The gene RPGR was previously identified in the RP3 region of Xp21.1 and shown to be mutated in 10-20% of patients with the progressive retinal degeneration X-linked retinitis pigmentosa (XLRP). The mutations predominantly affected a domain homologous to RCC1, a guanine nucleotide exchange factor for the small GTPase Ran, although they were present in fewer than the 70-75% of XLRP patients predicted from linkage studies. Mutations in the RP2 locus at Xp11.3 were found in a further 10-20% of XLRP patients, as predicted from linkage studies. Because the mutations in the remainder of the XLRP patients may reside in undiscovered exons of RPGR, we sequenced a 172-kb region containing the entire gene. Analysis of the sequence disclosed a new 3' terminal exon that was mutated in 60% of XLRP patients examined. This exon encodes 567 amino acids, with a repetitive domain rich in glutamic acid residues. The sequence is conserved in the mouse, bovine and Fugu rubripes genes. It is preferentially expressed in mouse and bovine retina, further supporting its importance for retinal function. Our results suggest that mutations in RPGR are the only cause of RP3 type XLRP and account for the disease in over 70% of XLRP patients and an estimated 11% of all retinitis pigmentosa patients.

X-linked retinitis pigmentosa (xlRP) is a severe progressive retinal degeneration which affects about 1 in 25,000 of the population. The most common form of xlRP, RP3, has been localised to the interval between CYBB and OTC in Xp21.1 by linkage analysis and deletion mapping. Identification of microdeletions within this region has now led to the positional cloning of a gene, RPGR, that spans 60 kg of genomic DNA and is ubiquitously expressed. The predicted 90 kD protein contains in its N-terminal half a tandem repeat structure highly similar to RCC1 (regulator of chromosome condensation), suggesting an interaction with a small GTPase. The C-terminal half contains a domain, rich in acidic residues, and ends in a potential isoprenylation anchorage site. The two intragenic deletions, two nonsense and three missense mutations within conserved domains provide evidence that RPGR (retinitis pigmentosa GTPase regulator) is the RP3 gene.

A primary feature of age-related macular degeneration (AMD) is the presence of extracellular deposits between the retinal pigment epithelium (RPE) and underlying Bruch's membrane, leading to RPE dysfunction, photoreceptor death and severe visual loss. AMD accounts for about 50% of blind registrations in Western countries and is a common, genetically complex disorder. Very little is known regarding its molecular basis. Late-onset retinal degeneration (L-ORD) is an autosomal dominant disorder with striking clinical and pathological similarity to AMD. Here we show that L-ORD is genetically heterogeneous and that a proposed founder mutation in the CTRP5 (C1QTNF5) gene, which encodes a novel short-chain collagen, changes a highly conserved serine to arginine (Ser163Arg) in 7/14 L-ORD families and 0/1000 control individuals. The mutation occurs in the gC1q domain of CTRP5 and results in abnormal high molecular weight aggregate formation which may alter its higher-order structure and interactions. These results indicate a novel disease mechanism involving abnormal adhesion between RPE and Bruch's membrane.

Ten new and seventeen previously reported Enhanced S Cone Syndrome (ESCS) subjects were used to search for genetic heterogeneity. All subjects were diagnosed with ESCS on the basis of clinical, psychophysical and/or electroretinography testing using published criteria. Mutation analysis was performed on the NR2E3 nuclear receptor gene by single strand conformation analysis and direct sequencing, which revealed either homozygous (N=13) or compound heterozygous (N=11) mutations in 24 subjects (89%), heterozygous mutations in 2 subjects (7%) and no mutations in 1 subject (4%). Fifteen different mutations were identified, including six not previously reported. The subject (Patient A) with no detected NR2E3 mutation had features not usually associated with ESCS, in particular moderate rod photoreceptor function in peripheral retina and an abnormally thick retinal nerve fibre layer. Mutation analysis of the NRL, CRX, NR1D1 and THRB genes in this individual revealed a heterozygous one base-pair insertion in exon 2 of the NRL gene, which results in a predicted truncation of the NRL protein. Loss-of-function NRL alleles have not been described previously in humans, but since the same mutation was present in unaffected family members, it raises the possibility that the abnormal ESCS phenotype in Patient A may result from a digenic mechanism, with a heterozygous NRL mutation and a mutation in another unknown gene.

The ORF15 isoform of RPGR (RPGR(ORF15)) and RPGR interacting protein 1 (RPGRIP1) are mutated in a variety of retinal dystrophies but their functions are poorly understood. Here, we show that in cultured mammalian cells both RPGR(ORF15) and RPGRIP1 localize to centrioles. These localizations are resistant to the microtubule destabilizing drug nocodazole and persist throughout the cell cycle. RPGR and RPGRIP1 also co-localize at basal bodies in cells with primary cilia. The C-terminal (C2) domain of RPGR(ORF15) (ORF15(C2)) is highly conserved across 13 mammalian species, suggesting that it is a functionally important domain. Using matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we show that this domain interacts with a 40 kDa shuttling protein nucleophosmin (NPM). The RPGR(ORF15)-NPM interaction was confirmed by (i) yeast two-hybrid analyses; (ii) binding of both recombinant and native HeLa cell NPM to RPGR(ORF15) fusion proteins in vitro; (iii) co-immunoprecipitation of native NPM, RPGR(ORF15) and RPGRIP1 from bovine retinal extracts and of native HeLa cell NPM and transfected RPGR(ORF15) from cultured cells and (iv) co-localization of NPM and RPGR(ORF15) at metaphase centrosomes in cultured cells. NPM is a multifunctional protein chaperone that shuttles between the nucleoli and the cytoplasm and has been associated with licensing of centrosomal division. RPGR and RPGRIP1 join a growing number of centrosomal proteins involved in human disease.