Skewed X-inactivation is associated with retinal dystrophy in female carriers of RPGR mutations.

Progressive degeneration of rod and cone photoreceptors frequently is caused by mutations in the X-chromosomal gene Retinitis Pigmentosa GTPase Regulator (RPGR). Males hemizygous for a RPGR mutation often are affected by Retinitis Pigmentosa (RP), whereas female mutation carriers only occasionally present with severe RP phenotypes. The underlying pathomechanism leading to RP in female carriers is not well understood. Here, we analyzed a three-generation family in which two of three female carriers of a nonsense RPGR mutation presented with RP. Among two cell lines derived from the same female family members, differences were detected in RPGR transcript expression, in localization of RPGR along cilia, as well as in primary cilium length. Significantly, these differences correlated with alterations in X-chromosomal inactivation patterns found in the patient-derived cell lines from females. In summary, our data suggest that skewed X-chromosomal inactivation is an important factor that determines the disease manifestation of RP among female carriers of pathogenic sequence alterations in the RPGR gene.

The Major Ciliary Isoforms of RPGR Build Different Interaction Complexes with INPP5E and RPGRIP1L.

X-linked retinitis pigmentosa (XLRP) is frequently caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. A complex splicing process acts on the RPGR gene resulting in three major isoforms: RPGRex1-19, RPGRORF15 and RPGRskip14/15. We characterized the widely expressed, alternatively spliced transcript RPGRskip14/15 lacking exons 14 and 15. Using the CRISPR/eSpCas9 system, we generated HEK293T cell lines exclusively expressing the RPGRskip14/15 transcript from the endogenous RPGR gene. RPGRex1-19 and RPGRORF15 were knocked out. Immunocytochemistry demonstrated that the RPGRskip14/15 protein localizes along primary cilia, resembling the expression pattern of RPGRex1-19. The number of cilia-carrying cells was not affected by the absence of the RPGRex1-19 and RPGRORF15 isoforms. Co-immunoprecipitation assays demonstrated that both RPGRex1-19 and RPGRskip14/15 interact with PDE6D, further supporting that RPGRskip14/15 is associated with the protein networks along the primary cilium. Interestingly, interaction complexes with INPP5E or RPGRIP1L were only detectable with isoform RPGRex1-19, but not with RPGRskip14/15, demonstrating distinct functional properties of the major RPGR isoforms in spite of their similar subcellular localization. Our findings lead to the conclusion that protein binding sites within RPGR are mediated through alternative splicing. A tissue-specific expression ratio between RPGRskip14/15 and RPGRex1-19 seems required to regulate the ciliary concentration of RPGR interaction partners.