Novel mutations in XLRS1 causing retinoschisis, including first evidence of putative leader sequence change

Juvenile retinoschisis is an X-linked recessive disease caused by mutations in the XLRS1 gene. We screened 31 new unrelated patients and families for XLRS1 mutations in addition to previously reported mutations for 60 of our families (Retinoschisis Consortium, Hum Mol Genet 1998;7:1185–1192). Twenty-three different mutations including 12 novel ones were identified in 28 patients. Mutations identified in this study include 19 missense mutations, two nonsense mutations, one intragenic deletion, four microdeletions, one insertion, and one intronic sequence substitution that is likely to result in a splice site defect. Two novel mutations, c.38T→C (L13P) and c.667T→C (C223R), respectively, present the first genetic evidence for the functional significance of the putative leader peptide sequence and for the functional significance at the carboxyl terminal of the XLRS1 protein beyond the discoidin domain. Mutations in 25 of the families were localized to exons 4–6, emphasizing the critical functional significance of the discoidin domain of the XLRS1 protein. Hum Mutat 14:423–427, 1999. © 1999 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/35178/1/8_ftp.pd

Hick and Radhakrishnan on Religious Diversity: Back to the Kantian Noumenon

We shall examine some conceptual tensions in Hick’s ‘pluralism’ in the light of S. Radhakrishnan’s reformulation of classical Advaita. Hick himself often quoted Radhakrishnan’s translations from the Hindu scriptures in support of his own claims about divine ineffability, transformative experience and religious pluralism. However, while Hick developed these themes partly through an adaptation of Kantian epistemology, Radhakrishnan derived them ultimately from Śaṁkara (c.800 CE), and these two distinctive points of origin lead to somewhat different types of reconstruction of the diversity of world religions. Our argument will highlight the point that Radhakrishnan is not a ‘pluralist’ in terms of Hick’s understanding of the Real. The Advaitin ultimate, while it too like Hick’s Real cannot be encapsulated by human categories, is, however, not strongly ineffable, because some substantive descriptions, according to the Advaitic tradition, are more accurate than others. Our comparative analysis will reveal that they differ because they are located in two somewhat divergent metaphysical schemes. In turn, we will be able to revisit, through this dialogue between Hick and Radhakrishnan, the intensely vexed question of whether Hick’s version of pluralism is in fact a form of covert exclusivism.This is the author accepted manuscript. The final version is available from Springer via http://dx.doi.org/10.1007/s11841-015-0459-

Rd9 Is a Naturally Occurring Mouse Model of a Common Form of Retinitis Pigmentosa Caused by Mutations in RPGR-ORF15

Animal models of human disease are an invaluable component of studies aimed at understanding disease pathogenesis and therapeutic possibilities. Mutations in the gene encoding retinitis pigmentosa GTPase regulator (RPGR) are the most common cause of X-linked retinitis pigmentosa (XLRP) and are estimated to cause 20% of all retinal dystrophy cases. A majority of RPGR mutations are present in ORF15, the purine-rich terminal exon of the predominant splice-variant expressed in retina. Here we describe the genetic and phenotypic characterization of the retinal degeneration 9 (Rd9) strain of mice, a naturally occurring animal model of XLRP. Rd9 mice were found to carry a 32-base-pair duplication within ORF15 that causes a shift in the reading frame that introduces a premature-stop codon. Rpgr ORF15 transcripts, but not protein, were detected in retinas from Rd9/Y male mice that exhibited retinal pathology, including pigment loss and slowly progressing decrease in outer nuclear layer thickness. The levels of rhodopsin and transducin in rod outer segments were also decreased, and M-cone opsin appeared mislocalized within cone photoreceptors. In addition, electroretinogram (ERG) a- and b-wave amplitudes of both Rd9/Y male and Rd9/Rd9 female mice showed moderate gradual reduction that continued to 24 months of age. The presence of multiple retinal features that correlate with findings in individuals with XLRP identifies Rd9 as a valuable model for use in gaining insight into ORF15-associated disease progression and pathogenesis, as well as accelerating the development and testing of therapeutic strategies for this common form of retinal dystrophy

Differential replication of circular DNA molecules co-injected into early Xenopus laevis embryos.

Replication of co-injected supercoiled DNA molecules in fertilized Xenopus eggs was monitored through the blastula stage of development. The extent of replication, as measured by 32P-dTMP incorporation into form I DNA, was directly proportional to the number of molecules, rather than the size, of the plasmid injected. Although only a small fraction of molecules of either template was replicated, incorporation was predominantly into full length daughter molecules. Over at least a 20-fold concentration range of microinjected DNA, injection of equal masses of DNA resulted in greater incorporation into the smaller form I DNA present in molar excess. The extent of incorporation into supercoiled DNA for a particular plasmid was apparently independent of the concentration of a second, co-injected plasmid. The relative extents of replication of co-injected supercoiled templates could be altered simply by changing the molar ratios of the templates

A long-term efficacy study of gene replacement therapy for RPGR-associated retinal degeneration

Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene account for >70% of X-linked retinitis pigmentosa (XLRP) and 15-20% of all inherited retinal degeneration. Gene replacement therapy for RPGR-XLRP was hampered by the relatively slow disease progression in mouse models and by difficulties in cloning the full-length RPGR-ORF15 cDNA that includes a purine-rich 3'-coding region; however, its effectiveness has recently been demonstrated in four dogs with RPGR mutations. To advance the therapy to clinical stage, we generated new stable vectors in AAV8 or AAV9 carrying mouse and human full-length RPGR-ORF15-coding sequence and conducted a comprehensive long-term dose-efficacy study in Rpgr-knockout mice. After validating their ability to produce full-length proteins that localize to photoreceptor connecting cilia, we evaluated various vector doses in mice during a 2-year study. We demonstrate that eyes treated with a single injection of mouse or human RPGR-ORF15 vector at an optimal dose maintained the expression of RPGR-ORF15 throughout the study duration and exhibited higher electroretinogram amplitude, thicker photoreceptor layer and better targeting of opsins to outer segments compared with sham-treated eyes. Furthermore, mice that received treatment at an advanced age also showed remarkable preservation of retinal structure and function. Retinal toxicity was observed at high vector doses, highlighting the importance of careful dose optimization in future clinical experiments. Our long-term dose-efficacy study should facilitate the design of human trials with human RPGR-ORF15 vector as a clinical candidate