CLONING OF DISEASE GENES FROM THE HUMAN XP22.3 REGION

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A "reverse genetics" approach is proposed for the cloning of disease genes from the distal short arm of the human X chromosome. This region is of particular interest since it shares homology with the Y chromosome, it contains genes escaping X inactivation, and it shows a very high frequency of deletions and translocations. Five disease gene have been assigned to specific intervals of an Xp22.3 deletion map which was constructed studying patients with contiguous gene syndromes. These diseases are: 1) X-linked ichthyosis, due to steroid sulfatase deficiency; 2) Kallmann syndrome, characterized by hypogonadotropic hypogonadism and anosmia and in some cases by unilateral renal hypoplasia; 3) X-linked recessive chondrodysplasia punctata, a bone dysplasia characterized by nasal and phalangeal hypoplasia and stippling of the epiphyses; 4) mental retardation; and 5) short stature. We plan to perform chromosome walking in the Xp22.3 region using yeast artificial chromosome (YAC) clones. YAC contigs have been assembled in the region and will be used as starting points. The available deletion and long range restriction maps of Xp22.3 will serve to orient our walks. Contig closure will be achieved by both walking and by the isolation of new YACs using sequences derived from a cosmid library constructed from an Xp21-pter hybrid and from a plasmid library of PCR products from a radiation hybrid retaining the Xp22.3 region. During our walk we will clone has been developed and will be used to reach the patients' deletion breakpoints. YAC clones found to be in the immediate proximity of disease genes will be screened for the presence of expressed sequences using a variety of traditional and new methods, including subcloning of the YACs in lambda phage and plasmid clones, followed by hybridization to Zoo blots, northern blots and cDNA libraries, subcloning of CpG islands from the YACs, and exon trapping. Once the disease genes are isolated, characterization of molecular defects in the patients will be performed and molecular diagnostic tests will be developed. The expression and function of Xp22.3 disease genes in man and other species will also be studied. In particular the expression of the gene for Kallmann syndrome, probably a neuronal migration developmental defect, will be studied during development of mouse embryos. In addition, mouse models of the diseases will be created by introducing defective genes in embryonic stem cells. The proposed studies will lead to a better understanding of Xp22.3 diseases and to more efficient diagnosis and possible therapy. They may also give insights into important factors and mechanisms involved in human development.