MOLECULAR GENETICS OF CYSTIC FIBROSIS

描述（申请人摘要）：常染色体隐性遗传 疾病囊性纤维化（CF）产生了新的见解的过程 上皮细胞电解质运动的分子水平。 患者与此 疾病改变了分泌物的粘度和抗菌特性 由于氯和钠转运缺陷， 穿过上皮细胞膜 这种疾病中的蛋白质缺陷， CF跨膜传导调节器（CFTR），作为一种 cAMP激活的氯离子通道，并在气道上皮细胞中作为 同一细胞中的其他离子通道。 CFTR的后一个作用解释了 CF细胞中几种不同离子通道的功能异常 患者，并表明这种分子是一个关键组成部分， 协调离子穿过气道细胞顶膜的途径。 它还表明，CFTR调节的通道和参与CFTR的蛋白质可能与CFTR有关。 这些调节途径可能能够独立影响肺功能 CFTR，因此可能是CF的治疗靶点。 总目标 这一建议的重要性是确定的调节功能， CFTR在肺上皮细胞电解质转运中的作用 这将通过以下方式实现 （1）确定是否保存 CFTR的调节功能与肺功能改善相关， 每个CFTR基因都携带突变的患者。 CFTR突变将是 在具有CFTR功能障碍的临床证据但未发现 肺部疾病和没有CFTR功能障碍证据但肺部 用变性梯度凝胶电泳检测类似CF的疾病 （DGGE）技术。 错义突变对CFTR的影响 将通过免疫沉淀和突变CFTR的大小测定来评估处理 在HEK 293细胞中瞬时表达的蛋白。 蚀变 调节功能将通过CFTR的膜片钳分析来确定 在非极化的人CF气道上皮中瞬时表达的突变体 电解质运动的细胞和Ussing室测量 稳定表达突变CFTR的极化上皮细胞。 2)以确定 CFTR以外的蛋白质缺陷是否会引起肺 表型与CF相似。 对独特CFTR突变的广泛搜索将 在DGGE未鉴定出突变的CF患者中进行。 将在无CFTR的患者中评估cAMP激活的Cl-传导 鼻电位差测试和膜片钳分析的突变， 鼻上皮细胞 最后， 没有CFTR突变，但有异常cAMP激活的Cl-传导， 用野生型CFTR cDNA转染，以确认提供 功能正常的CFTR不能纠正Cl-传导的缺陷。

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.