title: Orphan missense mutations in the cystic fibrosis transmembrane conductance regulator: A three-step biological approach to establishing a correlation between genotype and phenotype.
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 three-step 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.
authors.affiliation: Institute of Physiology and Cell Biology (IPCB), National Centre for Scientific Research (NCSR), Université de Poitiers, Poitiers, France.
authors.forename: Fleur
authors.initials: F
authors.lastname: Fresquet
authors.forename: Romain
authors.initials: R
authors.lastname: Clement
authors.forename: Caroline
authors.initials: C
authors.lastname: Norez
authors.forename: Adélaïde
authors.initials: A
authors.lastname: Sterlin
authors.forename: Patricia
authors.initials: P
authors.lastname: Melin
authors.forename: Frédéric
authors.initials: F
authors.lastname: Becq
authors.forename: Alain
authors.initials: A
authors.lastname: Kitzis
authors.forename: Vincent
authors.initials: V
authors.lastname: Thoreau
authors.forename: Frédéric
authors.initials: F
authors.lastname: Bilan
mesh_terms: D000328:Adult; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D019556:COS Cells; D002675:Child, Preschool; D002522:Chlorocebus aethiops; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000235:genetics*; D005260:Female; D056726:Genetic Association Studies / Q000379:methods*; D006801:Humans; D007231:Infant, Newborn; D015640:Ion Channel Gating; D008297:Male; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000235:genetics; D020125:Mutation, Missense / Q000235:genetics*; D010641:Phenotype; D016415:Sequence Alignment
chemical_list: D050505:Mutant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator