Nayudu, Manimekalai; (2000) Genetic analysis of inherited X-linked retinitis pigmentosa: Development of a transcriptional map of the RP2 region. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

X-linked retinitis pigmentosa is genetically heterogeneous with at least two loci on proximal Xp, known as RP2 and RP3. The RP2 locus is not well defined due to a lack of deletion patients; at the onset of this study the RP2 locus had been mapped to a 13cM region between markers DXS7 and DXS255 (Xp11.3-Xp11.23). The primary objective of this thesis was to further progress positional cloning and identification of the RP2 disease-causative gene. Initially, thirteen new XIRP families were analysed using twenty eight microsatellite markers spanning Xp22.13 to Xp11.22 with the aim of refining the genetic interval within which the RP2 gene lies. From this haplotype analysis, three families were found to be linked to the RP3 locus, one to the Choroideremia locus, seven could not be unambiguously assigned and two families were found to be linked to the RP2 locus with crossovers observed below the marker MAOA, further localising the RP2 gene proximal to MAOA and confirming previous linkage data. The TIMP-1 gene was considered to be a candidate for RP2 since mutations in the TIMP-3 gene were found to cause Sorsby's fundus dystrophy, a macular degeneration. Direct sequencing of the six exons and exon/intron boundaries of TIMP-1 in affected male RP2 patients did not reveal mutations associated with the disease phenotype, hence TIMP-1 is unlikely to be the RP2 disease-causative gene. Heterogeneity analysis suggests that the RP2 gene probably lies 2cM distal to DXS426. Well characterised YACs around DXS426 were utilised in the development of a transcriptional map of this region. The selection for enriched cDNAs using the magnetic bead capture technique was applied to four overlapping YACs which cover a region of 500kb. Two adult retinal cDNA libraries were used in the selection procedure. cDNA/YAC DNA hybrids were captured using streptavidin coated magnetic beads and two rounds of selection were performed. The A1220 and 33CA11 YACs contain the TIMP-1 and properdin (PFC) genes respectively, and these genes were also present in the starting retinal cDNA libraries with the TIMP-1 gene shown to be especially rare. The YACs chosen from the region of interest therefore contain these internal positive control genes. A rhodopsin YAC was included for use as an external positive control for the technique. YAC specific sub-libraries created for enriched cDNAs were screened for contaminating sequences such as ribosomal genes, yeast DNA and p-actin cDNAs and showed depletion for such unwanted trancripts. The successful establishment of the selection procedure for the efficient enrichment of low abundance retinal trancripts was demonstrated by a 1,000 fold enrichment for the rare positive control gene TIMP-1. A selection of enriched cDNA clones (40-50 per YAC) were sequenced and compared to each other in order to identify overlapping clones, and to database entries using the Gapped BLAST search program. Twelve interesting clones were used in mapping back studies to the region investigated.

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