Nanette Kälin

ΔF508 CFTR protein expression in tissues from patients with cystic fibrosis

Heterologous expression of the cystic fibrosis transmembrane conductance regulator (CFTR) provided evidence that the major cystic fibrosis (CF) mutation ΔF508 leads to defective protein folding in the endoplasmic reticulum, which prevents its processing and targeting to the cell surface. In this study, we investigated endogenous CFTR expression in skin biopsies and respiratory and intestinal tissue specimens from ΔF508 homozygous and non-CF patients, using immunohistochemical and immunoblot analyses with a panel of CFTR antibodies. CFTR expression was detected at the luminal surface of reabsorptive sweat ducts and airway submucosal glands, at the apex of ciliated cells in pseudostratified respiratory epithelia and of isolated cells of the villi of duodenum and jejunum, and within intracellular compartments of intestinal goblet cells. In ΔF508 homozygous patients, expression of the mutant protein proved to be tissue specific. Whereas ΔF508 CFTR was undetectable in sweat glands, the expression in the respiratory and intestinal tracts could not be distinguished from the wild-type by signal intensity or localization. The tissue-specific variation of ΔF508 CFTR expression from null to apparently normal amounts indicates that ΔF508 CFTR maturation can be modulated and suggests that determinants other than CFTR mislocalization should play a role in ΔF508 CF respiratory and intestinal disease.

CFTR and differentiation markers expression in non-CF and delta F 508 homozygous CF nasal epithelium.

Human nasal polyps from non-CF and delta F 508 homozygous CF patients were used to compare the expression of CFTR and markers epithelial differentiation, such as cytokeratins (CK) and desmoplakins (DP), at the transcriptional and translational levels. mRNA expression was assessed by semiquantitative RT/PCR kinetic assays while the expression and distribution of proteins were evaluated by immunofluorescence analysis. In parallel, for each nasal tissue specimen, the importance of surface epithelium remodeling and inflammation was estimated after histological observations. Our results show that the steady-state levels of CFTR, CK13, CK18, CK18, CK14, or DP 1 mRNA transcripts in delta F 508 CF nasal polyps were not significantly different from those of non-CF tissues. A variability in the CFTR mRNA transcript level and in the pattern of CFTR immunolabeling has been observed between the different tissue samples. However, no relationship was found between the level of CFTR mRNA transcripts and the CFTR protein expression and distribution, either in the non-CF or in the CF group. The histological observations of non-CF and CF nasal polyp tissue indicated that the huge variations in the expression and distribution of the CFTR protein were associated with the variations in the degree of surface epithelium remodeling and inflammation in the lamina propria. A surface epithelium, showing a slight basal cell hyperplasia phenotype associated with diffuse inflammation, was mainly characterized by a CFTR protein distribution at the apex of ciliated cells in both non-CF and CF specimens. In contrast, in a remodeled surface epithelium associated with severe inflammation, CFTR protein presented either a diffuse distribution in the cytoplasm of ciliated cells, or was absent. These results suggest that abnormal expression and distribution of the CFTR protein of CF airways is not only caused by CFTR mutations. Airway surface epithelium remodeling and inflammation could play a critical role in the posttranscriptional and/or the posttranslational regulation of the CFTR protein expression in non-CF and CF airways.

Detection of more than 50 different CFTR mutations in a large group of German cystic fibrosis patients.

We have conducted a comprehensive study of the molecular basis of cystic fibrosis (CF) in 350 German CF patients. A screening approach based on single-strand conformation analysis and direct sequencing of genomic polymerase chain reaction products has allowed us to detect the molecular defects on 95.4% of the CF chromosomes within the coding region and splice sites of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The spectrum of sequence changes comprises 54 different mutations, including 17 missense mutations, 14 nonsense mutations, 11 frameshift mutations, 10 splice site variants and two amino acid deletions. Eleven of these mutations have not previously been described. Our results reflect the marked mutational heterogeneity of CF in a large sample of patients from a non-isolated population.

Intra- and extragenic marker haplotypes of CFTR mutations in cystic fibrosis families

SummaryIn order to facilitate the screening for the less common mutations in the cystic fibrosis (CF) gene viz., the CF transmembrane conductance regulator gene (CFTR), marker haplotypes were determined for German nonCF (N) and CF chromosomes by polymerase chain reaction analysis of four polymorphisms upstream of the CF gene (XV-2c, KM.19, MP6-D9, J44) and six intragenic polymorphisms (GATT, TUB9, M470V, T854T, TUB18, TUB20) that span the CFTR gene from exon 6 through exon 21. Novel informative sequence variants of CFTR were detected in front of exons 10 (1525-61 A or G), 19 (3601-65 C or A), and 21 (4006-200 A or G). The CF locus exhibits strong long-range marker-marker linkage disequilibrium with breakpoints of recombination between XV-2c and KM.19, and between exons 10 and 19 of CFTR. Marker alleles of GATT-TUB9 and TUB18-TUB20 were found to be in absolute linkage disequilibrium. Four major haplotypes encompass more than 90% of German N and CF chromosomes. Fifteen CFTR mutations detected on 421 out of 500 CF chromosomes were each identified on one of these four predominant 7-marker haplotypes. Whereas all analysed ΔF508 chromosomes carried the same KM.19-D9-J44-GATT-TUB9-M470V-T854T haplotype, another frequent mutation in Germany, R553X, was identified on two different major haplotypes. Hence, a priori haplotyping cannot exclude a particular CF mutation, but in combination with population genetic data, enables mutations to be ranked by decreasing probability.

Applicability of Different Antibodies for Immunohistochemical Localization of CFTR in Sweat Glands from Healthy Controls and from Patients with Cystic Fibrosis

The hereditary disease cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Understanding of the consequences of CFTR gene mutations is derived chiefly from in vitro studies on heterologous cell cultures and on cells hyperexpressing CFTR. Data from ex vivo studies on human tissue are scarce and contradictory, a fact which is in part explained by secondary tissue destruction in most affected organs. The purpose of this study was to establish conditions under which wild-type and mutated CFTR can be studied in affected human tissue. Sweat glands carry the basic defect underlying CF and are not affected by tissue destruction and inflammation. Therefore, we used this tissue to test a panel of eight different CFTR antibodies under various fixation techniques. The antibodies were tested on skin biopsy sections from healthy controls, from CF patients homozygous for the most common mutation, ΔF508, and from patients carrying two nonsense mutations. Of the eight CFTR antibodies, only three—M3A7, MATG 1104, and cc24—met the criteria necessary for immunolocalization of CFTR in sweat glands. The labeling pattern in the CF sweat glands was consistent with the postulated processing defect of ΔF508 CFTR. The antibodies exhibited different sensitivities for detecting ΔF508 CFTR.

A termination mutation (2143delT) in the CFTR gene of German cystic fibrosis patients

German patients with cystic fibrosis (CF) were screened for molecular lesions in exon 13 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by single strand conformation polymorphism (SSCP) and chemical cleavage of mismatch analyses. Direct sequencing of four samples that displayed the same SSCP pattern and that were susceptible to cleavage of heteroduplexes by osmium tetroxide revealed, in all cases, a deletion of a single T residue at nucleotide position 2143 within codon 671 of the CFTR gene. As a result, leucine codon 671 is changed into a termination codon. In total, the 2143delT mutation was confirmed in 6 out of 271 German non-ΔF508 CF chromosomes by artificial restriction fragment length polymorphism analysis, indicating that this frameshift mutation accounts for about 2% of German non-Δ508 mutations. The 6 pancreas insufficient patients who are compound heterozygous for 2143-delT suffer from the typical features of pulmonary and gastrointestinal CF disease. The 2143delT mutation completes the panel of the more frequent CFTR mutations that reside on the “ΔF508 haplotype” and that contribute to its overpresentation among German non-ΔF508 alleles that are associated with severe forms of disease.

Cystic fibrosis: the impact of analytical technology for genotype-phenotype studies

The generalized exocrinopathy cystic fibrosis (CF) is the most common severe genetic disease in Caucasian populations. A panel of more than 700 chromosomes from German and Turkish CF patients was screened for disease-causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene by chemical cleavage of mismatch, single strand conformation polymorphism, restriction analysis and direct sequencing of genomic DNA amplified by polymerase chain reaction. Besides the major 3-bp deletion, delta F508 that was found on 73% of German CF chromosomes, more than 50 other missense, nonsense, frame-shift, and splice-site mutations have already been identified. In general, a CFTR mutation is linked with a single 10-marker haplotype which indicates that in most cases a particular mutation spread from a common ancestor. The comparison of mutation genotypes with the disease phenotype emphasized the causative role of the type and localization of the CFTR mutation for clinical course and prognosis. Pancreatic status and the risk of colonization of airways with opportunistic pathogens are genetically determined. Most patients who are harbouring mutations in the nucleotide binding folds were suffering from severe CF disease. Mild or even aberrant forms of CF were observed for many missense mutations located in the putative transmembrane domains or for mutations that are expected to result in a truncated protein of half of wild-type CFTR.