MOLECULAR BIOLOGY OF THE MENKES SYNDROME GENE

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We propose to clone and characterize the gene responsible for Menkes syndrome. Menkes syndrome is an X-linked recessive disorder of copper metabolism characterized by early growth retardation and severe neurological impairment. There is no effective treatment for the disease and its exact cause at the biochemical level is unknown. Recently, a female with the disease and a de novo X-autosome translocation was identified. The translocation breakpoint at Xql3 coincides with a previous linkage assignment of the Menkes locus and with the probable location of the homologous mottled locus in the mouse. It therefore almost certainly disrupts proper expression of the Menkes syndrome gene and may very likely directly interrupt it. Our cloning strategy is based on the physical identification and cloning of DNA sequences at this translocation breakpoint. The location of the translocation breakpoint with respect to a number of Xql3 probes has been determined by utilizing somatic cell hybrids containing the translocation chromosome. The translocation was found to break the X chromosome just proximal to the PGK-1 locus. With this knowledge, a long range physical map of the region was begun in attempt to detect the translocation breakpoint. The breakpoint was found to be within 300kb of the PGK-1 locus on Sfil digested DNA. Efforts have been initiated to obtain yeast artificial chromosomes (YACS) that span this region. In this revised application, we propose to identify YAC clones that cross the Menkes syndrome translocation breakpoint and will very likely contain sequences from within or closely linked to the Menkes syndrome gene. A physical map of the region will be constructed and CpG islands identified. Both total YACs and lambda subclones will be used to screen cDNA libraries for identification of candidate genes. Candidate clones will be used to analyze DNA and RNA from Menkes syndrome patients and from mottled mice for mutations. The gene will be characterized by sequence analysis, expression in different tissues and possibly by expression studies in vitro. The Menkes translocation provides an invaluable resource as a "signpost" for the gene and is one of but a few X-linked and autosomal translocations at a human disease gene locus available for studies of this kind. The proposed studies should not only lead to better understanding of the basic defect in Menkes syndrome, but also to new findings concerning copper metabolism in general.