F. Vermeulen

Ataluren (PTC124) induces cystic fibrosis transmembrane conductance regulator protein expression and activity in children with nonsense mutation cystic fibrosis.

RATIONALE Nonsense (premature stop codon) mutations in mRNA for the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis (CF) in approximately 10% of patients. Ataluren (PTC124) is an oral drug that permits ribosomes to readthrough premature stop codons in mRNA to produce functional protein. OBJECTIVES To evaluate ataluren activity, safety, and pharmacokinetics in children with nonsense mutation CF. METHODS Patients were assessed in two 28-day cycles, comprising 14 days on and 14 days off ataluren. Patients took ataluren three times per day (morning, midday, and evening) with randomization to the order of receiving a lower dose (4, 4, and 8 mg/kg) and a higher dose (10, 10, and 20 mg/kg) in the two cycles. MEASUREMENTS AND MAIN RESULTS The study enrolled 30 patients (16 male and 14 female, ages 6 through 18 yr) with a nonsense mutation in at least one allele of the CFTR gene, a classical CF phenotype, and abnormal baseline nasal epithelial chloride transport. Ataluren induced a nasal chloride transport response (at least a -5-mV improvement) or hyperpolarization (value more electrically negative than -5 mV) in 50% and 47% of patients, respectively, with more hyperpolarizations at the higher dose. Improvements were seen in seven of nine nonsense mutation genotypes represented. Ataluren significantly increased the proportion of nasal epithelial cells expressing apical full-length CFTR protein. Adverse events and laboratory abnormalities were infrequent and usually mild. Ataluren pharmacokinetics were similar to those in adults. CONCLUSIONS In children with nonsense mutation CF, ataluren can induce functional CFTR production and is well tolerated.

What’s new in cystic fibrosis? From treating symptoms to correction of the basic defect

Chronic relentless lung infection and pancreatic insufficiency are the cardinal features of cystic fibrosis (CF), a life-shortening autosomal recessive disease. Mutations in the ’cystic fibrosis transmembrane conductance regulator’ (CFTR) are currently classified into five groups according to their repercussion on CFTR protein synthesis and its chloride channel function. Stop codon mutations (class I) result in a truncated nonfunctional CFTR, class II mutations consist of aberrantly folded CFTR protein that is degraded by the cell quality control system, while class III mutations lead to defective regulation of the CFTR protein and, consequently, the absence of CFTR function. These three classes usually lead to a classic CF phenotype with pancreatic insufficiency. CFTR mutations that lead to defective chloride conductance are grouped together in class IV. Class V mutations interfere with normal transcription, thereby reducing the amount of otherwise normal CFTR. These latter two classes are mostly associated with a milder expression of the disease. In the absence of CFTR function, unrestrained Na+ absorption and the failure of active Cl- secretion lead to a decreased airway surface liquid (ASL) volume and subsequent failure of normal mucociliary clearance. This review highlights recent therapeutic strategies that either target the underlying defect or the early steps in CF pathophysiology. To date, gene therapy has failed to demonstrate a clinical benefit after repeated administration. Mutation-specific chloride channel correction pharmacotherapy is currently being developed, an example of which is PTC124, a new chemical compound that selectively induces read-through of premature stop codons. However, clinical efficacy for most of the compounds still has to be proven in large clinical trials. The positive effect of nebulised hypertonic saline on mucociliary clearance is based on the restoration of ASL height. Recent advances in the current treatment of lung infection and inflammation are highlighted in this review. Lung transplantation should be considered in terminally ill patients, but the timing of the transplantation is crucial: transplanting too early shortens survival, while transplanting too late results in patients dying on the waiting list.

New clinical diagnostic procedures for cystic fibrosis in Europe

In the majority of cases, there is no difficulty in diagnosing Cystic Fibrosis (CF). However, there may be wide variation in signs and symptoms between individuals which encourage the scientific community to constantly improve the diagnostic tests available and develop better methods to come to a final diagnosis in patients with milder phenotypes. This paper is the result of discussions held at meetings of the European Cystic Fibrosis Society Diagnostic Network supported by EuroCareCF. CFTR bioassays in the nasal epithelium (nasal potential difference measurements) and the rectal mucosa (intestinal current measurements) are discussed in detail including efforts to standardize the techniques across Europe. New approaches to evaluate the sweat gland, future of genetic testing and methods on the horizon like CFTR expression in human leucocytes and erythrocytes are discussed briefly.

Mutations in the Amiloride-Sensitive Epithelial Sodium Channel in Patients With Cystic Fibrosis-Like Disease

We investigated whether mutations in the genes that code for the different subunits of the amiloride‐sensitive epithelial sodium channel (ENaC) might result in cystic fibrosis (CF)‐like disease. In a small fraction of the patients, the disease could be potentially explained by an ENaC mutation by a Mendelian mechanism, such as p.V114I and p.F61L in SCNN1A. More importantly, a more than three‐fold significant increase in incidence of several rare ENaC polymorphisms was found in the patient group (30% vs. 9% in controls), indicating an involvement of ENaC in some patients by a polygenetic mechanism. Specifically, a significantly higher number of patients carried c.–55+5G>C or p.W493R in SCNN1A in the heterozygous state, with odds ratios (ORs) of 13.5 and 2.7, respectively.The p.W493R‐SCNN1A polymorphism was even found to result in a four‐fold more active ENaC channel when heterologously expressed in Xenopus laevis oocytes. About 1 in 975 individuals in the general population will be heterozygous for the hyperactive p.W493R‐SCNN1A mutation and a cystic fibrosis transmembrane conductance regulator (CFTR) gene that results in very low amounts (0–10%) functional CFTR. These ENaC/CFTR genotypes may play a hitherto unrecognized role in lung diseases. Hum Mutat 30:1–11, 2009.

Primary ciliary dyskinesia: critical evaluation of clinical symptoms and diagnosis in patients with normal and abnormal ultrastructure

BackgroundPrimary ciliary dyskinesia (PCD) is a rare disorder with variable disease progression. To date, mutations in more than 20 different genes have been found. At present, PCD subtypes are described according to the ultrastructural defect on transmission electron microscopy (TEM) of the motile cilia. PCD with normal ultrastructure (NU) is rarely reported because it requires additional testing. Biallelic mutations in DNAH11 have been described as one cause of PCD with NU.The aim of our study was to describe the clinical characteristics of a large population of patients with PCD, in relation to the ultrastructural defect. Additionally, we aimed to demonstrate the need for biopsy and cell culture to reliably diagnose PCD, especially the NU subtype.MethodsWe retrospectively analyzed data from 206 patients with PCD. We compared the clinical characteristics, lung function, microbiology and imaging results of 68 patients with PCD and NU to those of 90 patients with dynein deficiencies and 41 patients with central pair abnormalities. In addition, we aimed to demonstrate the robustness of the diagnosis of the NU subtype in cell culture by data from genetic analysis.ResultsPCD with NU comprised 33% (68/206) of all patients with PCD. Compared to other subtypes, patients with PCD and NU had a similar frequency of upper and lower respiratory tract problems, as well as similar lung function and imaging. With the currently widely applied approach, without cell culture, the diagnosis would have been missed in 16% (11/68) of patients with NU. Genetic analysis was performed in 29/68 patients with PCD and NU, and biallelic mutations were found in 79% (23/29) of tested patients.ConclusionsWe reported on the clinical characteristics of a large population of patients with PCD and NU. We have shown that systematic performance of biopsy and cell culture increases sensitivity to detect PCD, especially the subtype with NU.PCD with NU has similar clinical characteristics as other PCD types and requires biopsy plus ciliogenesis in culture for optimal diagnostic yield.

Lung clearance index predicts pulmonary exacerbations in young patients with cystic fibrosis

Rationale The lung clearance index (LCI) is a promising endpoint for use in cystic fibrosis (CF) clinical trials, but correlations with validated clinical endpoints have not yet been established. Objective This study aimed to demonstrate that, in young patients with CF, baseline LCI predicts subsequent pulmonary exacerbation (PE) and correlates with the respiratory domain of the CF Questionnaire-Revised (CFQ-Rresp). Methods Baseline LCI, forced expiratory volume in 1 s (FEV1), CFQ-Rresp and PEs over the subsequent year were prospectively recorded in 63 patients aged 5–19 years. The ability of baseline LCI to predict PE was assessed using negative binomial regression models and Kaplan–Meier plots. Results Twenty-six patients (41%) experienced 48 PEs. Baseline LCI and FEV1 were predictors of PE. Compared with the quartile with the lowest LCI, the annual PE rate in increasing LCI quartiles was 2.9 (95% CI 0.5 to 16.5, p=0.238), 5.4 (95% CI 1.0 to 29.0, p=0.045) and 13.6 (95% CI 2.8 to 67.1, p=0.001). Similarly, time to first PE decreased with worsening LCI quartiles (log-rank test for trend, p<0.001). Furthermore, LCI correlated with CFQ-Rresp (r=−0.43, p<0.001). In the subgroup of 53 patients with normal FEV1, LCI was a predictor of PE. In this subgroup, LCI also correlated with CFQ-Rresp (r=−0.282, p=0.043). Conclusions Baseline LCI predicts PE in young patients with CF and correlates with CFQ-Rresp, a validated patient-reported outcome, even in the subgroup with normal FEV1. These data further support the use of LCI as a surrogate outcome measure in CF clinical trials.

Lung structure–function correlation in patients with primary ciliary dyskinesia

Background Primary ciliary dyskinesia (PCD) is a rare disease, characterised by chronic airway infection. In cystic fibrosis, FEV1 is insensitive to detect patients with structural damage, and Lung Clearance Index (LCI) was proposed as a better marker of early lung damage. In PCD, the relationship between functional and structural abnormalities has been less studied. We aimed to re-examine this in a cohort of children and adults with mild to moderate PCD. Methods Thirty-eight patients with PCD (5.2–25.0 years) and 70 healthy controls (4.4–25.8 years) were recruited to compare LCI, measured by N2 multiple breath washout and FEV1 in a prospective observational trial. In a subset of 30 patients who underwent chest imaging, structural abnormalities were evaluated with cystic fibrosis computed tomography (CFCT) scores. Results LCI was abnormal in 28 of 38 patients and a moderate correlation was observed between LCI and FEV1 (r=−0.519, p=0.001). Moreover, LCI correlated well with CFCT total score (r=0.800, p<0.001) and also with subscores for airway wall thickening (r=0.809, p<0.001), mucus plugging (r=0.720, p<0.001) and bronchiectasis (r=0.494, p<0.001). Concordance was seen between LCI and CFCT in 25 of 30 (83%) patients, but between FEV1 and CFCT in only 16 of 30 (53%) patients. LCI was more sensitive (90.9%, 95% CI 70.8 to 98.6) to detect patients with structural abnormalities than FEV1 (36.4%, 95% CI 17.2 to 59.3). Conclusions We demonstrated that measuring LCI in patients with PCD is of clinical relevance; it was more frequently abnormal than FEV1, correlated well with CFCT and was more sensitive than FEV1 to detect patients with structural abnormalities.

Non-invasive liver elastography (Fibroscan) for detection of cystic fibrosis-associated liver disease

BACKGROUND Cystic fibrosis-associated liver disease (CFLD) is the second cause of mortality in CF. The prevalence is estimated to be 26-45%, but sensitive diagnostic tools are lacking. We investigated whether non-invasive liver elastography (Fibroscan) could serve as a screening tool. METHODS Fibroscan measurements were performed in 66 CF patients. Age-specific cutoff values were determined in a control population (n=59). The measurements were compared to clinical data, bi-yearly biochemistry and ultrasound. RESULTS Fibroscan was easy to perform in this patient population. There were 14 patients (21%) with abnormal liver stiffness measurements. Liver stiffness was significantly increased in patients with clinical CFLD (11.2 kPa versus 5.1 kPa), biochemical CFLD (7.4 kPa versus 5.4 kPa) or ultrasonographical CFLD (8.2 versus 4.3 kPa) (p<0.02 for all). CONCLUSIONS Fibroscan is an objective measure and is easy to perform in CF patients, even in children and could provide a valuable tool to detect, and quantify CFLD.

An International Randomized Multicenter Comparison of Nasal Potential Difference Techniques

BACKGROUND The transepithelial nasal potential difference (NPD) is used to assess cystic fibrosis transmembrane conductance regulator (CFTR) activity. Unreliability, excessive artifacts, and lack of standardization of current testing systems can compromise its use as a diagnostic test and outcome measure for clinical trials. METHODS To determine whether a nonperfusing (agar gel) nasal catheter for NPD measurement is more reliable and less susceptible to artifacts than a continuously perfusing nasal catheter, we performed a multicenter, randomized, crossover trial comparing a standardized NPD protocol using an agar nasal catheter with the same protocol using a continuously perfusing catheter. The data capture technique was identical in both protocols. A total of 26 normal adult subjects underwent NPD testing at six different centers. RESULTS Artifact frequency was reduced by 75% (P < .001), and duration was less pronounced using the agar catheter. The measurement of sodium conductance was similar between the two catheter methods, but the agar catheter demonstrated significantly greater CFTR-dependent hyperpolarization, because Δ zero Cl- + isoproterenol measurements were significantly more hyperpolarized with the agar catheter (224.2 ± 12.9 mV with agar vs 18.2 ± 9.1 mV with perfusion, P < .05). CONCLUSIONS The agar nasal catheter approach demonstrates superior reliability compared with the perfusion nasal catheter method for measurement of NPD. This nonperfusion catheter method should be considered for adoption as a standardized protocol to monitor CFTR activity in clinical trials.

Impact of Air Pollution on Cystic Fibrosis Pulmonary Exacerbations: A Case-Crossover Analysis

BACKGROUND Pulmonary exacerbations in cystic fibrosis (CF) contribute to the burden of disease, with a negative impact on quality of life, costs, and lung function. Our aim was to evaluate whether exacerbations, defi ned by antibiotic use, were triggered by daily fl uctuations in air pollution. METHODS In a case-crossover analysis, we evaluated 215 patients with CF and pollution data from January 1, 1998, to December 31, 2010. Exacerbation was defi ned as the start of IV or oral antibiotic use in a home or hospital setting. We calculated regional background levels of particulate matter with a diameter , 10 m m (PM 10 ), ozone, and nitrogen dioxide (NO 2 ) on the day of the event and on the 2 days prior to the event at each patient’s home address. We matched for day of the week and controlled for temperature on the day of the event and the 2 preceding days. In the month where antibiotic treatment was started, all days with the same temperature ( 2°C) as the event day served as control days, excluding 3 days before and after the start of treatment. RESULTS A total of 215 patients (male sex, 49%, mean age, 21 13 years) had 2,204 antibiotic treatments (1,107 IV and 1,097 oral). Over a period of 12 years, an increase in risk of antibiotic use was associated with increasing concentrations of PM 10 , NO 2 , and ozone on the event day and for NO 2 on the day before. A tendency toward signifi cance was seen the day before antibiotic use for PM 10 and ozone. Overall, a rise in OR was seen from 2 days before until the day of the start of antibiotics. CONCLUSIONS In patients with CF and exacerbations, ambient concentrations of ozone, PM 10 , and NO 2 play a role in triggering an exacerbation.