Characterisation of molecular DNA rearrangements within the Xq12 - q13.1 region, in three patients with X-linked hypohidrotic ectodermal dysplasia (EDA)

A panel of somatic cell hybrids and X-linked hypohidrotic ectodermal dysplasia (EDA) patient-derived cell lines, containing different rearranged X chromosomes, have been used to refine the physical map of the Xq12-q13.1 region. The patient-derived material included genomic DNA from an EDA male (EDA family 1015) with an interstitial deletion , and a cell line GM0705A, obtained from an isolated female patient with a de novo balanced (X;9) translocation, and the somatic hybrid, AnLy, derived from this cell line. This map subdivides the region into at least 6 mapping-intervals. DNA probes from DXS732 and DXS453, identified as the closest flanking marker loci to the EDA locus, were used to identify homologous Yeast Artificial Chromosome (YAC) clones. Two of the DXS732-specific YACs were shown by fluorescent in situ hybridisation (FISH) analysis to bridge the (X;9) translocation breakpoint. These two YACs were also screened against the ICRF human X chromosome cosmid library and identified 36 cosmid clones. Direct cosmid-cosmid hybridisation analysis placed subsets of these clones within four different cosmid contigs. Mapping of anchor clones from each contig, against the mapping panel, localised all these contigs within the Xql2-qi3.1 region. One cosmid, ICRFc104C03.1 84, identified potential junctional-fragments in several restriction digests of AnLy hybrid DNA. This was confirmed by FISH analysis of the GMO705A cell line with total cosmid ICRFc104C03.184, In which both chromosomal elements of the (X;9) translocation were identified. A single-copy probe pC03.184F5, derived from this cosmld, also Identified the der(9)- derived junctional fragment when hybridised against AnLy DNA. Screening a large panel of genomic DNAs from 80 unrelated affected EDA males with pC03.184F5 identified, in addition to the original EDA 1015 deletion, a molecular deletion in a second unrelated EDA male (EDA family 1003). Characterisation of these two EDA deletions has shown that the proximal endpoint of the EDA 1003 deletion lies distal, to both the DXS452 locus and to the proximal endpoint of the EDA 1015 deletion. The distal endpoint of the EDA 1015 deletion has been located within the most distal of the four cosmid contigs. The distal endpoint of the EDA 1003 deletion has still to be established, but must be located proximal of the DXS453 locus. Determination of the extent of the EDA 1003 deletion and further analysis of the (X;9) translocation-fianking cosmids, should greatly facilitate the positional cloning of the EDA gene.