Thiselton, Dawn Louise; (1997) A genetic and physical mapping study of the retinitis pigmentosa critical region on the proximal short arm of the X chromosome. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Retinitis Pigmentosa (RP) is an inherited disease of the retina, in which progressive degeneration of the photoreceptors leads to night blindness and ultimately complete loss of vision. Genetic linkage studies have identified two major loci (RP2 and RP3) mapping to the short arm of the X chromosome which are implicated in the X-linked form of the disease: RP2 maps to Xp11.3-Xp11.22 and RP3 maps to Xp21. The precise localisation of RP3 in Xp21 has been facilitated by the identification of cytogenetic abnormalities in affected patients and extensive physical mapping studies to a 150kb region flanked by OTC and DXS1110. At the outset of this study, several groups were actively engaged in analysing transcripts from this region as potential candidates for the disease and a gene RPGR has since been established as the site of a proportion of RP3 mutations. The focus of this thesis was to more definitively characterise the RP2 gene critical region in Xp11 by a combination of genetic and physical mapping methods, as a first step toward positional cloning of the defective gene. The precise localisation of RP2 has been hampered since its initial assignment to Xp11 by a lack of associated cytogenetic anomalies, few informative recombination events and a paucity of polymorphic markers in proximal Xp. In order to generate novel genetic markers mapping to the region, 13 human X chromosome-specific cosmids known to contain (CA)n microsatellites were FISH mapped to X chromosome sub-regions and polymorphic microsatellites isolated from 6 that mapped to proximal Xp. This thesis describes the isolation and characterisation of one such microsatellite (DXS556) from cosmid HX20 that mapped to Xp11.4. To determine the relationship between the new microsatellites and existing genetic markers mapping to proximal Xp, these markers were used to genetically characterise a panel of 14 XLRP families by linkage and haplotype analysis. A combination of multiply informative crossovers and physical mapping via YAC STS-content analysis enabled the successful positioning and ordering of the new microsatellites and their incorporation into the well-established framework map of proximal Xp. In an attempt to determine the particular XLRP locus segregating in each of the XLRP families, further genotyping was performed using additional markers as they became available through the Genome Database (GDB). Key recombination events detected by haplotype analysis resulted in the assignment of one family as RP3 and four as RP2. Finer localisation of the RP2 gene was achieved by extensive genetic analysis incorporating the most recently available CEPH/Genethon markers to generate dense haplotypes spanning Xp11.3-Xp11.22. Two multiply informative crossovers in one family defined new proximal and distal boundaries for the RP2 critical region and significantly reduced its size from ~13cM to ~5cM. During the course of this study, mutations in the TIMP-3 (tissue inhibitor of metalloproteinases-3) gene were found to cause the retinal degenerative disease Sorsby's Fundus Dystrophy. TIMP-3 is a member of a family of proteins that play an integral role in connective tissue homeostasis. A related gene, TIMP-1, lay within the RP2 critical interval and thus immediately became a positional candidate for RP2. The genomic organisation of TIMP-1 was completed prior to mutation screening by PCR and direct sequencing of the entire coding region, exon-intron boundaries and 5' untranslated region, in affected and unaffected males from three RP2 families. Although a neutral polymorphism was discovered in exon 5, no disease-associated sequence alterations were found, suggesting that TIMP-1 is unlikely to play a causal role in the etiology of XLRP. (Abstract shortened by ProQuest.)

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