The spectrum of CFTR mutations in south-west German cystic fibrosis patients

The cystic fibrosis transmembrane conductance regulator (CFTR) gene of 110 cystic fibrosis (CF) patients from the south-west of Germany was screened for 12 different mutations. This analysis resulted in an identification of 79% of all CF mutations and a complete genotype in 66% of the families. The most common mutation found was F508 (67%). Another 5 mutations accounted for a further 12.5% (4% G542X; 3% R553X; 3% N1303K; 2% 1717-1 GA; 0.5% G551D) whereas 6 mutations (R117H, A455E, I507, S549I, S549N, and R1162X) were not found. Fifty-four (49%) patients were AF508 homozygotes and 18 (16.5%) were compound heterozygotes for F508 and one of the rarer mutations. These frequencies differ slightly from those found in the north of Germany and considerably from those reported from the south of Europe, which seems to be consistent with a north to south decline of the relative abundance of F508. Two patients, age 6 and 25 years, were compound heterozygotes for G542X and N1303K. The clinical features of the 6 year old were characterised by severe gastrointestinal and as yet only mild pulmonary complications whereas the 25 year old manifested severe pulmonary and gastrointestinal symptoms indicating that the N1303K mutation of the C-terminal CFTR nucleotide binding fold significantly impairs protein function in both the pancreas and the lungs.     

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

Summary In 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 F508 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.     

Exon 9 of the CFTR gene: splice site haplotypes and cystic fibrosis mutations

The alternatively spliced exon 9 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene codes for the initial part of the amino-terminal nucleotide-binding fold of CFTR. A unique feature of the acceptor splice site preceding this exon is a variable length polymorphism within the polypyrimidine tract influencing the extent of exon 9 skipping in CFTR mRNA. We investigated this repeat for its relationship to CFTR mutations and intragenic markers on 200 chromosomes from German patients with cystic fibrosis (CF). Four frequent length variations were strongly associated with the four predominant haplotypes previously defined by intragenic marker dimorphisms. One of these alleles displayed absolute linkage disequilibrium to the major CF mutation F508. Other frequent CFTR mutations were linked to one particular splice site haplotype indicating that differential exon 9 skipping contributes little to the clinical heterogeneity among CF patients with an identical mutation. We also identified a novel missense mutation (V456F) and a novel nonsense mutation (Q414X) within the coding region of exon 9. The missense mutation V456F adjacent to Walker motif A was present in a pancreas-sufficient CF patient. In contrast, the pancreas-insufficient Q414X/F508 compound heterozygote suffered from a severe form of the disease, indicating that alternative splicing of exon 9 does not overcome the deleterious effect of a stop codon within this exon.     

Heterogeneity in the severity of cystic fibrosis and the role of CFTR gene mutations

Cystic fibrosis is a common, fatal disorder caused by abnormalities in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR encodes a chloride channel that regulates secretion in many exocrine tissues. The presentation of cystic fibrosis is highly variable as measured by the age of onset of disease, the presence of pancreatic insufficiency, or the progression of lung disease. Over 400 mutations in the CFTR gene have been described in cystic fibrosis patients and considerable effort has focused on the correlation between specific mutations and genotypes and clinical characteristics. Individual tissues display variation in their sensitivity to CFTR mutations. The vas deferens is functionally disrupted in nearly all males, whereas mild and severe pancreatic involvement is determined by the patient's genotype. The severity of pulmonary disease is poorly correlated with genotype, suggesting that there are other important genetic and/or environmental factors that contribute to lung infections and the subsequent disruption of lung function.     

CFTR mutations in Turkish and North African cystic fibrosis patients in Europe: implications for screening

AIMS: To obtain more insight into the variability of the CFTR mutations found in immigrant cystic fibrosis (CF) patients who are living in Europe now, and to estimate the test sensitivity of different frequently used methods of DNA analysis to detect CF carriers or patients among these Turkish or North African immigrants. METHODS: A survey among 373 European CF centers asking which CFTR mutations had been found in Turkish and North African CF patients. RESULTS: 31 and 26 different mutations were reported in Turkish and North African patients, identifying 64.2% (113/176) and 87.4% (118/135) alleles, respectively (p < 0.001). The mean sensitivity (detection rate) of three most common CFTR mutation panels to detect these mutations differed between Turkish and North African people, 44.9% (79/176) versus 69.6% (94/135) (p < 0.001), and can be increased to 57.4% (101/176) and 79.3% (107/135) (p < 0.001), respectively, by expanding these panels with 13 mutations which have been found on two or more alleles. CONCLUSION: 35.8% and 12.6%, respectively, of CF alleles in Turkish and North African patients living in Europe now had not been identified. Among these populations, the test sensitivity of common CFTR mutation panels is insufficient for use in screening programs in Europe, even after expansion with frequent Turkish and North African mutations. This raises questions about whether and how to implement CF carrier and neonatal screening in a multiethnic society.     

The cystic fibrosis and is product CFTR

Cystic fibrosis is the commonest, fatal, inherited disease of caucasian populations occurring with a frequency of 1 in 2000 live births. The CF gene spans about 230 kb of genomic DNA and encodes a protein of 1480 amino acids named the cystic fibrosis transmembrane conductance regulator (CFTR). The primary sequence predicts that CFTR is an ABC type protein with twelve transmembrane spans, two nucleotide binding domains and a cytoplasmic regulatory domain. CFTR functions as a cyclic AMP-regulated, low conductance, chloride channel in epithelial cells, but other roles are possible. Failure of the CFTR channel in CF reduces epithelial salt and water secretion, leading to a dehydration of epithelial surfaces which initiates the pathology of the disease.     

CFTR mutations impart elevated immune reactivity in a murine model of cystic fibrosis related diabetes

Increased life expectancy in cystic fibrosis (CF) is accompanied by an increasing incidence of CF related diabetes (CFRD). Altered immune reactivity occurs in CF, which we hypothesize, is exacerbated by hyperglycemia. Cystic fibrosis transmembrane conductance regulator deficient (CFTR-/-) mice were rendered hyperglycemic by streptozotocin (STZ) to test this hypothesis. CFTR-/-, C57BL/6J, and FVB/NJ mice received either STZ or lactated ringers (LR) (n=5-10). Four weeks later, splenocytes were harvested, mitogen stimulated, and analyzed for cytokine production (IL-2, IL-4, and IL-10) along with stimulation indices (SI). SI of STZ-treated CFTR-/- were elevated compared to LR-treated mice, although both were greater than C57BL/6J and FVB/NJ (p<0.05). Fasting glucose levels of STZ-treated CFTR-/- mice correlated with SI (p<0.003). Stimulated IL-10 concentrations were elevated in STZ-treated CFTR-/- compared to LR-treated animals and controls (p<0.05). IL-2 levels were greater in CFTR-/- mice compared to controls (p<0.05), but unrelated to STZ. Reinforcing generalized cytokine up-regulation in CFTR-/-, IL-4 levels were greater in CFTR-/- mice compared to C57BL/6J, but FVB/NJ mice demonstrated greatest concentrations following STZ. These results suggest that, hyperglycemia may exacerbate the clinical course in CF by impacting immune reactivity. There is clear need to maximize metabolic management in CFRD.     

CFTR inhibition mimics the cystic fibrosis inflammatory profile

Primary airway epithelial cells grown in air-liquid interface differentiate into cultures that resemble native epithelium morphologically, express ion transport similar to those in vivo, and secrete cytokines in response to stimuli. Comparisons of cultures derived from normal and cystic fibrosis (CF) individuals are difficult to interpret due to genetic differences besides CFTR. The recently discovered CFTR inhibitor, CFTR(inh)-172, was used to create a CF model with its own control to test if loss of CFTR-Cl(-) conductance alone was sufficient to initiate the CF inflammatory response. Continuous inhibition of CFTR-Cl(-) conductance for 3-5 days resulted in significant increase in IL-8 secretion at basal (P = 0.006) and in response to 10(9) Pseudomonas (P = 0.0001), a fourfold decrease in Smad3 expression (P = 0.02), a threefold increase in RhoA expression, and increased NF-kappaB nuclear translocation upon TNF-alpha/IL-1beta stimulation (P < 0.000001). CFTR inhibition by CFTR(inh)-172 over this period does not increase epithelial sodium channel activity, so lack of Cl(-) conductance alone can mimic the inflammatory CF phenotype. CFTR(inh)-172 does not affect IL-8, IL-6, or granulocyte/macrophage colony-stimulating factor secretion in two CF phenotype immortalized cell lines: 9/HTEo(-) pCEP-R and 16HBE14o(-) AS, or IL-8 secretion in primary CF cells, and inhibitor withdrawal abolishes the increased response, so CFTR(inh)-172 effects on cytokines are not direct. Five-day treatment with CFTR(inh)-172 does not affect cells deleteriously as evidenced by lactate dehydrogenase, trypan blue, ciliary activity, electron micrograph histology, and inhibition reversibility. Our results support the hypothesis that lack of CFTR activity is responsible for the onset of the inflammatory cascade in the CF lung.     

New insights into cystic fibrosis: molecular switches that regulate CFTR

Cystic fibrosis transmembrane conductance regulator (CFTR), a Cl(-)-selective ion channel, is a prototypic member of the ATP-binding cassette transporter superfamily that is expressed in several organs. In these organs, CFTR assembles into large, dynamic macromolecular complexes that contain signalling molecules, kinases, transport proteins, PDZ-domain-containing proteins, myosin motors, Rab GTPases, and SNAREs. Understanding how these complexes regulate the intracellular trafficking and activity of CFTR provides a unique insight into the aetiology of cystic fibrosis and other diseases.     

Spectrum of mutations in cystic fibrosis

Cystic fibrosis (CF) is a disorder characterized by elevated sweat electrolytes and thick mucous secretions due to abnormal chloride permeability in epithelial tissues. The gene responsible for this disease, the CF transmembrane conductance regulator (CFTR) was identified by a positional cloning approach 3 years ago. Since that time, over two hundred mutations have been found in CFTR genes from affected individuals. Analysis of these disease-associated mutations has provided new insight into the etiology of this disease and into the mechanisms of epithelial electrolyte secretion.     

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.     

Three point mutations in the CFTR gene in French cystic fibrosis patients: Identification by denaturing gradient gel electrophoresis

Summary The cystic fibrosis (CF) gene was recently identified as a gene spanning 250 kilobases (kbp) and coding for a 1480 amino acid protein, cystic fibrosis transmembrane conductance regulator (CFTR). Approximately 70% of CF mutations involve a three-base-pair deletion in CFTR exon 10, resulting in the loss of a phenylalanine at position 508 in the gene product (ΔF508). In order to screen for other molecular defects, we have used a strategy based on denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified gene segments. This method, which permits rapid detection of any sequence change in a given DNA stretch, was used successfully to analyse 61 non-ΔF508 CF chromosomes from French CF patients. A study of CFTR exons 10, 11, 14a, 15 and 20 detected three mutations located in exons 14a, 15 and 20, along with several nucleotide sequence polymorphisms. These nucleotide changes were identified by direct sequencing of PCR fragments displaying altered electrophoretic behaviour, together with some of the polymorphisms and mutations previously characterized by others. The strategy presented here constitutes a valuable tool for the development of carrier testing for individuals or couples with a family history of cystic fibrosis, and will contribute to deciphering the functionally important regions of the CFTR gene.     

Retrospective study of the cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in Guthrie cards from a large cohort of neonatal screening for cystic fibrosis

The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes a cAMP-activated chloride channel, and in individuals with both alleles of the gene mutated, symptoms of CF disease are manifest. With more than 300 mutations so far described in the gene the profile of mutant alleles in a population is specific to its ethnic origin. For an analysis with an unbiased recruitment of the CF alleles in neonates of similar origin (Normandy, France), we have retrospectively analyzed the Guthrie cards of affected newborns, diagnosed by the immunoreactive trypsinogen (IRT) assay. Analysis of the 27 exons of the CFTR gene using a GC clamp denaturing gradient gel electrophoresis (DGGE) assay has enabled us to identify over 96% of the mutated alleles. Two of these were novel mutations. We would like to propose this strategy as an efficient method of retrospective molecular genetic diagnosis that can be performed wherever Guthrie cards can be obtained. Knowledge of rare alleles could be a prerequisite for CF therapy in the future.     

Cystic fibrosis in Lebanon: distribution of CFTR mutations among Arab communities

Cystic fibrosis (CF) is thought to be rare among the Arab populations from the Middle East and little data have been reported so far. We have studied a sample of 20 families living in Lebanon for several generations and who have at least one child with CF. These families are mainly from the Maronite, Greek Catholic, Greek Orthodox, Shiite or Sunnite groups. We found a 50% rate of consanguineous marriage, independent of the community of origin. The distribution of CF genotypes was determined through the screening of all exons of the CFTR (cystic fibrosis transmembrane conductance regulator) gene by the technique of denaturing gradient gel electrophoresis combined with asymmetric amplification DNA sequencing. A total of ten different mutations accounting for 87.5% of 32 unrelated CF alleles was identified, including two novel putative mutations (E672del and IVS21-28G→A). Three mutations, ΔF508 (37.5%), W1282X (15.6%), and N1303K (9.4%) accounted for 62.5% of CF alleles. Interestingly, in the Maronite group, 66.7% of the ΔF508 chromosomes were found to be associated with allele 7 of the IVS8(T)tract, contrasting with the absolute linkage disequilibrium between European ΔF508 chromosomes and allele 9. During this study, two previously undescribed polymorphisms (IVS14a + 17del5 and 2691T/C) were also identified. Received: 2 January 1997 / Accepted: 16 March 1997     

Molecular characterization of cystic fibrosis: 16 novel mutations identified by analysis of the whole cystic fibrosis conductance transmembrane regulator (CFTR) coding regions and splice site junctions.

The spectrum of cystic fibrosis (CF) mutations was determined in 105 patients by using denaturing gradient gel electrophoresis to screen the entire coding regions and adjacent cystic fibrosis transmembrane conductance regulator (CFTR) gene sequences. The nucleotide substitutions detected included 16 novel mutations, 11 previously described defects, and 11 nucleotide sequence polymorphisms. Among the novel mutations, 6 were of the missense type, 4 were nonsense mutations, 4 were frameshift defects, and 2 affected mRNA splicing. The mutations involved all the CFTR domains, including the R domain. Of the 61 non-delta F508 CF chromosomes studied, mutations were found on 36 (59%), raising the proportion of CF alleles characterized in our patient cohort to 88%. Given the efficacy of the screening method used, the remaining uncharacterized mutations probably lie in DNA sequences outside the regions studied, e.g., upstream-promoter sequences, the large introns, or putative regulatory regions. Our results further document the highly heterogeneous nature of CF mutations and provide the information required for DNA-based genetic testing.     

The spectrum of cystic fibrosis mutations.

Although the major mutation causing cystic fibrosis accounts for almost 70% of mutant chromosomes screened, almost 300 sequence alterations have been identified in the gene during the past two and a half years. At least 230 of these mutations are probably associated with disease. This rapid accumulation of data is in part due to the highly coordinated effort by members of the Cystic Fibrosis Genetic Analysis Consortium. The information is not only essential to genetic diagnosis, but also will aid in understanding the structure and function of the protein, and possibly in correlating genotype with phenotype.