Retinitis pigmentosa (RP) is an inherited group of retinal degenerations that are both clinically and genetically heterogeneous. Autosomal dominant form of RP is caused by mutations in at least 10 loci. The research described in this thesis is focused upon the adRP locus on chromosome 19q13.4 (RP11), which was found by a genome wide linkage analysis on a large British pedigree in our laboratory. At the start of this project RP11 was confined by markers D19S180 and D19S926 to a genetic interval of approximately 11cM and 3 adRP families was linked to RP11 (ADRP5, ADRP29 and RPl907). To further refine this localisation and in order to obtain an estimate of the frequency of RP caused by mutations at this locus 12 more adRP families were genotyped for markers from the disease interval. This led to the identification of two additional RP11 families (ADRP2 and ADRP11) and haplotype analysis in these two families confined the disease region to a 5.8 cM interval between D19S921 and D19S418 and the frequency of the RP11 locus was estimated at approximately 20%. The gene for protein kinase gamma (PRKCG) is localised in the refined RP11 interval and was considered as a positional candidate gene worthy of a thorough investigation. Therefore the genomic organisation of this gene was determined and exon-intron structure was characterised. PRKCG gene was found to consist of 18 exons and a genomic span of at least 16 kb. Following its characterisation this gene was subjected to a thorough mutation analysis. The exons, 5' promoter region, and 3'UTR of this gene was screened in 6 families linked to RP11 locus (ADRP5, ADRP29, ADRP2, RPl907, ADRP24 and a Japanese family). In this analysis a mutation was identified in two adRP families (RP 1907 and ADRP24) and two sporadic RP cases, which were subsequently identified as being founded upon a common ancestor and therefore a single mutation event rather than several independent events. This mutation resulted in the non-conservative replacement of a basic arginine with an uncharged serine at codon 659. Arg659Ser mutation was not found in over 500 normal chromosomes indicating it to be a rare polymorphism. Unfortunately the absence of PRKCG mutations in 7 other RP11 linked pedigrees led to the exclusion of this gene. In order to further refine the RP11 locus novel microsatellite markers were isolated from the distal end of the RP interval using cosmid clones FISH mapped to the region between D19S927 and D19S418, and analysed in the pedigree ADRP2, which presented the distal flanking cross over with D19S418. In this endeavour a total of four microsatellite markers and 5 useful STSs were generated. One of the novel markers, D19S781.2N, recombined with the disease phenotype in ADRP2 and superseded D19S418 as the distal flanking marker. At this stage another group linked three additional families of North American origin to 19ql3.4 and refined the locus proximally replacing D19S921 with D19S572 as the novel proximal flanking marker of the RP11 disease interval. Therefore through the combined genetic data generated both at our laboratory and elsewhere, the RP11 locus was confined between the markers D19S572 (proximal) and D19S781.2N (distal). According to the physical mapping data generated at the Lawrence Livermore National Laboratory (LLNL) Genome Centre the physical distance between these two markers approximate 0.7 Mb. Concurrent to the work described above construction of a YAC contig was also initiated across the RP11 interval, which includes YAC clones from three different YAC libraries. However STS content mapping revealed deletions in several YAC clones that were meant to provide coverage across the RP11 interval, thus indicating a region of instability or a region unclonable in YACs. Therefore PAC clones have replaced YACs as the cloning vector of choice and the current on going work of the project involves the construction of a complete PAC contig across the RP11 interval. Future work will involve the mapping of retinally or brain expressed ESTs, already placed in the 19q13.4 region by the EST mapping consortium (Schuler et al., 1996), to the RP11 critical interval to facilitate the identification of the RP11 gene
