Woodward, Karen Jane; (1995) Characterisation of the TSC1 candidate region on human chromosome 9q34. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Tuberous sclerosis (TSC) is an autosomal dominant disorder with clinical symptoms ranging from minor skin lesions to more serious manifestations such as mental retardation and seizures. Linkage analysis established heterogeneity and assigned genes to 9q34 (TSC1) and 16p13.3 (TSC2). Detection of TSC-associated deletions on 16p13.3 refined the localisation of the gene and TSC2 has now been identified. Meiotic recombination events in TSC families have defined a consensus candidate region for TSC1 between D9S149 and D9S114, but further refinement gives conflicting positions for the gene. A positional cloning strategy has been applied to characterise the TSC 1 candidate region and identify the gene. Radiation hybrids retaining markers from the target interval were characterised and used as a resource for region specific DNA. The clones isolated plus ones from other sources were confirmed to map to 9q34 by FISH. Localisations were refined by using three lymphoblastoid cell lines with translocations breakpoints within 9q34, thereby subdividing the band into 4 intervals. A total of 32 loci from 28 contigs, and 43 anonymous contigs were mapped enabling a FISH map to be constructed. Cosmids mapping to the TSC1 candidate region were subsequently screened for transcribed sequences by exon amplification. The initial study led to the identification VAV2, a good candidate for TSC1. It was followed by more extensive analysis of 6 contigs mapping to the same FISH interval as D9S149 and D9S114. A total of 171 putative exons were cloned, 48 potentially different products were sequenced and 34 were inferred to be authentic exons. A subset from 4 contigs were confirmed to be conserved sequences by cross-species hybridisation and cDNA clones were isolated and partially characterised. The exons and cDNAs isolated are important resources for construction of a 9q34 transcription map and may facilitate identification of the TSC1 gene.

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