencing allows for thorough genetic analysis of candidate genes as well as novel gene discovery. It allows for an unbiased analysis of genetic variants to reduce the chance that the pathogenic mutation will be missed due to incomplete or inaccurate family history or analysis at the early stage of a syndromic form of retinal degeneration.

Dataset Information

Transcriptional profile analysis of RPGRORF15 frameshift mutation identifies novel genes associated with retinal degeneration.

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Transcriptional profile analysis of RPGRORF15 frameshift mutation identifies novel genes associated with retinal degeneration.

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