The broad, long term objectives are to use somatic cell and molecular techniques to define with molecular probes and to physically map a region of chromosome 13q whose deletion appears cytogenetically to be responsible for the 13q- syndrome. This region is thus assumed to contain genes critical for brain, eye and limb development. Probes which map to this region will be useful clinically for detecting deletions in patients with suggestive phenotype but normal karyotype. The physical map and associated cloned DNA segments can be used to locate important developmental genes within this region. The ultimate goal is to understand several human malformations at the molecular level. We will carry out the following specific aims in order to begin progress towards these long term goals. 1) Use lymphoblastoid cell lines (LCL's) derived from a number of patients with deletions in the distal long arm of chromosome 13 to create a panel of somatic cell hybrids each of which contains a deleted chromosome 13. A selectable marker gene will be inserted into the chromosomes of the LCL's using a retrovirus vector. Microcell mediated chromosome transfer will be used to make hybrids and the marker will be selected for. 2) Map all of the existing distal chromosome 13q probes to this panel of patient derived chromosome 13 deletions. This is accomplished with standard Southern blotting. 3) Make a hybrid cell line which contains the undeleted distal portion of chromosome 13 as its only human component. UV irradiation will be used to fragment the chromosome and a UV repair gene located on 13q is selected for. 4) Use a pulsed field gel electrophoresis based technique to isolate large restriction fragments which arise from the critical 13q region. A human specific Alu probe is used to detect large fragments which are present in normal but not in deleted DNA in Southern transfers of DNA from deleted and normal chromosomes. Probes which recognize these bands are made by cloning sequences from the bands themselves. 5) Isolate probes which distinguish between deletion panel chromosomes by mapping random clones from a 13q phage library against the deletion panel. 6) Begin a long range restriction map of the regions defined by the deletion panel. This is accomplished with PFGE of normal DNA which is then transferred and hybridized with a battery of mapped 13q probes.
| Brown, S; Russo, J; Chitayat, D et al. (1995) The 13q- syndrome: the molecular definition of a critical deletion region in band 13q32. Am J Hum Genet 57:859-66 |
| Brown, S; Gersen, S; Anyane-Yeboa, K et al. (1993) Preliminary definition of a ""critical region"" of chromosome 13 in q32: report of 14 cases with 13q deletions and review of the literature. Am J Med Genet 45:52-9 |
