Novel and recurrent rearrangements in the CFTR gene: clinical and laboratory implications for cystic fibrosis screening

Because standard techniques used to detect mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene do not detect single or multiple exonic rearrangements, the importance of such rearrangements may be underestimated. Using an in-house developed, single-tube, semi-quantitative fluorescent PCR (SQF PCR) assay, we analyzed 36 DNA samples submitted for extensive CFTR sequencing and identified ten samples with rearrangements. Of 36 patients with classic CF, 10 (28%) harbored various deletions in the CFTR gene, accounting for 14% of CF chromosomes. A deletion encompassing the CFTR promoter and exons 1 and 2 was detected in a sample from one proband, and in the maternal DNA as well. In another family, a deletion of the promoter and exon 1 was detected in three siblings. In both of these cases, the families were African American and the 3120+1G>A splice site mutation was also identified. These promoter deletions have not been previously described. In a third case, a deletion of exons 17a, 17b, and 18 was identified in a Caucasian female and the same mutation was detected in the paternal DNA. In the other seven cases, we identified the following deletions: exons 2 and 3 (n=2); exons 4, 5, and 6a; exons 17a and 17b; exons 22 and 23; and exons 22, 23, and 24 (n=2). In our series, the frequency of CFTR rearrangements in classic CF patients, when only one mutation was identified by extensive DNA sequencing, was >60% (10/16). Screening for exon deletions and duplications in the CFTR gene would be beneficial in classic CF cases, especially when only one mutation is identified by standard methodologies. An erratum to this article can be found at     

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.     

CFTR in cystic fibrosis and cholera: from membrane transport to clinical practice

We have used a brief analysis of transport via cystic fibrosis (CF) transmembrane conductance regulators (CFTRs) in various organ systems to highlight the importance of basic membrane transport processes across epithelial cells for first-year medical students in physiology. Because CFTRs are involved in transport both physiologically and pathologically in various systems, we have used this clinical correlation to analyze how a defective gene leading to defective transport proteins can be directly involved in the symptoms of cholera and CF. This article is a "Staying Current" approach to transport via CFTRs including numerous helpful references with further information for a teaching faculty member. The article follows our normal presentation which begins with a discussion of the involvement of CFTR transport in the intestine and how cholera affects intestinal transport, extends to CFTR transport in various organ systems in CF, and concludes with the logic behind many of the treatments that improve CF. Student learning objectives are included to assist in assessment of student understanding of the basic concepts.     

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.     

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.     

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.     

Molecular basis of cystic fibrosis in Lithuania: incomplete CFTR mutation detection by PCR-based screening protocols

Mutational analysis of the cystic fibrosis transmembrane regulator (CFTR) gene was performed in 98 unrelated CF chromosomes from 49 Lithuanian CF patients through a combined approach in which the p.F508del mutation was first screened by allele-specific PCR while CFTR mutations in nonp.F508del chromosomes have been screened for by denaturing gradient gel electrophoresis analysis. A CFTR mutation was characterized in 62.2% of CF chromosomes, two of which (2.0%) have been previously shown to carry a large gene deletion CFTRdele2,3(21 kb). The most frequent Lithuanian CF mutation is p.F508del (52.0%). Seven CFTR mutations, p.N1303K (2.0%), p.R75Q (1.0%), p.G314R (1.0%), p.R553X (4.2%), p.W1282X (1.0%), and g.3944delGT (1.0%), accounted for 10.1% of Lithuanian CF chromosomes. It was not possible to characterize 35.8% of the CF Lithuanian chromosomes. Analysis of intron 8 (TG)mTn and M470V polymorphic loci did not permit the characterization of the CFTR dysfunction underlying the CF phenotype in the patients for which no CFTR mutation was identified. Thus, screening of the eight CFTR mutations identified in this study and of the large deletion CFTRdele2,3(21 kb) allows the implementation of an early molecular or confirmatory CF diagnosis for 65% of Lithuanian CF chromosomes.     

Discrimination between recurrent mutation and identity by descent: application to point mutations in exon 11 of the cystic fibrosis (CFTR) gene

Summary A total of 75 non-F508 chromosomes from 59 German cystic fibrosis patients was screened for mutations in exon 11 of the cystic fibrosis (CFTR) gene. These Caucasian patients were found to possess an identical haplotype background for two common mutations (G551D, R553X) constistent with their being identical by descent. However, a different R553X associated haplotype found in American black patients was suggestive of recurrent mutation, a postulate supported by the location of the R553X alteration in a hypermutable CpG dinucleotide. Likelihood estimates for recurrent mutation and identity by descent were compared and strongly supported the hypothesis of recurrent R553X mutation. The ability to distinguish between these two alternatives provides an indication of whether or not the search for mutations should be restricted to chromosomes with similar haplotypes.     

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.     

Frequency of the F508 deletion in the CFTR gene in Turkish cystic fibrosis patients

Summary The F508 deletion in the cystic fibrosis transmembrane conductance regulator (CFTR) gene was found in 8 out of 30 Turkish cystic fibrosis (CF) chromosomes (27%). Five Turkish ΔF508 CF chromosomes were associated with the risk haplotype B in KM19 (2 allele)/XV2c (1 allele). In the Turkish population, cystic fibrosis is predominantly caused by mutations other than the F508 deletion.     

Neonatal screening for cystic fibrosis: result of a pilot study using both immunoreactive trypsinogen and cystic fibrosis gene mutation analyses

We have evaluated a two-tier neonatal cystic fibrosis (CF) screening of immunoreactive trypsinogen (IRT) followed by CFTR gene mutation analysis using a systematic scanning of exons 7, 10, and 11, and, if necessary, by direct DNA sequencing. Over an 18-month period we screened 32,300 neonates born in the western part of Britanny. The first tier, involving IRT screening at 3 days of age, utilizes a low elevation of the trypsinogen level (600 ng/ml), which is highly sensitive. The second tier, which corresponds to the exhaustive screening for mutations in three exons of the gene, is highly specific for this population (Britanny). The false positive rate is very low, and no false negatives have been reported to date. This strategy has allowed the identification of five novel alleles (V322A, V317A, 1806 del A, R553G, G544S). Moreover the test can be adapted to other countries in which the distribution of mutations is established.     

A frequent large rearrangement in the CFTR gene in cystic fibrosis patients from Reunion Island

Reunion Island is a French province, 800 km east of Madagascar and 200 km west of Mauritius. On Reunion Island, the birth prevalence of cystic fibrosis (CF) is particularly high in the population of European origin, approximately 1:1000. In a previous study, we demonstrated that the screening of the 27 exons of the CF transmembrane conductance regulator (CFTR) gene by denaturing high-pressure liquid chromatography (DHPLC) in 114 CF families allowed the detection of about 93% of the molecular defects present on Reunion Island. Unidentified CF mutations may lie in introns or in regulatory regions that are not routinely investigated, or may correspond to gene rearrangements such as large, heterozygous deletions that escape detection using current PCR-based techniques. Using a combination of different methods (such as multiplex ligation-dependent probe amplification), 6 of the 13 unidentified CF alleles (46%) were found to harbor a deletion of 5288 bp, spanning from exon 17a to 18. Identification and examination of the breakpoint sequences showed that this deletion is different from the 3120+1kbdel8.6Kb previously found in the Palestinian Arabs. The chromosomes bearing IVS16+3316_IVS18+644del5288 did not have a common extragenic haplotype. Clinical evaluation of homozygotes (2 unrelated patients) and compound heterozygotes indicated that this deletion represents a severe mutation associated with positive sweat chloride test, pancreatic insufficiency, and early age at diagnosis.