MOLECULAR GENETICS OF CYSTIC FIBROSIS

描述（申请人摘要）：常染色体隐性遗传的研究

DESCRIPTION (Applicant's abstract): Study of the autosomal recessive disorder cystic fibrosis (CF) has produced novel insights into the process of epithelia electrolyte movement at a molecular level. Patients with this disorder have altered viscosity and anti-bacterial properties of secretions in the lungs and pancreas due to defective chloride and sodium transport across epithelial cell membranes. The protein defective in this disease, the CF transmembrane conductance regulator (CFTR), functions as a cAMP-activated chloride channel and, in airway epithelia, as a regulator of other ion channels in the same cell. The latter role of CFTR explains abnormal function of several different ion channels in cells from CF patients, and indicates that this molecule is a critical component of a pathway coordinating ion movement across apical membranes of airway cells. It also suggests that channels regulated by CFTR an the proteins involved in these regulatory pathways may be able to influence lung function independent of CFTR and could therefore be therapeutic targets for CF. The overall goal of this proposal is to determine the importance of th regulatory function of CFTR in pulmonary epithelial electrolyte transport. Thi will be achieved by pursuit of the following aims: 1) to determine whether preservation of the regulatory function of CFTR correlates with improved lung function in patients carrying mutations in each CFTR gene. CFTR mutations will be identified in patients with clinical evidence of CFTR dysfunction but absen lung disease and patients without evidence of CFTR dysfunction but with lung disease similar to CF using the denaturing gradient-gel electrophoresis (DGGE) technique. The consequence of missense mutations upon CFTR processing will be assessed by immunoprecipitation and sizing of mutant CFTR protein transiently expressed in HEK 293 cells. Alteration in the regulatory function will be determined by patch-clamp analysis of CFTR mutants transiently expressed in non-polarized human CF airway epithelial cells and Ussing chamber measurements of electrolyte movement across polarized epithelial cells stably expressing mutant CFTR. 2) To determine whether defects in proteins other than CFTR can give rise to pulmonary phenotype similar to CF. An extensive search for unusua CFTR mutations will be performed in CF patients that have no mutations identified by DGGE. CAMP-activated Cl- conduction will be assessed in patients without CFTR mutations by nasal potential difference testing and patch-clamp analysis of their nasal epithelial cells. Finally, epithelial cells from patients without CFTR mutations but with abnormal cAMP-activated Cl- conductio will be transfected with the wild-type CFTR cDNA to confirm that provision of normally functioning CFTR does not correct the defect in Cl- conduction.