Wijesuriya, Sujeewa D.; (1997) Molecular analysis of inherited choroidoretinal dystrophies. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Inherited choroidoretinal dystrophies significantly contribute to the proportion of dystrophies that affect the retina, resulting in the early or late loss of vision. Although numerous retinal genes have been isolated, few have been implicated in disease pathogenesis. This is profoundly apparent when considering those diseases that principally affect the macular region of the retina and thus affect central vision. The principle concerns of this thesis therefore centres upon the molecular genetic investigation of three autosomal dominantly inherited choroidoretinal diseases, namely Sorsby's fundus dystrophy (SFD), Doyne's honeycomb retinal dystrophy (DHRD) and Cone-rod dystrophy (CRD) which principally affect the central retina with the aim of elucidating the underlying molecular pathologies. One of the most dynamic areas of human genetic research, centres upon the precise placement of genes on particular chromosomes using the technique of linkage analysis. Such an approach was undertaken for SFD and DHRD, both being late onset diseases that principally affect the macula region of the retina. Following the exclusion of a number of candidate disease and gene loci, a systematic search of the entire genome was initiated in a large British DHRD pedigree utilising 230 microsatellite markers, and finally resulted in the localisation of DHRD to a 5 cM region on chromosome 2p21-16. A similar linkage approach was directed towards the search for the SFD causative gene for which a large British pedigree was available. Candidate disease and gene loci were initially excluded. SFD was localised to a 24 cM region on chromosome 22ql3-qter (Weber et al, 1994a). This localisation was confirmed and the disease region considerably refined to a 4 cM interval on 22ql3.1 by linkage and haplotype analysis. TIMP-3 was subsequently implicated as the SFD causative gene and confirmed in our family by the presence of the Ser181Cys mutation of this gene. Fifteen SFD families from diverse parts of the British Isles with a cumulative number of 78 affected individuals were recruited for mutation screening purposes. Identification of the same Ser181Cys mutation cosegregating across all families led to the postulation of a founder effect for this mutation. Haplotype analysis in conjunction with statistical evaluation using conventional chi squared analysis and a complex likelihood ratio test provided the statistical significance required to confirm the existence of a founder effect for this mutation of TIMP-3 in the British SFD population and it was possible to trace the mutation back to 300 years based on the available family information. Mapping of a disease to a chromosome is a prerequisite for its positional cloning, leading to the characterisation of the gene, its putative protein, its functional role and finally to the pathophysiology of the disease phenotype with which it is associated. A variety of strategies can be employed to isolate and clone a human disease gene on identification of its chromosomal location. Where plausible candidates are absent, complete dissection of the genetically defined region is required. Such positional cloning strategies were employed in the study of an early onset blinding disease, Cone-rod dystrophy which had previously been localised to chromosome 19ql3.3 and assigned the CORD2 locus. To refine the disease interval which approximated 4.5 Mb at the onset of this project, cosmids mapping within the disease region were screened for the presence of (CA)n repeats using hybridisation techniques and five polymorphic markers and several STSs were identified. The allele frequencies and heterozygosity values of these polymorphic markers have been calculated and registered in GenBank. Analysis of these markers in the CORD2 family identified the first informative marker which displayed no recombination with disease and whose physical localisation was known. A second highly informative marker displaying 14 alleles in the population was found to map proximal to the CORD2 region by analysis of recombination events in the family but will be of immense use for disease traits mapping in the 19ql3. 1-13.2 region. With the analysis of novel markers from newly published genomic maps the disease region was refined to approximately 1.6 Mb. The physical mapping endeavours were extended to the characterisation of a preliminary YAC contig across the disease interval. YACs were isolated from several libraries, STS content mapped and end-sequenced allowing overlapping YACs to be identified and novel YACs to be isolated. This YAC contig which has been saturated with available STSs and novel STSs discovered from cosmids in the region has contributed towards detailed characterisation of the region and provides a resource for identification of disease causative genes.

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