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.

Figure 1

L102P and L167R amino acid substitutions impair CFTR protein maturation. A: Fluorescent confocal imaging of mutant GFP-tagged CFTR proteins (green) expressed in COS-7 cells. CFTR partially colocalizes with calreticulin (red). Scale bar = 20 μm. B: Maturation status of mutant CFTR visualized with GFP-specific Western blot analysis. C: CFTR channel activity measured by iodide efflux assays using stimulation cocktail (10 μmol/L forskolin and 30 μmol/L genistein). The null activation observed when the CFTR-specific inhibitor (10 μmol/L CFTR-inh172) is added shows that the anionic efflux is because of CFTR channel activity. Data are presented as the mean ± SEM (n = 3 to 4 for each experimental condition). ***P < 0.001.

Figure 2

P574S amino acid substitution decreases CFTR protein maturation. A: Fluorescent confocal imaging of mutant GFP-tagged CFTR proteins (green) expressed in COS-7 cells. CFTR partially colocalizes with calreticulin (red). Scale bar = 20 μm. B: Maturation status of mutant CFTR visualized with GFP-specific Western blot analysis. Histogram showing the band B/(band B + band C) ratio. C: CFTR channel activity measured by the iodide efflux assay. Data are presented as the mean ± SEM (n = 3 to 4 for each experimental condition). **P < 0.01, ***P < 0.001.

Figure 3

K696R and P841R amino acid substitutions affect CFTR channel activity. A: Fluorescent confocal imaging of mutant GFP-tagged CFTR proteins (green) expressed in COS-7 cells. Phalloidin staining (red) shows partial colocalization of CFTR with cortical actin (arrows). Scale bar = 20 μm. B: GFP-specific Western blot analyses. C: CFTR channel activity measured by iodide efflux assays. Data are presented as the mean ± SEM (n = 3 to 4 for each experimental condition). **P < 0.01.

Figure 4

The V562I amino acid substitution does not alter CFTR phenotype. A: Fluorescent confocal imaging of mutant GFP-tagged CFTR protein (green) expressed in COS-7 cells. Phalloidin staining (red) shows partial colocalization of CFTR with cortical actin (arrows). Scale bar = 20 μm. B: GFP-specific Western blot analysis. C: CFTR channel activity measured by iodide efflux assay. Data are presented as the mean ± SEM (n = 3 to 4 for each experimental condition). ns indicates nonsignificant difference.

Figure 5

Local alignment, centered on the position 562 residue, of CFTR protein sequences from different animal species. We used the PolyPhen (now upgraded to PolyPhen-2) tool, designed to achieve polymorphism phenotyping.