Cystic fibrosis in the year 2020: A disease with a new face

The autosomal recessive disease cystic fibrosis (CF) was once untreatable and deadly in childhood, but now most patients survive to adulthood. Many countries have instituted CF newborn screening because early diagnosis improves outcome. CF research has greatly intensified following the discovery of the CF transmembrane conductance regulator (CFTR) gene, which has more than 2000 different mutations. For patients with common mutations like F508del, CFTR modulators are life transforming and may even prevent major complications if started early in childhood. For some patients with rare CFTR mutations, a treatment path still needs to be developed. Conclusion: This review provides a general update on CF, including screening and current and future treatment.     

Rare CFTR mutation 1525-1G>A in a Pakistani patient

Cystic fibrosis (CF) is rare in non-Caucasian populations, and in such populations little is known about the spectrum of mutations and polymorphisms in the cystic fibrosis transmembrane conductance (CFTR) gene. We report the detection of a very rare CFTR mutation 1525-1G>A in intron 9 in a 5-year-old Pakistani child with typical clinical features of CF. It remains to be seen whether mutation 1525-1G>A is characteristic of Pakistani ethnicity with CF or associated with severe phenotypic features.     

Role of <Emphasis Type="Italic">CFTR</Emphasis> mutation analysis in the diagnostic algorithm for cystic fibrosis

Background:The cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation identification is being used with increased frequency to aid in the diagnosis of cystic fibrosis (CF) in those suspected with CF.Aim of this study was to identify diagnostic outcomes when CFTR mutational analysis was used in CF diagnosis.CFTR mutational analysis results were also compared with sweat chloride results.Methods:This study was done on all patients at our institution who had CFTR mutation analysis over a sevenyear period since August 2006.Results:A total of 315 patients underwent CFTR mutational analysis.Fifty-one (16.2％) patients had two mutations identified.Among them 32 had positive sweat chloride levels (≥60 mmol/L),while seven had borderline sweat chloride levels (40-59 mmol/L).An additional 70 patients (22.3％) had only one mutation identified.Among them eight had positive sweat chloride levels,and 17 had borderline sweat chloride levels.Fifty-five patients (17.5％) without CFTR mutations had either borderline (n=45) or positive (n=10) sweat chloride results.Three patients with a CF phenotype had negative CFTR analysis but elevated sweat chloride levels.In eighty-three patients (26.4％) CFTR mutational analysis was done without corresponding sweat chloride testing.Conclusions:Although CFTR mutation analysis has improved the diagnostic capability for CF,its use either as the first step or the only test to diagnose CFTR dysfunction should be discouraged and CF diagnostic guidelines need to be followed.     

Molecular biological analysis of cystic fibrosis--a model example for the strategy of "reverse genetics"]

The elucidation of the basic defect causing cystic fibrosis (CF) is a paradigm for the application of "reverse genetics" to the analysis of human genetic disease. Following this strategy, linkage analysis localized the responsible gene for CF on chromosome 7. Chromosome mediated gene transfer and chromosome walking and jumping led to the isolation of the CFTR-gene and its cDNA. A major 3 bp deletion mutation (DeltaF508) and more than 100 other mutations of this gene have been identified as molecular basis of cystic fibrosis. The CFTR-amino acid sequence, obtained by conversion of the cDNA-sequence, indicates that CFTR belongs to a group of integral membrane transport proteins (ABC-proteins). The normal cAMP-stimulated chloride-transport, lacking in CF-cells is restored by transfer and expression of CFTR-cDNA-recombinants in these cells. CFTR is most likely itself a chloride channel. The molecular identification of this gene has already led to substantial advances in diagnosis and prevention of this disease. New therapeutic approaches by pharmacological means or gene therapy are expected from the further molecular and functional analysis of the CFTR-gene.     

Calcium-modulated chloride pathways contribute to chloride flux in murine cystic fibrosis-affected macrophages

Cystic fibrosis (CF), a common lethal inherited disorder defined by ion transport abnormalities, chronic infection, and robust inflammation, is the result of mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein, a cAMP-activated chloride (Cl-) channel. Macrophages are reported to have impaired activity in CF. Previous studies suggest that Cl- transport is important for macrophage function; therefore, impaired Cl- secretion may underlie CF macrophage dysfunction. To determine whether alterations in Cl- transport exist in CF macrophages, Cl- efflux was measured using N-[ethoxycarbonylmethyl]- 6-methoxy-quinolinium bromide (MQAE), a fluorescent indicator dye. The contribution of CFTR was assessed by calculating Cl- flux in the presence and absence of cftr(inh)-172. The contribution of calcium (Ca(2+))-modulated Cl- pathways was assessed by examining Cl- flux with varied extracellular Ca(2+) concentrations or after treatment with carbachol or thapsigargin, agents that increase intracellular Ca(2+) levels. Our data demonstrate that CFTR contributed to Cl- efflux only in WT macrophages, while Ca(2+)-mediated pathways contributed to Cl- transport in CF and WT macrophages. Furthermore, CF macrophages demonstrated augmented Cl- efflux with increases in extracellular Ca(2+). Taken together, this suggests that Ca(2+)-mediated Cl- pathways are enhanced in CF macrophages compared with WT macrophages.     

What's new in CF airway inflammation: an update

Neutrophilic airway inflammation is a characteristic feature of cystic fibrosis (CF) lung disease and present in most patients with pulmonary manifestations of the disease. Here we discuss the ongoing controversy whether the CFTR mutation itself causes a pro-inflammatory milieu in the airways or whether inflammation is always secondary to infection. Since the presence of inflammation has been shown to be a risk factor for subsequent lung function decline, noninvasive tests to monitor airway inflammation are urgently needed. Induced sputum is currently being assessed as a clinical and research tool, but unfortunately is only feasible in cooperative children. While nonspecific treatment approaches that decrease infection or improve clearance of airway secretions were found to positively affect airway inflammation, specific anti-inflammatory treatment strategies have been less successful. Since any intervention that decreases inflammation may potentially have a detrimental effect by promoting airway infection, a better understanding of the factors regulating inflammation in the CF lung will form the basis for more targeted treatment strategies in the future.     

Buccal cell DNA mutation analysis for diagnosis of cystic fibrosis in newborns and infants inaccessible to sweat chloride measurement

OBJECTIVES: To assess the application of DNA-based cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation analysis as a primary cystic fibrosis (CF) diagnostic test in preterm and term newborns and infants for whom the quantitative pilocarpine iontophoresis test (QPIT) cannot be used. DESIGN: Retrospective survey. SETTING: DNA Diagnostic Laboratory, Children's Hospital, Boston, Massachusetts. Buccal cell DNA samples were received from inpatients, outpatients, and three neonatal intensive care units. OUTCOME MEASURE: Detection of at least 1 of 12 CFTR mutations. PATIENTS: Between November 1, 1992, and April 30, 1994, 28 newborns and infants under 12 months of age at risk for CF had CFTR DNA mutation analysis performed because a sweat chloride (SC) value could not be obtained. QPIT was either not performed (infant weight <2 kg, QPIT not available at site of hospitalization, or infant not accessible to QPIT laboratory) or was inconclusive (sweat volume <75 mg or indeterminate SC [>/=40, <60 mEq/L]). The postnatal age at time of testing ranged from 1 day to 11 months, and gestational age at birth from 25 to 40 weeks. RESULTS: Six (21%) of 28 infants with unobtainable or indeterminate QPIT had 1 or 2 CFTR mutations detected. Immediate CF diagnosis by direct detection of 2 CFTR mutations was made in 5 of these 6 patients. Definitive CF diagnosis in the infant with 1 CFTR mutation was delayed until an elevation in SC could be documented. The patients with no CFTR mutations detected had a low likelihood of CF. CONCLUSIONS: For infants in whom CF is suspected but QPIT cannot be obtained, buccal cell DNA-based CFTR mutation analysis can be used as a rapid, noninvasive primary diagnostic test. This simple mode of DNA collection may aid in the diagnosis of other inherited disorders in newborns.     

Somatische Gentherapie der Mukoviszidose Aktueller Forschungsstand

Background. Numerous gene mutations associated with hereditary disorders have been identified. In cystic fibrosis the hereditary defect is attributed to mutations in one single gene, the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). Conventional therapies of CF have dramatically increased the life expectancy of afflicted individuals. However, the ultimate incurability of this disease calls for novel and better therapeutic strategies. As cystic fibrosis is believed to be caused by mutations in one single gene, it has appeared to be the ideal candidate for one of the most tempting approaches in clinical therapy, namely gene therapy. Laboratory protocols for the introduction of genes into various tissues have been developed and applied over the last years. The ease of gene transfer under laboratory conditions gave rise to the hope that rapid advances in gene transfer protocols under clinical settings could be achieved as well. Clinical trials. 25 clinical trials of gene therapy for cystic fibrosis have been initiated using viral and non-viral vectors for gene transfer. The outcome of the CF gene therapy studies as well as of those for other diseases have clearly demonstrated that gene transfer and gene therapy in humans is a much more complex and challenging task than originally thought. Still, the encouraging results achieved in animal models and the rapid progress in vector technology justify the hope that the novel genetic therapies will be applied successfully to the benefit of patients suffering from cystic fibrosis.     

Radiological analysis of children with cystic fibrosis who are homozygous for cystic fibrosis transmembrane conductance regulator mutation S549R (T-->G).

Genotype-phenotype analyses in cystic fibrosis (CF) have shown that cystic fibrosis transmembrane conductance regulator (CFTR) genotypes can predict pancreatic status but that correlations with pulmonary status remain elusive. We investigated the extent and severity of lung disease associated with CFTR mutation S549R (T-->G). This mutation is localized in intron 11 (nucleotide-binding fold 1 of the CFTR protein) and had so far been described as a private mutation only. It is associated with an extremely severe overall CF phenotypic expression. Detailed radiological analyses were performed by a single observer in 12 children with CF from the United Arab Emirates who were homozygous for CFTR mutation S549R (T-->G). A diversity of pulmonary changes included marked hyperinflation in early infancy in conjunction with inflammation of the interstitium. After 2 years of age, signs of central airway involvement occurred in association with early signs of pulmonary hypertension. In conclusion, although there is some diversity in the radiological findings of these CF patients, R549 is a very severe allele associated with extreme lung disease and rapid pulmonary decline.     

Phenotypic and genetic characterization of patients with features of "nonclassic" forms of cystic fibrosis

OBJECTIVE: To determine which features of incomplete or "nonclassic" forms of cystic fibrosis (CF) are associated with deleterious CF transmembrane conductance regulator gene ( CFTR ) mutations, and to explore other etiologies for features not associated with deleterious CFTR mutations. STUDY DESIGN: Clinical features were compared between 57 patients with deleterious mutations in each CFTR and 63 with no deleterious mutations. The Shwachman Bodian Diamond syndrome gene ( SBDS ) was sequenced to search for mutations in patients with no deleterious CFTR mutations and steatorrhea to determine if any had unrecognized Shwachman-Diamond syndrome (SDS). RESULTS: The presence of a common CF-causing mutation, absence of the vas deferens, and Pseudomona aeruginosa in the sputum correlated with the presence of two deleterious CFTR mutations, whereas sweat chloride concentration, diagnostic criteria for CF, and steatorrhea did not. However, sweat chloride concentration correlated with CFTR mutation status in patients infected with P aeruginosa. One patient had disease-causing mutations in each SBDS . CONCLUSIONS: Presence of a common CF-causing mutation, absence of the vas deferens and/or P aeruginosa infection in a patient with features of nonclassic CF are predictive of deleterious mutations in each CFTR , whereas steatorrhea in the same context is likely to have etiologies other than CF transmembrane conductance regulator (CFTR) dysfunction.     

Faecal elastase-1 concentration in cystic fibrosis patients with CFTR I1234V mutation

Aim: To assess the exocrine pancreatic function among cystic fibrosis patients with cystic fibrosis trans-membrane conductance regulator (CFTR) I1234V mutation. Methods: Cross-sectional study of 40 cystic fibrosis patients with homozygous CFTR I1234V mutation belonging to a large Arab kindred family and 25 healthy subjects as a control group over a period of 12 mo. Assessment of their exocrine pancreatic function was performed by measuring faecal elastase-1 (FE1) concentration with a commercial ELISA kit using polyclonal antibodies (BioServ Diagnostics) in CF patients compared to healthy subjects. The results were compared with those obtained from a second laboratory using another commercial ELISA (ScheBo; Biotech, Germany) that uses two monoclonal antibodies against different specific epitopes of human pancreatic elastase. Results: All CF patients with CFTR I1234V mutation had normal levels of faecal elastase 1. No significant difference was found between the two methods for the CF groups or between the CF patients with and without pancreatic enzyme replacement.

Conclusion: Cystic fibrosis with homozygous CFTR I1234V mutation is associated with pancreatic sufficiency. Assessment of exocrine function using polyclonal antibodies does not significantly differ from that using two monoclonal antibodies against different specific epitopes of human pancreatic elastase.     

Hepatobiliary disease in cystic fibrosis

Cystic Fibrosis (CF) is an inherited, multisystem disease characterised by defective salt and water transport across the secretory epithelia of the respiratory and gastrointestinal tracts. Much of the morbidity and mortality in CF results from inflammation and infection of the airways. However, since the introduction of more intensive therapies and Centre-based care, life expectancy has increased significantly. This has led to the recognition of other life limiting conditions in CF including liver disease which is now the 2^nd commonest cause of death in CF patients. Defective function of the cystic fibrosis transmembrane conductance regulator (CFTR) gene impairs bile secretion at the hepatocellular and cholangiocellular levels leading to cholestasis. However, this alone fails to explain the broad spectrum of hepatobiliary problems seen in cystic fibrosis, and why only a proportion of patients develop clinically significant liver disease. It is now believed that the development of CF-related liver disease may reflect the influence of modifier genes on CFTR function.The characteristic lesion is focal biliary cirrhosis, resulting from biliary obstruction and progressive periportal fibrosis. The focal fibrogenic process progresses in about 10% of patients to multilobular biliary cirrhosis, portal hypertension and eventually liver failure. Careful evaluation of all CF patients needs to be made to ascertain the presence of liver disease with the majority of patients developing liver disease by 10 years of age. Aggressive treatment of malnutrition and portal hypertension is required but some children may progress to liver transplant.     

Genetically determined male infertility caused by the CFTR gene mutations

One of the reasons for the genetically determined male infertility is congenital bilateral absence of the vas deferens (CBAVD). CBAVD until recently classified as a separate disease, is now recognised to be a form of atypical CF (cystic fibrosis). The above is based on the fact that both CF and CBAVD result from mutations in the same CFTR gene. Assisted reproduction techniques (ART), including the ICSI (IntraCytoplasmic Sperm Injection) technique, is an acknowledged chance of having offspring for males with extremely bad semen quantitative parameters, including CBAVD patients. Molecular analysis of the CFTR gene along with genetic counselling enables to estimate the risk of having offspring with classical or atypical CF after ICSI procedure.     

Cystic Fibrosis and Chiari Type I Malformation: Autopsy Study of Two Infants with a Rare Association

Cystic fibrosis (CF), an epithelial cell transport disorder caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, is not generally associated with malformations of the central nervous system (CNS). This report describes two African-American children who died at less than 2 years of age with known Chiari I malformations and were found, unexpectedly at autopsy, to have the classic pancreatic and respiratory changes of CF. Both patients had suffered from failure to thrive that had been attributed to their CNS malformations. One child also had recurrent pneumonia and died with Pseudomonas sepsis. Mutational analysis for > 70 common CFTR mutations identified a single delta F508 mutation in one patient and a single 3120+1G to A mutation in the other. Their second CFTR mutations were not identified. The association of CF with Chiari I malformation is not likely to be purely coincidental, as the probability of such an occurrence in African-Americans is greater than one in 7,500,000 patients. It is possible that the CFTR gene may play a previously unrecognized role in CNS development. Alternatively, this CNS abnormality may be acquired due to the metabolic and electrolyte imbalances that characteristically occur in CF.     

Phenotype of CF and the effects of possible modifier genes

Cystic fibrosis is an inherited multi-system disease, characterised by progressive lung disease and pancreatic insufficiency that is classically attributed to the dysfunction of a single gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). The widely diverse phenotypic expression of CF is likely influenced by other genetic traits separate from the CFTR locus or modifier genes. Many of the genes currently under study as potential modifiers of CF, particularly those which influence the severity of lung disease, are involved in the control of infection, immunity and inflammation. Some of these include HLA class II antigens, mannose-binding lectin, alpha(1)-antitrypsin and alpha(1)-antichymotrypsin, glutathione-S-transferase, nitric oxide synthase type I, TNF-alpha, TGF-beta, IL-1beta and IL-1Ra.     

Markedly elevated neonatal immunoreactive trypsinogen levels in the absence of cystic fibrosis gene mutations is not an indication for further testing.

AIMS: To investigate the immunoreactive trypsinogen (IRT) values above the usual 99th centile laboratory cut-off and determine the value of offering further testing to those infants with a markedly elevated IRT but no cystic fibrosis transmembrane regulator (CFTR) gene mutation identified by the screening programme. METHODS: All babies born in Victoria, Australia, between 1991 and 2003, were screened by IRT followed by CF gene mutation analysis. RESULTS: Of the 806,520 babies born, 9268 with the highest IRT levels had CFTR mutation analysis. There were 123 DeltaF508 homozygotes and 703 heterozygotes (86 with CF, 617 carriers). A total of 8442 babies had no CFTR gene mutation, of whom 18 (0.21%) had CF. The total number of CF babies with IRT greater than the laboratory cut-off was 227 (2.4%). The IRT results of the CF patients were distributed normally, with the majority above the laboratory cut-off of newborn IRT results. There was no evidence of an excess of babies with CF in the very highest levels of IRT above the 99th centile. CONCLUSIONS: Only a small proportion of babies with a neonatal IRT >99th centile have CF. Additional CF testing for infants with an elevated IRT but no CFTR gene mutation has an extremely low yield, no matter how high the IRT result.     

Molekulargenetische Grundlagen der Zystischen Fibrose

Cystic Fibrosis (CF) is the second most common autosomal-recessive disorder in Caucasians with an incidence of 1 in 2500 to 1 in 1600 and a carrier frequency of 4 to 5%. More than 900 different disease-causing mutations within the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been reported so far. The large size of the CFTR gene (250 kb of genomic DNA, 6100 bp of coding sequences) as well as the multiplicity of mutations complicate molecular genetic diagnostics. Therefore, a specific and efficient SSCP/HD technique has been established detecting 97% of all CF mutations and enabeling reliable diagnosis, carrier detection and prenatal analyses in patients, family members and partners. However, how the different types of mutations (nonsense, missense, frameshift, splice site mutations, in-frame deletions and insertions) disrupt the protein function is not predictable by the gene defect alone but has to be evaluated for each single mutation by functional investigations. Characterization of the CFTR gene and identification of the pathogenic mutations as well as genotype phenotype association studies offer the basic knowledge for the development of new diagnostic, prognostic and therapeutic perspectives and extensive insight into the pathogenesis of the disease.     

Respiratory exacerbations in childhood associated with compound heterozygosity Phe508del/Arg117His-7T of the cystic fibrosis transmembrane regulator gene

Debate continues regarding the clinical implications for compound heterozygotes identified with Phe508del and Arg117His-7T mutations of the cystic fibrosis transmembrane regulator (CFTR) gene. We report respiratory exacerbations and airway culture of Staphylococcus aureus and Pseudomonas aeruginosa in a child with this genotype. Conclusion: The compound heterozygote cystic fibrosis (CF) mutation Phe508del with Arg117His-7T should not necessarily be considered benign in childhood.     

The cystic fibrosis gene: mutation and the function of CFTR protein]

The spectrum of mutations identified in cystic fibrosis patients includes a major defect (delta F508), found in 70% of CF chromosomes from French patients, and a large number of infrequent mutations. This significant heterogeneity of molecular defects precludes large scale screening for the disease at the time being. The presumptive structure of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR), the membrane protein encoded by the CF gene, is briefly recalled. The role this protein is thought to play in the regulation of chloride ion transport across epithelial membranes is discussed, along with the new insights into the molecular mechanisms of CF gained as a result of identification of the CF gene.