Char syndrome is an inherited heart-hand disorder comprising patent ductus arteriosus (PDA), abnormal 5th fingers, and facial dysmorphism. We initiated a positional cloning effort to discover the disease gene by linking the disease to 6p12-p21.1 and completing a physical map of the region. The gene was tentatively identified by discovery of two missense mutations in a neural crest cell-related transcription factor. Final proof that this is the Char syndrome gene awaits demonstration that the mutant proteins function abnormally. We propose to study the role of the disease gene and a novel related gene on the development of the ductus arteriosus and other affected structures in vitro and in vivo. To investigate the hypothesis that Char syndrome mutations are acting in a dominant negative manner, mutant proteins will be expressed in vitro and then tested functionally using electrophoretic mobility shift assays and cross-linking studies. Mutants will also be expressed transiently in cell culture and tested for their ability to transactivate gene expression. Other gene defects will be sought among Char syndrome patients lacking coding region mutations. To examine the hypothesis that PDA in Char syndrome results from abnormal development of the vessel's medial layer, a mouse model of Char syndrome will be created by transgenesis. This model will be phenotyped and abnormal development characterized. Migration of cardiac neural crest cells, particularly their presence in the developing ductus arteriosus, will be assessed. Ductal histology and gene expression in the medial smooth muscle cells will be determined. To test the hypothesis that a novel, homologous gene at 6p12 is important for ductal development and may be a second Char syndrome gene, the full length gene will be cloned. Its protein will be characterized for DNA binding, dimerization and transactivation. Expression of this gene during development will be assessed in the mouse by in situ hybridization. This homologous gene will be scanned for mutations that cause the second, milder Char syndrome phenotype. To consider the hypothesis that there are eye, dental and other skeletal defects in Char syndrome, patients will be evaluated with a skeletal survey, dental X-rays and a complete ophthalmologic assessment. In sum, the proposed research will delineate the effects of mutations causing Char syndrome at the molecular, cellular, and organ levels. Aside from illuminating one pathway toward the clinical endpoint of PDA, it will provide the starting place for identifying genes critical to ductal development that are activated by Char syndrome-related genes.
