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S. Gilbert-Gregory, B. Zangerl, G. D. Aguirre; Tissue and Isoform Specific Expression of Canine Rpgr. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2295.
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© ARVO (1962-2015); The Authors (2016-present)
XLRP is a severe form of inherited blindness primarily caused by mutations in RPGR; some mutations in this gene are also associated with disorders in tissues with functional cilia. Retinal phenotypes often result from mutations in the alternatively spliced, retinal specific, C-terminal exonORF15. Two naturally occurring canine models, both with exonORF15 mutations, have been identified with a phenotype mimicking XLRP. Despite reports of tissue-specific splicing, or implications of isoform-specific expression patterns on disease phenotypes, detailed studies explaining genotype-phenotype correlations are absent. Use of canine models to investigate tissue specific isoforms and expression levels will provide insights into the influence of individual mutations in RPGR on associated phenotypes.
RNA was extracted from tissues with functional cilia (retina, kidney, testis, trachea) and reference tissues (brain, heart, liver, spleen) by standard TRIzol procedure. RT-PCR was performed on retinal RNA, and specific primers used to identify RPGR isoforms. qRT-PCR with isoform-specific probes established RPGR expression in different tissues. Total RNA northern blot analysis is used to support and confirm results.
Cloning of RPGR confirmed both common isoforms (with/without exonORF15) in the retina. Two new isoforms were cloned; one skips exons 14-15a, the other exons 5-11. Tissue specific expression levels were assessed for total RPGR expression using a probe specific to exons 1-2. RPGR is most abundant in retina; expression in other tissues is reduced ~3-5 fold, and is even lower in heart and liver (12-14 fold). Isoform specific expression patterns are being investigated.
To date, we verified total RPGR expression is most prevalent in retinal tissue, and may explain the majority of retinal phenotypes related to mutations in the gene. Two new isoforms were identified; it has yet to be determined if the expression profile of these may be key to tissue specificity and/or disease phenotypes. We have established a system to not only investigate these differences, but to also relate them to disease models relevant to XLRP. These studies will elucidate genotype-phenotype correlations of disorders caused by mutations in RPGR.
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