MOLECULAR GENETICS OF CYSTIC FIBROSIS

囊性纤维化（CF）是由CF跨膜突变引起的

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The protein encoded by this gene functions as a cAMP-activated chloride channel. However it has recently been shown that CFTR also regulates the function of another distinct chloride channel in airway epithelia. Which functions of CFTR are affected by disease associated mutations is not clear. This is problematic since pulmonary: disease continues to be the major cause of mortality and the lungs are the primary target for current gene therapy efforts. The overall goal of this proposal is to determine which function of CFTR is abnormal in individuals with lung disease. By using this disease model, these studies will increase not only our understanding of CF pathophysiology but also of the normal mechanisms of electrolyte transport in epithelial tissues. We propose to achieve this goal by pursuit of the following aims: 1) to identify regions of CFTR protein that contribute to its normal function by identification of deleterious-mutations in four groups of individuals with unusual phenotypes: CF patients without pancreatic disease, patients without lung disease but with features suggestive of CF patients with pulmonary disease similar to CF but without pancreatic disease or elevated sweat chloride levels and African-American CF patients because they have been reported to have milder lung disease. 2) to assess the effect of mutations identified in aim 1 by: a) analyzing CFTR expression in respiratory epithelia obtained by nasal brushing from selected patients. CFTR transcription and splicing will be determined by RNA-PCR. CFTR protein processing and localization will be assessed by Western blotting and immunocytochemistry. b) analyzing CFTR function in human epithelial cell lines stably expressing mutant CFTR. Select mutations will be created in the CFTR cDNA within the adeno-associated virus-CFTR expression vector (AAV-CFTR). Human bronchial epithelial cells (IB3) will be co-transfected with the mutagenized AAV-CFTR vector and on AAV vector containing the neomycin resistance gene. Neomycin resistant cell lines with AAV-CFTR integration (assessed by PCR and Southern analysis of genomic DNA) will be assessed for CFTR expression by Northern and Western blotting. Chloride channel and regulatory functions of mutant CFTR will be analyzed by 36-Cl efflux and patch clamp studies in collaboration with Dr. William Guggino (Johns Hopkins University).