Orphan Missense Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator

Abstract

More than 1860 mutations have been found within the human cystic fibrosis transmembrane conductance regulator (CFTR) gene sequence. These mutations can be classified according to their degree of severity in CF disease. Although the most common mutations are well characterized, few data are available for rare mutations. Thus, genetic counseling is particularly difficult when fetuses or patients with CF present these orphan variations. We describe a threestep in vitro assay that can evaluate rare missense CFTR mutation consequences to establish a correlation between genotype and phenotype. By using a green fluorescent protein-tagged CFTR construct, we expressed mutated proteins in COS-7 cells. CFTR trafficking was visualized by confocal microscopy, and the cellular localization of CFTR was determined using intracellular markers. We studied the CFTR maturation process using Western blot analysis and evaluated CFTR channel activity by automated iodide efflux assays. Of six rare mutations that we studied, five have been isolated in our laboratory. The cellular and functional impact that we observed in each case was compared with the clinical data concerning the patients in whom we encountered these mutations. In conclusion, we propose that performing this type of analysis for orphan CFTR missense mutations can improve CF genetic counseling. Cystic fibrosis (CF) is the most common severe autosomal recessive genetic disorder in the white population, with an incidence of approximately 1 in 3500. It results from mutations in the CF transmembrane conductance regulator (CFTR) gene 1,2 that encodes a chloride channel normally expressed at the apical membrane of epithelial cells. 3,4 CFTR is a member of the ATP-binding cassette transporter family. It contains two repeated units composed of a membrane-spanning domain, which includes six transmembrane helices, and a nucleotide-binding domain, which harbors an ATP-binding and hydrolysis site. 5 Both halves are joined by a specific cytoplasmic regulator (R) domain, whose phosphorylation by protein kinase A activates the outward anionic channel activity. 6,7 To date, Ͼ1860 mutations identified within the human CFTR sequence are listed in the CF mutation database (http://www.genet.sickkids.on.ca/Home.html, last accessed March 8, 2011). These mutations can be divided into six classes, according to the mechanism that disrupts CFTR function 8,9 : absence of the protein at the apical plasma membrane because of i) defective protein synthesis or ii) impaired maturation leading to protein degradation, iii) defective regulation of CFTR channel activity, iv) altered ionic selectivity and conductance, v) lowered CFTR mRNA amount, and vi) decreased protein stability.The most common mutation, F508del, is a class II mutation. It is carried by 90% of patients with CF, on at least one allele, and it leads to a severe CF phenotype. In our laboratory, we use the Elucigene CF30 Kit (Gen-Probe, Inc., San Diego, CA), which has been approved in the French national neonatal CF screening program for rou...