Patel, Reshma J.; (1998) Physical and transcriptional mapping studies within the retinitis pigmentosa critical region on chromosome 7p. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Revolutionary developments in molecular genetic procedures in the past decade have accelerated the discovery of the genetic cause for numerous single gene disorders. This project has focused on employing positional cloning approaches to identify genes that are implicated in disease. These were applied to one form of autosomal dominant retinitis pigmentosa (RP9), an inherited disease of the retina, in which progressive degeneration of the photoreceptors leads to night blindness and ultimately the complete loss of vision. RP9 had previously been genetically linked to chromosome 7, and genetic refinement located the disease within an interval of approximately 1.6 cM between D7S795 and D7S484. With the absence of candidate genes in the critical region, the focus of this study primarily involved physical mapping procedures. These were used to provide a cloned resource onto which genes and STSs could be mapped, and for use in gene identification methods to identify transcribed sequences that lie within the disease interval. These could then be treated as potential candidate genes for further analysis. Physical mapping in the RP9 disease region on 7p14-15 was initiated by screening YAC libraries with genetically linked markers and integrating YACs which were available from the chromosome 7 YAC resource. Alu-PCR. and STS content strategies were used to identify overlaps of clones. The isolation of YAC terminal sequences permitted the progression of chromosome walking. This work contributed to a detailed contig that spans the RP9 critical region and extends distally. The contig was saturated with additional STSs which allowed the placement and ordering of six transcripts distal to the critical region. YAC clones were sized by PFGE to provide a physical size estimation of approximately 5,900 kb for the contig and 3,200 kb for the disease interval. Since no genes in the disease region had been identified at the commencement of this study, two strategies for detecting and isolating genes were then employed. The positional candidate gene approach is rapidly becoming the favoured approach to identify genes within critical intervals. To serve the RP9 project and to benefit other chromosome 7 positional cloning endeavours, 30 known chromosome 7 ESTs and several genes were mapped to sub-regional locations by PCR through a chromosome 7 human-mouse somatic cell hybrid panel. Five of the ESTs revealed substantial nucleotide and amino acid homology with known gene sequences in database records. One was identified as a homologue of a rat gene. Those ESTs that mapped to the 7p14-15 sub-region were tested by PCR assay for their presence in the RP9 YAC contig. Two ESTs mapped onto the contig, one within (EST02120) and one distal to the disease region. Further analysis was carried out by members of the group to determine whether EST02120 was truly part of a gene. A second strategy involved testing a novel cDNA hybrid selection method. The sandwich selection hybridisation method (Yan and Swaroop 1994) is based on solution hybridisation of cDNAs to target genomic clones. As the sandwich selection method was new and had not been widely discussed in the literature, the main aim was to test this system using a control strategy. A cosmid containing the blue opsin gene was used to isolate its cDNA counterpart as a test for the method's efficiency and sensitivity. This demonstrated that the technique may be sensitive to the size of the genomic source used in the procedure. The advantages, limitations and certain refinements are discussed. This technique was also applied to cosmid clones from the RP9 region in a further attempt to identify and isolate transcribed sequences within the disease region. Selected cDNAs were tested to ascertain whether any mapped back to their respective target genomic clone, and to assess their nature.

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