CHARACTERIZATION OF MOLECULAR DNA REARRANGEMENTS WITHIN THE XQ12-Q13.1 REGION, IN 3 PATIENTS WITH X-LINKED HYPOHIDROTIC ECTODERMAL DYSPLASIA (EDA)

A panel of somatic cell hybrids and X-linked hypohidrotic ectodermal d ysplasia (EDA) patient-derived cell lines, containing different rearra nged X chromosomes, have been used to refine the physical map of the X q12 - q13.1 region. The patient-derived material included genomic DNA from an EDA male (EDA family 101 5) with an interstitial deletion , an d a cell line GM0705A, obtained from an isolated female patient with a de novo balanced (X;9) translocation, and the somatic hybrid, AnLy, d erived from this cell line. This map subdivides the region into at lea st 6 mapping-intervals. DNA probes from DXS732 and DXS453, identified as the closest flanking marker loci to the EDA locus, were used to ide ntify 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 li brary and identified 36 cosmid clones. Direct cosmid-cosmid hybridisat ion analysis placed subsets of these clones within four different cosm id contigs. Mapping of anchor clones from each contig, against the map ping panel, localised all these contigs within the Xq12 - ql 3.1 regio n. One cosmid, ICRFc104C03.184, identified potential junctional-fragme nts in several restriction digests of AnLy hybrid DNA. This was confir med by FISH analysis of the GM0705A cell line with total cosmid ICRFc1 04C03.184, in which both chromosomal elements of the (X;9) translocati on were identified. A single-copy probe pC03.184E2, derived from this cosmid, also identified the der(9)-derived junctional fragment when hy bridised against AnLy DNA. Screening a large panel of genomic DNAs fro m 80 unrelated affected EDA males with pC03.184F5 identified, in addit ion to the original EDA 1015 deletion, a molecular deletion in a secon d unrelated EDA male (EDA family 1003). Characterisation of these two EDA deletions has shown that the proximal endpoint of the EDA 1003 del etion lies distal, to both the DXS732 locus and to the proximal endpoi nt of the EDA 101 5 deletion. The distal endpoint of the EDA 101 5 del etion has been located within the most distal of the four cosmid conti gs. The distal endpoint of the EDA 1003 deletion has still to be estab lished, but must be located proximal of the DXS453 locus. Determinatio n of the extent of the EDA 1003 deletion and further analysis of the ( X;9) translocation-flanking cosmids, should greatly facilitate the pos itional cloning of the EDA gene.