MOLECULAR GENETICS OF CYSTIC FIBROSIS

描述(申请人摘要)：常染色体隐性遗传研究 无序性囊性纤维化(CF)对这个过程有了新的见解 在分子水平上研究上皮细胞的电解质运动。患有这种病的患者 紊乱改变了分泌物的粘度和抗菌特性 在肺和胰腺由于氯化物和钠的转运缺陷 穿过上皮细胞膜。在这种疾病中有缺陷的蛋白质， CF跨膜电导调节器(CFTR)，其功能是 CAMP激活的氯通道，并在呼吸道上皮细胞中作为一种调节因子 同一细胞中的其他离子通道。CFTR的后一种作用解释说 几种不同离子通道在CFs细胞中的功能异常 患者，并表明这种分子是一个关键的组成部分 通过呼吸道细胞顶膜协调离子运动的途径。 这也表明，受CFTR调控的通道和参与其中的蛋白质 这些调节通路可能能够独立影响肺功能。 因此，它可能成为治疗CF的靶点。总目标 这项建议的目的是确定监管职能的重要性 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 追求以下目标：1)确定是否保存 CFTR的调节功能与改善肺功能有关 携带每个CFTR基因突变的患者。CFTR突变将是 在有临床证据的患者中发现CFTR功能障碍，但Absen 肺部疾病和没有CFTR功能障碍证据但有肺的患者 变性梯度凝胶电泳法检测类似于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的大小来评估加工 在HEK 293细胞中瞬时表达的蛋白质。更改 调控功能将通过对CFTR的膜片钳分析来确定 突变体在非极化的人CF呼吸道上皮细胞中瞬时表达 电池和电池室中电解质迁移的测量 极化上皮细胞稳定表达突变的cftr。2)确定 Cftr以外的蛋白质缺陷是否会导致肺 表型与CF型相似。对unusua CFTR突变的广泛搜索将 在DGGE未发现突变的CF患者中进行。 CAMP激活的氯离子传导将在没有CFTR的患者中进行评估 鼻电势差试验和膜片钳技术检测鼻咽癌突变 它们的鼻腔上皮细胞。最后，患者的上皮细胞 没有cftr突变但cAMP激活的氯电导异常的人将 将野生型cftr基因导入小鼠体内，证实 正常工作的CFTR不能纠正氯离子传导的缺陷。

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.