Understanding the mechanism(s) of retinal degeneration in X -linked retinitis pigmentosa.

Abstract

X-linked retinitis pigmentosa (XLRP) is a clinically and genetically heterogeneous degenerative disease of the retina. <italic>RP2 </italic> and <italic>RP3</italic> account for 10--20% and 70--90% of genetically identifiable disease, respectively. However, mutations in the corresponding genes, <italic>RP2</italic> and <italic>RPGR</italic>, were detected in only 10% and 20% of XLRP families. To understand the pathogenesis of XLRP, a comprehensive analysis was undertaken using genetic, molecular, and biochemical methods. To determine where the remaining mutations lie, families with no apparent <italic>RPGR</italic> or <italic>RP2</italic> mutation were genetically characterized. Haplotype analysis provided evidence of a distinct XLRP locus <italic>RP6</italic> that is tightly linked to <italic>RP2</italic> and <italic>RP3</italic>. A large cohort of 234 North American families with RP and apparent X-linked inheritance was screened for all known exons of <italic> RP2</italic> and <italic>RPGR</italic>, including a novel mutational hotspot in <italic>RPGR-ORF15</italic>. Mutations were detected in 60% of XLRP patients; however, a considerable number of mutations were also identified in simplex RP males. This is significant because simplex cases account for 50% of all RP and this data suggests that <italic>RP2</italic> and <italic>RPGR</italic> are responsible for 30% of these. RP2 and RPGR are ubiquitous proteins, yet the mutations result only in photoreceptor degeneration. To determine the function of RP2 in the retina, two distinct approaches were undertaken. Specific monoclonal and polyclonal antibodies were produced against a RP2-GST fusion protein expressed in <italic>E. coli</italic>. Immunocytochemistry and biochemical fractionation methods localized the RP2 protein to synaptic regions in the retina. RP2 interacting proteins were identified by the two-hybrid method using three different baits to screen a bovine retinal prey cDNA library. Two members of the ADP-ribosylation factor family, ARL2 and ARL3, were identified as specific interacting proteins. Localization and interaction results suggest that the RP2 protein has a role in subcellular trafficking in retinal synapses. These studies have broad implications on research not only for XLRP, but also for understanding photoreceptor biology and function.