Characterization and exploration of the Wilson disease locus on chromosome 13

Wilson disease (WD) is an autosomal recessive disorder of copper metabolism that has been previously mapped to chromosome 13q14.3. In order to more finely map the locus as well as begin physical cloning of the region three closely linked RFLP loci were converted into STSs and used to screen a CEPH YAC library. Two YACs were isolated, one each for the flanking proximal and distal markers (D13S31 and D13S59 respectively). Highly informative PCR-based polymorphic microsatellites closely linked to the WD locus (WND) were derived from the YAC containing D13S31. These markers, along with others in the region were placed on a genetic linkage map of chromosome 13 and typed in 74 multiplex WD families from a variety of geographic origins (166 affected members). Multipoint analysis provided very high odds that the location of WND is between D13S31 and D13S59. Previous odds with RFLP-based markers were only 7:1 more likely than any other location. Current odds are ;Additional markers in the region between D13S31 and D13S59 further defined the region and two markers showed linkage disequilibrium with WD. Cosmids derived from a YAC containing these markers were used in two gene search strategies: exon trapping and cDNA selection. 20 exon trapped fragments were studied. Six clones show no significant identities to known sequences (analysis done by BLASTn against the nucleotide non-redundant database). The rest of the clones were derived from vector sequences, centromeric sequences or E.coli DNA. Thirty-six cDNA clones were analyzed: three clones show no significant identities to known sequences, and two clones have been mapped back to cosmids in the WD region. Two other clones showed significant identity to known human (pregnancy specific beta-1-glycoprotein/chromosome 19) or rat (prolactin/human homolog on chromosome 6) genes. The rest of the clones were vector fragments which had contaminated the cDNA library.;The WD locus has now been mapped with greater confidence and physical cloning of the region was begun with the first two YACs isolated in the region. Nine putative transcribed DNA clones have been isolated which may prove to be useful in the search for the Wilson disease gene.