Physical Mapping on the Human X Chromosome and Its Application to the Positional Cloning of the XLP Gene

Résumé du livre

The work described in this thesis addresses some of the major questions posed in the study of the human genome. Among the most important goals has been the coverage of the complete genome in physical maps using cloned DNA, and the derivation of detailed gene maps from them to enable the determination of molecular defects underlying genetic disease. -- The feasibility of long-range mapping by using STS content analysis of yeast artificial chromosomes (YACs) was evaluated using the dystrophin gene as a model. Sequence tagged sites (STSs) designed from the dystrophin cDNA and genomic sequence provided a sufficient density (1 STS per 150 kb) to detect overlaps between clones over the entire 2.3 Mb gene, except across intron 1. -- An alternative approach, YAC fingerprinting using colony Alu-PCR enabled completion of the contig. This method was evaluated further on both established contigs, and a whole chromosome project. Whilst the method itself produced reproducible and accurate data, the approach was hampered by the extensive chimaerism of the YAC clones. -- As increasing numbers of landmarks became available, it became possible to construct physical maps of genomic regions with the aim of finding genes involved in disease. X-linked lymphoproliferative disease (XLP) is characterised by an extreme sensitivity to Epstein-Barr virus. Previous genetic linkage studies localised the XLP locus to Xq24-q25. The critical region was refined, and a complete YAC contig across it was constructed. The YAC map was used as a framework on which to assemble large insert bacterial clone contigs, minimum sets of clones from which were sequenced. Analysis of the sequence revealed the presence of two candidate genes in the critical region. Mutations in seven unrelated XLP patients were found in one gene, SH2D1A, which encodes a novel SH2-domain-containing protein, thus demonstrating its involvement in XLP.