Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice.

It is often challenging for the clinician interested in cystic fibrosis (CF) to interpret molecular genetic results, and to integrate them in the diagnostic process. The limitations of genotyping technology, the choice of mutations to be tested, and the clinical context in which the test is administered can all influence how genetic information is interpreted. This paper describes the conclusions of a consensus conference to address the use and interpretation of CF mutation analysis in clinical settings. Although the diagnosis of CF is usually straightforward, care needs to be exercised in the use and interpretation of genetic tests: genotype information is not the final arbiter of a clinical diagnosis of CF or CF transmembrane conductance regulator (CFTR) protein related disorders. The diagnosis of these conditions is primarily based on the clinical presentation, and is supported by evaluation of CFTR function (sweat testing, nasal potential difference) and genetic analysis. None of these features are sufficient on their own to make a diagnosis of CF or CFTR-related disorders. Broad genotype/phenotype associations are useful in epidemiological studies, but CFTR genotype does not accurately predict individual outcome. The use of CFTR genotype for prediction of prognosis in people with CF at the time of their diagnosis is not recommended. The importance of communication between clinicians and medical genetic laboratories is emphasized. The results of testing and their implications should be reported in a manner understandable to the clinicians caring for CF patients.

A sweat test centered protocol for the disclosure and diagnosis of cystic fibrosis in a newborn screening program.

We describe the development of a sweat test centered protocol for disclosure and diagnosis of Cystic Fibrosis. Our protocol aims to identify infants early, minimizes the time of uncertainty for the parents, and yet gives them time to begin to come to terms with the possibility of diagnosis. Over a 9-year period 295,247 newborn infants were screened for CF in Wales, of whom 121 infants were diagnosed as having CF. During this period there were four false negatives (3.3%). Parental satisfaction with the process appears very high 6 months after disclosure.

Reduced iC3b-mediated phagocytotic capacity of pulmonary neutrophils in cystic fibrosis.

Cystic fibrosis (CF) is characterized by a neutrophil-dominated chronic inflammation of the airways with persistent infections. In order to investigate whether neutrophils contribute to an inadequacy in the pulmonary defence mechanism, the phagocytic activity of pulmonary and peripheral blood neutrophils from CF and non-CF respiratory patients were compared. Neutrophils were isolated from both the blood and bronchoalveolar lavage fluid of 21 patients with CF (12 male, 9 female; mean age 7.5 years, range 0.25-16.4 years) and 17 non-CF subjects (9 male, 8 female; mean age 5.4 years, range 0.2-13.1 years). The ex vivo phagocytic rate of normal pulmonary neutrophils to internalize zymosan particles opsonized with iC3b was faster than that of circulating neutrophils (P < 0.05), but the maximum capacity (9 particles/cell) was similar. In contrast, pulmonary neutrophils from patients with CF had a lower phagocytic capacity than circulating neutrophils either from the same patients or from normal subjects. This deficiency could not be attributed to (i) the cell surface density of CR3 (CD18/CD11b) receptors, which were not significantly different between the other groups (ii) the signalling ability of the CR3 receptors, using cytosolic free Ca(2+) signalling as the receptor activity read-out or (iii) a decrease in cellular ATP concentration. As CFTR was not detectable on neutrophils from any source by either histochemistry or Western blotting, it was concluded that the reduced phagocytic capacity was not the direct result of a CFTR mutation, but was attributed to a failure of neutrophil phagocytic priming during translocation into the CF lung.

Sildenafil (Viagra) corrects DeltaF508-CFTR location in nasal epithelial cells from patients with cystic fibrosis.

BACKGROUND: Most patients with cystic fibrosis (CF) have a DeltaF508 mutation resulting in abnormal retention of mutant gene protein (DeltaF508-CFTR) within the cell. This study was undertaken to investigate DeltaF508-CFTR trafficking in native cells from patients with CF with the aim of discovering pharmacological agents that can move DeltaF508-CFTR to its correct location in the apical cell membrane. METHOD: Nasal epithelial cells were obtained by brushing from individuals with CF. CFTR location was determined using immunofluorescence and confocal imaging in untreated cells and cells treated with sildenafil. The effect of sildenafil treatment on CFTR chloride transport function was measured in CF15 cells using an iodide efflux assay. RESULTS: In most untreated CF cells DeltaF508-CFTR was mislocalised within the cell at a site close to the nucleus. Exposure of cells to sildenafil (2 hours at 37 degrees C) resulted in recruitment of DeltaF508-CFTR to the apical membrane and the appearance of chloride transport activity. Sildenafil also increased DeltaF508-CFTR trafficking in cells from individuals with CF with a single copy DeltaF508 (DeltaF508/4016ins) or with a newly described CF trafficking mutation (R1283M). CONCLUSIONS: The findings provide proof of principle for sildenafil as a DeltaF508-CFTR trafficking drug and give encouragement for future testing of sildenafil and related PDE5 inhibitors in patients with CF.

Effect of a microaerosol filter on spirometry in children with cystic fibrosis.

AIM: To measure the effect of an in-line microaerosol filter on spirometric values in cystic fibrosis (CF). METHODS: Twenty-six subjects with CF undertook a randomized, open, cross-over comparison of spirometry with and without an in-line filter. RESULTS: The filter had no significant effect on spirometric parameters nor was there any order effect. Measurement error was unrelated to the magnitude of the measurement. CONCLUSION: In-line microaerosol filters do not affect spirometric values or variability in children with CF.

Correction of delF508-CFTR activity with benzo(c)quinolizinium compounds through facilitation of its processing in cystic fibrosis airway cells.

A number of genetic diseases, including cystic fibrosis, have been identified as disorders of protein trafficking associated with retention of mutant protein within the endoplasmic reticulum. In the presence of the benzo(c)quinolizinium drugs, MPB-07 and its congener MPB-91, we show the activation of cystic fibrosis transmembrane conductance regulator (CFTR) delF508 channels in IB3-1 human cells, which express endogenous levels of delF508-CFTR. These drugs were without effect on the Ca(2+)-activated Cl- transport, whereas the swelling-activated Cl- transport was found altered in MPB-treated cells. Immunoprecipitation and in vitro phosphorylation shows a 20% increase of the band C form of delF508 after MPB treatment. We then investigated the effect of these drugs on the extent of mislocalisation of delF508-CFTR in native airway cells from cystic fibrosis patients. We first showed that delF508 CFTR was characteristically restricted to an endoplasmic reticulum location in approximately 80% of untreated cells from CF patients homozygous for the delF508-CFTR mutation. By contrast, 60-70% of cells from non-CF patients showed wild-type CFTR in an apical location. MPB-07 treatment caused dramatic relocation of delF508-CFTR to the apical region such that the majority of delF508/delF508 CF cells showed a similar CFTR location to that of wild-type. MPB-07 had no apparent effect on the distribution of wild-type CFTR, the apical membrane protein CD59 or the ER membrane Ca(2+),Mg-ATPase. We also showed a similar pharmacological effect in nasal cells freshly isolated from a delF508/G551D CF patient. The results demonstrate selective redirection of a mutant membrane protein using cell-permeant small molecules of the benzo(c)quinolizinium family and provide a major advance towards development of a targetted drug treatment for cystic fibrosis and other disorders of protein trafficking.

Recent advances in cystic fibrosis.

The median life expectancy for cystic fibrosis is now over 30 years, and it is projected that in newborn infants it will become more than 40 years. The identification of the cystic fibrosis gene and its product, cystic fibrosis transmembrane conductance regulator (CFTR), has widened the spectrum of the disease from the classical case of the infant with cystic fibrosis to the elderly childless man with unexplained bronchiectasis. There is increasing evidence of the advantages of newborn screening for cystic fibrosis and subsequent specialist care. Management concentrates on optimising nutritional status and preventing lung infection and inflammation.

Cystic fibrosis-related deaths in infancy and the effect of newborn screening.

Although newborn screening for cystic fibrosis (CF) is widely advocated, hard evidence in its favor is difficult to obtain, partly because of a dramatically improved life expectancy. Between 1985--1989 infants, born in Wales and the West Midlands were randomized to newborn CF screening by heel-prick immunoreactive trypsin (IRT) measurement or diagnosis by clinical presentation. Eligible children with CF who died in the first 5 years of life were identified from the local pediatricians and from the National UK CF Survey. In all, 230,076 infants were randomized to be screened, while 234,510 were unscreened. One hundred seventy-six CF children were identified, of whom 7 died in the first 5 years of life, 3 having presented with meconium ileus. Median age of diagnosis in the screened group was 8 weeks. On an intention to treat analysis, all 4 nonmeconium ileus-related deaths occurred in the unscreened group (Fisher's exact test, P < 0.05). However, the clinical presentation of 2 of these infants led to them being diagnosed prior to 8 weeks, i.e., earlier than would have been likely by screening. In conclusion, newborn screening has the potential to decrease infant CF deaths, but if it is to be successful, identification and treatment must occur as soon as possible after birth.

Localisation of wild-type and DeltaF508-CFTR in nasal epithelial cells.

Wild-type and the DeltaF508 mutation of the cystic fibrosis transmembrane conductance regulator (DeltaF508-CFTR) were localised by confocal imaging in DeltaF508/DeltaF508 native airway epithelial cells using a well-characterised CFTR antibody. Surface nasal epithelial cells from three control and three CF individuals were obtained from nasal brushings. Cells were fixed, permeabilised and incubated with first antibody for 18 h at 4 degrees C. Following labelling with second antibody, cells were viewed with the confocal microscope. Wild-type CFTR was localised predominantly apically, whereas DeltaF508-CFTR was located mainly inside the cell in a region close to the nucleus. Incubation of cells with MPB-07 (250 microM) at 37 degrees C for 2 h resulted in pronounced movement of DeltaF508-CFTR to the cell periphery, but did not change the localisation of wild-type CFTR. The results show that DeltaF508-CFTR is mislocalised in native nasal epithelial cells and that its distribution is altered in response to the new CFTR activator, MPB-07. The findings should lead to development of a rational drug treatment for CF patients carrying the DeltaF508 mutation.

Maintenance of growth in cystic fibrosis despite reduction in pancreatic enzyme supplementation.

Twenty one children with cystic fibrosis were advised to decrease their pancreatic enzyme supplement (PES) dose to less than 10,000 units lipase/kg/day. Mean PES dosage was significantly decreased in 15 patients from 18,380 to 8647 units lipase/kg/day. There were no significant changes in energy or fat intake, but there were significant increases in weight SD score, height SD score, and weight/height ratio.

Tracheobronchomalacia in preterm infants with chronic lung disease.

Tracheobronchomalacia is a treatable cause of persisting ventilatory requirements in the preterm neonate, and warrants a high index of suspicion. Five preterm infants with persisting ventilatory requirements with evidence of tracheobronchomalacia are reported. Four were diagnosed by tracheobronchogram and one by flexible endoscopy. All were successfully managed by continuous positive airway pressure (CPAP) via a tracheostomy. One infant died of unrelated causes. The oldest child in this series at the age of 2 years requires no further ventilatory support. Tracheobronchial anomalies should be considered in all preterm infants with persisting ventilatory requirements.

Asthma: early predisposing factors.

The significance of factors affecting the development of asthma and atopy in children must be judged against their ability to contribute to the increase in childhood asthma. Although genetic factors are clearly important to the development of asthma and atopy, they can not explain the increased prevalence of these conditions.

Flexible fibre-optic bronchoscopy in the management of lung complications in cystic fibrosis.

Flexible fibre-optic bronchoscopy was performed under sedation in 24 children with cystic fibrosis. In eight cases bronchoscopy was carried out as a therapeutic procedure. Most children were subjectively improved, but objective evidence of change was minimal. Useful information was obtained in 9/16 children who underwent the procedure for diagnostic purposes. Information included sensitive identification of organisms responsible for lower respiratory symptoms in non-expectorating cases and identification of unsuspected gastro-oesophageal reflux.

Bronchoscopic appearances of congenital lobar emphysema.

Although decreased bronchial cartilage is found in 50% of cases of congenital lobar emphysema (CLE), it can only be surmised that this defect produces a ball valve effect with consequent overinflation. We describe the flexible bronchoscopic features of CLE in a 3-year-old child. The observed airway patency during inspiration, and dynamic airway collapse on expiration suggests that bronchomalacia contributes to lung overinflation in these cases.

Exclusion from proximal 11q of a common gene with megaphenic effect on atopy.

We have typed three markers on proximal 11q in 131 random families with three or more children studied for atopy. A summary map that includes the FCER1B candidate was constructed. Using a 2-locus disease model, we performed combined segregation and linkage analysis of three models, none of which suggested linkage. Nine marker loci on other chromosomes were also negative. In the regions swept by these 12 markers we cannot rule out a rare gene, perhaps of large effect, nor a common gene of small effect. However, a common gene of large effect is excluded. These results and alternative strategies are discussed in the perspective of inconsistent evidence for a major atopy gene.

Intraosseous infusion for resuscitation.

An 11 week old infant who had a cardiac arrest secondary to gastrointestinal haemorrhage and was successfully treated using intraosseous infusion is reported. The child was discharged with no apparent neurological deficit.