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The last decade has witnessed developments in the CF drug pipeline which are both exciting and unprecedented, bringing with them previously unconsidered challenges. The Task Force group was brought together to consider these challenges and possible strategies to address them. Over the last 18 months, we have discussed internally and gathered views from a broad range of individuals representing patient organisations, clinical and research teams, the pharmaceutical industry and regulatory agencies. In this and the accompanying article, we discuss two main areas of focus: i) optimising trial design and delivery for speed and efficiency; ii) drug development for patients with rare CFTR mutations. We propose some strategies to tackle the challenges ahead and highlight areas where further thought is needed. We see this as the start of a process rather than the end and hope herewith to engage the wider community in seeking solutions to improved treatments for all patients with CF.

BACKGROUND: Most patients with cystic fibrosis (CF) suffer from pancreatic insufficiency, leading to fat malabsorption, malnutrition and abdominal discomfort. Until recently, no specific tool was available for assessing gastro-intestinal related quality of life (GI QOL) in patients with CF. As the Horizon2020 project MyCyFAPP aims to improve GI QOL by using a newly designed mobile application, a sensitive and reliable outcome measure was needed. We aimed to study the applicability of the existing child-specific Pediatric Quality of Life Inventory, Gastrointestinal Symptoms Scales and Module (PedsQL GI) in children with CF. METHODS: A multicenter, prospective observational study was performed in 6 European centers to validate the PedsQL GI in children with CF during 3 months. RESULTS: In total, 248 children and their parents were included. Within-patient variability of PedsQL GI was low (24.11), and there was reasonable agreement between children and parents (ICC 0.681). Nine of 14 subscales were informative (no ceiling effect). The PedsQL GI and the median scores for 4 subscales were significantly lower in patients compared to healthy controls. Positive associations were found between PedsQL GI and age (OR = 1.044, p = 0.004) and between PedsQL GI and BMI z-score (OR = 1.127, p = 0.036). PedsQL GI correlated with most CFQ-R subscales (r 0.268 to 0.623) and with a Visual Analogue Scale (r = 0.20). CONCLUSIONS: PedsQL GI is a valid and applicable instrument to assess GI QOL in children with CF. Future research efforts should examine the responsiveness of the CF PedsQL GI to change in the context of clinical interventions and trials. ispartof: PLOS ONE vol:14 issue:12 ispartof: location:United States status: published

MosheAshkenazi,1,2SaraySity,2 IfatSarouk,1,2Bat El Bar Aluma,1,2AdiDagan,1,2YaelBezalel,1,2LeaBentur3 KrisDe Boeck,4 OriEfrati1,2 1Pediatric Pulmonology and National CF Center, Edmond and Lilly Safra Children’s Hospital, Sheba Medical Center, Ramat Gan, Israel; 2Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; 3Pediatric Pulmonology Institute, Ruth Rappaport Children’s Hospital, Rambam Health Care Campus, Haifa, Israel; 4Pediatric Pulmonology, Department of Pediatrics, University of Leuven, Leuven, Belgium Background: Allergic bronchopulmonary aspergillosis (ABPA) is a condition characterized by a Th2 response, serum eosinophilia, and increased total serum IgE to Aspergillus fumigatus. ABPA occurs in cystic fibrosis (CF) and asthma. Omalizumab is a humanized recombinant monoclonal antibody against IgE. Previous studies reported borderline results when treating ABPA with omalizumab. Methods: A retrospective study to investigate the efficacy of omalizumab in the treatment of ABPA in CF patients was conducted at 3 CF centers in Israel and Belgium. Data were obtained from the digital archive. We measured 4 outcome parameters: forced expiratory volume in 1 second, body mass index, pulmonary exacerbations, and steroid sparing. Results: The database was composed on the records of 9 patients. None of the outcome parameters showed any improvement. A favorable outcome was observed in patients with higher levels of posttreatment total IgE than those with lower levels. CF-related diabetes and male gender showed trends for poorer outcomes. Conclusion: No benefits were detected on treating ABPA in CF with omlaizumb. Monitoring the total IgE was not helpful. A prospective randomized double-blind study is needed. Keywords: ABPA, Omalizumab, CF, IgE

The last decade has witnessed developments in the CF drug pipeline which are both exciting and unprecedented, bringing with them previously unconsidered challenges. The Task Force group came together to consider these challenges and possible strategies to address them. Over the last 18 months, we have discussed internally and gathered views from a broad range of individuals representing patient organizations, clinical and research teams, the pharmaceutical industry and regulatory agencies. In this and the accompanying article, we discuss two main areas of focus: i) optimising trial design and delivery for speed/efficiency; ii) drug development for patients with rare CFTR mutations. We propose some strategies to tackle the challenges ahead and highlight areas where further thought is needed. We see this as the start of a process rather than the end and hope herewith to engage the wider community in seeking solutions to improved treatments for all patients with CF.

Background A method to adjust Pancreatic Enzyme Replacement Therapy in Cystic Fibrosis is not currently available. Objectives To assess the in vivo efficacy of a method to adjust the dose of enzymatic supplement in CF extrapolated from previous in vitro digestion studies (theoretical optimal dose, TOD). Secondly, to assess how individual patient characteristics influence the expected coefficient of fat absorption (CFA) and thus to identify an individual correction factor to improve TOD. Methods A prospective interventional study in 43 paediatric patients with CF from 5 European centres. They followed a 24h fixed diet with the theoretical optimal dose for each meal. Faecal collection was carried out between colorimetric markers in order to include all the faeces corresponding to the fixed diet. Beta regression models were applied to assess the associations of individual patient characteristics with the CFA. Results Median CFA was 90% (84, 94% 1st, 3rd Q.) with no significant differences among centres. Intestinal transit time was positively associated with CFA (p = 0.007), but no statistical associations were found with and age, gender, phenotype or BMI. Regression model showed no improvement of the in vitro predicted theoretical optimal dose when taking individual patient characteristics into account. Conclusion Strict adherence to the theoretical optimal dose of enzymatic supplement for a prescribed meal, led to median CFA levels at the clinical target of 90% with a low variability between patients. The proposed method can be considered as a first approach for an evidence-based method in PERT dosing based on food characteristics. Results have to be confirmed in free dietary settings.

BACKGROUND: A method to adjust Pancreatic Enzyme Replacement Therapy in Cystic Fibrosis is not currently available. OBJECTIVES: To assess the in vivo efficacy of a method to adjust the dose of enzymatic supplement in CF extrapolated from previous in vitro digestion studies (theoretical optimal dose, TOD). Secondly, to assess how individual patient characteristics influence the expected coefficient of fat absorption (CFA) and thus to identify an individual correction factor to improve TOD. METHODS: A prospective interventional study in 43 paediatric patients with CF from 5 European centres. They followed a 24h fixed diet with the theoretical optimal dose for each meal. Faecal collection was carried out between colorimetric markers in order to include all the faeces corresponding to the fixed diet. Beta regression models were applied to assess the associations of individual patient characteristics with the CFA. RESULTS: Median CFA was 90% (84, 94% 1st, 3rd Q.) with no significant differences among centres. Intestinal transit time was positively associated with CFA (p = 0.007), but no statistical associations were found with and age, gender, phenotype or BMI. Regression model showed no improvement of the in vitro predicted theoretical optimal dose when taking individual patient characteristics into account. CONCLUSION: Strict adherence to the theoretical optimal dose of enzymatic supplement for a prescribed meal, led to median CFA levels at the clinical target of 90% with a low variability between patients. The proposed method can be considered as a first approach for an evidence-based method in PERT dosing based on food characteristics. Results have to be confirmed in free dietary settings. ispartof: PLOS ONE vol:14 issue:3 ispartof: location:United States status: published

Background: Consensus guidelines recommend early treatment to eradicate newly acquired Pseudomonas aeruginosa (Pa) infection in cystic fibrosis (CF) patients although there is no single preferred regimen. Aztreonam for inhalation solution (AZLI) significantly reduces sputum Pa density in CF patients with chronic Pa infection and has been well tolerated in the pediatric population. This single-arm, open-label Aztreonam Lysine for Pseudomonas Infection Eradication (ALPINE) study was conducted to evaluate the safety and efficacy of a 28-day treatment course of AZLI to eradicate newly acquired Pa infection in pediatric CF patients. Methods: CF patients (3 months to b18 years) with new onset Pa infection were treated with AZLI 75 mg 3 times daily for 28 days. New onset Pa infection was defined as first lifetime Pa-positive respiratory tract culture (throat swab, sputum) or Pa-positive culture after a ≥2-year history of Pa-negative cultures (≥2 cultures/year). Sputum or throat swab cultures were collected at study entry (baseline) and at weeks 4 (end of treatment), 8, 16, and 28. Primary endpoint was the percentage of patients with cultures negative for Pa at all post-treatment time points. Results: A total of 105 pediatric CF patients enrolled (3 months to b2 years, n = 24; 2 to b6 years, n = 25; 6 to b18 years, n = 56). Of the 101 patients who completed treatment, 89.1% (n = 90) were free of Pa at the end of treatment and 75.2% (n = 76) were free of Pa 4 weeks after the end of treatment. Of the 79 patients evaluable for the primary endpoint, 58.2% were free of Pa at all post-treatment time points. Conclusions: AZLI was effective and well tolerated in eradicating Pa from newly infected pediatric patients with CF. These eradication rates are consistent with success rates reported in the literature for various antibiotic regimens, including other inhaled antibiotics studied for eradication. ClinicalTrials.gov: NCT01375049. © 2014 Published by Elsevier B.V. on behalf of European Cystic Fibrosis Society.

Access to immunization varies greatly across the world. In order to increase vaccine coverage, the required vaccines need to be able to reach the targeted population. However, in developing countries, this often turns out to be a challenging task. This article provides a literature review on vaccine distribution supply chains in developing countries and consists of two main parts. The first part elaborates on the characteristics and challenges inherent to such distribution supply chains. In order to obtain a complete overview, both quantitative and qualitative papers are included. In the second part, relevant operations research and operations management literature is structured according to seven classification criteria: decision level, methodology, part of the VDSC modelled, uncertainties and characteristics covered, performance measures, real-life applicability, and countries and vaccines covered. Throughout these classifications, a comparison is made between issues reported in practice and those investigated in operations research and operations management literature. Based on this analysis, we identify trends and conclude that several gaps exist, providing a promising avenue for future research. ispartof: FEB Research Report KBI_1815 status: published

Introduction: For the optimal management of children with cystic fibrosis, there are currently no efficient tools for the precise adjustment of pancreatic enzyme replacement therapy, either for advice on appropriate dietary intake or for achieving an optimal nutrition status. Therefore, we aim to develop a mobile application that ensures a successful nutritional therapy in children with cystic fibrosis. Methods and analysis: A multidisciplinary team of 12 partners coordinate their efforts in 9 work packages that cover the entire so-called 'from laboratory to market' approach by means of an original and innovative co-design process. A cohort of 200 patients with cystic fibrosis aged 1-17 years are enrolled. We will develop an innovative, clinically tested mobile health application for patients and health professionals involved in cystic fibrosis management. The mobile application integrates the research knowledge and innovative tools for maximising self-management with the aim of leading to a better nutritional status, quality of life and disease prognosis. Bringing together different and complementary areas of knowledge is fundamental for tackling complex challenges in disease treatment, such as optimal nutrition and pancreatic enzyme replacement therapy in cystic fibrosis. Patients are expected to benefit the most from the outcomes of this innovative project. Ethics and dissemination: The project is approved by the Ethics Committee of the coordinating organisation, Hospital Universitari La Fe (Ref: 2014/0484). Scientific findings will be disseminated via journals and conferences addressed to clinicians, food scientists, information and communications technology experts and patients. The specific dissemination working group within the project will address the wide audience communication through the website (http://www.mycyfapp.eu), the social networks and the newsletter.

Background: The New European guidelines have established the most updated recommendations on nutrition and pancreatic enzyme replacement therapy (PERT) in CF. In the context of MyCyFAPP project - a European study in children with CF aimed at developing specific tools for improvement of self-management - the objective of the current study was to assess nutritional status, daily energy and macronutrient intake, and PERT dosing with reference to these new guidelines. Methods: Cross sectional study in paediatric patients with CF from 6 European centres. SD-scores for weight-for-age (WFA), height-for-age (HFA) and body mass index-for-age (BMI) were obtained. Through a specific 4-day food and enzyme-dose record, energy and macronutrients intake and PERT-use (LU/g lipids) were automatically calculated by the,MyCyFAPP system. Comparisons were made using linear regression models. Results: The lowest quartiles, for BMI and HFA were between 0 and -1SD in all the centres with no significant differences, and 33.5% of the patients had a SD-score

Ashkenazi M, Sity S, Sarouk I, et al. J Asthma Allergy. 2018;11:101–107.On page 101, in the Conclusion section, the text “omlaizumb” should have been “omalizumab”.Read the original article.&nbsp

OBJECTIVES: The aim of this study was to assess the feasibility of fetal tracheal injection in the late-gestational pig to target the airways. METHODS: Following laparotomy and hysterotomy, fetoscopy was performed in pregnant sows to access the fetal trachea. Two volumes of fluospheres were injected (1 and 3 mL). Fluosphere distribution to the different lung lobes was investigated by microscopy. Possible fetal airway injury, caused by the surgical procedure or intratracheal injection, was investigated. Lung morphology and fetal lung volumes were calculated by micro computed tomography (μCT). RESULTS: Intratracheal administration was successfully performed in 20/21 fetuses. Analysis by confocal microscopy demonstrated that 3 mL, and not 1 mL, most efficiently targeted all lung lobes. On high-resolution μCT, total airway volume was estimated at 2.9 mL; strengthening that 3 mL is appropriate to target all lung lobes. No procedural damage was evidenced in the lungs or trachea. CONCLUSIONS: Intratracheal injection of nanoparticles is feasible in the pregnant pig and does not cause procedural lung damage. Using an injection volume of 3 mL, all lung lobes were efficiently targeted. This nanoparticle delivery model to fetal airways opens perspectives for therapeutic interventions. © 2016 John Wiley & Sons, Ltd. ispartof: PRENATAL DIAGNOSIS vol:36 issue:10 pages:926-934 ispartof: location:England status: published

In Belgian cystic fibrosis (CF) clinics, sputum samples are evaluated on selective MAST medium routinely every 3 months. In this study, in 1993 and 1999, isolates were further examined by recA restriction fragment length polymorphism analysis and pulsed-field gel electrophoresis of genomic DNA restricted with SpeI. In 1993, 12 patients were colonised with Burkholderia cepacia complex (Bcc): B. cenocepacia (n=6), B. multivorans (n=3), B. stabilis (n=3). Four patients were colonised with the same B. cenocepacia strain; two with the same B. stabilis strain. After 5 yrs, three B. cenocepacia- and one B. multivorans-colonised patients had died. In 1999, Bcc was isolated in 12 patients: B. multivorans (n=9), B. stabilis (n=1) and B. cenocepacia (n=2). Three patients were colonised by the same B. multivorans strain. Compared to matched controls, the 5 yr outcome was poor; four B. cepacia patients died and none of the control patients died. Lung-function evolution was poor. In conclusion, the rate of colonisation in Belgian cystic fibrosis patients is stable and low. Burkholderia cenocepacia was most prevalent in 1993; Burkholderia multivorans in 1999. The cross-infection rate is low. Three patients had transient colonisation. The impact of Burkholderia cepacia complex on morbidity in the Belgian cystic fibrosis population is high and not limited to Burkholderia cenocepacia.

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RATIONALE: New treatment strategies are needed to improve airway clearance and reduce the morbidity and the time burden associated with cystic fibrosis (CF). OBJECTIVES: To determine whether long-term treatment with inhaled mannitol, an osmotic agent, improves lung function and morbidity. METHODS: Double-blind, randomized, controlled trial of inhaled mannitol, 400 mg twice a day (n = 192, "treated" group) or 50 mg twice a day (n = 126, "control" group) for 26 weeks, followed by 26 weeks of open-label treatment. MEASUREMENTS AND MAIN RESULTS: The primary endpoint was absolute change in FEV(1) from baseline in treated versus control groups, averaged over the study period. Secondary endpoints included other spirometric measurements, pulmonary exacerbations, and hospitalization. Clinical, microbiologic, and laboratory safety were assessed. The treated group had a mean improvement in FEV(1) of 105 ml (8.2% above baseline). The treated group had a relative improvement in FEV(1) of 3.75% (P = 0.029) versus the control group. Adverse events and sputum microbiology were similar in both treatment groups. Exacerbation rates were low, but there were fewer in the treated group (hazard ratio, 0.74; 95% confidence interval, 0.42-1.32; P = 0.31), although this was not statistically significant. In the 26-week open-label extension study, FEV(1) was maintained in the original treated group, and improved in the original control group to the same degree. CONCLUSIONS: Inhaled mannitol, 400 mg twice a day, resulted in improved lung function over 26 weeks, which was sustained after an additional 26 weeks of treatment. The safety profile was also acceptable, demonstrating the potential role for this chronic therapy for CF. Clinical trial registered with www.clinicaltrials.gov (NCT 00630812).

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Several diseases have been clinically or genetically related to cystic fibrosis (CF), but a consensus definition is lacking. Here, we present a proposal for consensus guidelines on cystic fibrosis transmembrane conductance regulator (CFTR)-related disorders (CFTR-RDs), reached after expert discussion and two dedicated workshops.A CFTR-RD may be defined as "a clinical entity associated with CFTR dysfunction that does not fulfil diagnostic criteria for CF" .The utility of sweat testing, mutation analysis, nasal potential difference, and/or intestinal current measurement for the differential diagnosis of CF and CFTR-RD is discussed. Algorithms which use genetic and functional diagnostic tests to distinguish CF and CFTR-RDs are presented.According to present knowledge, congenital bilateral absence of vas deferens (CBAVD), acute recurrent or chronic pancreatitis and disseminated bronchiectasis, all with CFTR dysfunction, are CFTR-RDs. © 2011 European Cystic Fibrosis Society.

Background: The use of involved-field radiotherapy after chemotherapy for advanced Hodgkin's lymphoma is controversial. Methods: We randomly assigned patients with previously untreated stage III or IV Hodgkin's lymphoma who were in complete remission after hybrid chemotherapy with mechlorethamine, vincristine, procarbazine, prednisone, doxorubicin, bleomycin, and vinblastine (MOPP-ABV) to receive either no further treatment or involved-field radiotherapy. Radiotherapy consisted of 24 Gy to all initially involved nodal areas and 16 to 24 Gy to all initially involved extranodal sites. Patients in partial remission were treated with 30 Gy to nodal areas and 18 to 24 Gy to extranodal sites. Results: Of 739 patients, 421 had a complete remission; 161 of these patients were assigned to no further treatment, and 172 to involved-field radiotherapy. The median follow-up was 79 months. The five-year event-free survival rate was 84 percent in the group that did not receive radiotherapy and 79 percent in the group that received involved-field radiotherapy (P=0.35). The five-year overall survival rates were 91 and 85 percent, respectively (P=0.07). Among the 250 patients in partial remission after chemotherapy, the five-year event-free and overall survival rates were 79 and 87 percent, respectively. Conclusions: Involved-field radiotherapy did not improve the outcome in patients with advanced-stage Hodgkin's lymphoma who had a complete remission after MOPP-ABV chemotherapy. Radiotherapy may benefit patients with a partial response after chemotherapy.

Editor—Cystic fibrosis (CF, MIM 219700) is a common, severe, autosomal recessive disease caused by mutations in the CF transmembrane conductance regulator ( CFTR ) gene cloned in 1989.1-3The disease, characterised by chronic lung disease which is the main cause of morbidity and mortality, pancreatic dysfunction, raised electrolyte levels in sweat, and male infertility, is caused by altered chloride (Cl−) secretion across the apical membrane of epithelial cells.4 There is, however, substantial variability in the clinical manifestations affecting the various organs.4 5 One single mutation, F508del, generally associated with severe disease, accounts for about 70% of CF chromosomes world wide, although with a heterogeneous geographical distribution.5 Patients homozygous for the F508del mutation have the classical severe form of the disease which includes chronic mucous obstruction of the lung and conducting airways, followed by recurrent infections mostly by Pseudomonas aeruginosa (Pa) and Staphylococcus aureus (Sa), exocrine pancreatic insufficiency (PI), resulting in failure to gain weight and height, and raised levels of Cl−, sodium, and potassium in exocrine sweat.5 However, almost 1000 genetic alterations have been detected in the CFTR gene ( CFTR Mutation Database), most presumed to be disease causing mutations. About half of these are amino acid substitutions (missense mutations) and about 20% are splicing mutations. The remainder are nonsense, frameshift (including small deletions and insertions), and a small proportion of promoter mutations. The relationship between genotype, that is, the mutations in the CFTR gene, and the clinical phenotype of CF patients has been difficult to establish, in particular for lung disease. It was previously shown that the 3272-26A>G mutation leads to the creation of an alternative acceptor splice site competing with the normal one during RNA processing and resulting in the occurrence of an alternatively spliced mRNA with 25 extra nucleotides from …

Mucociliary transport is one of the most important local defense mechanisms of the airways, but it is prone to many and frequent acquired abnormalities and to inherited abnormalities. These abnormalities result in basic physiologic disturbances leading to a number of respiratory symptoms and signs. In order to critically evaluate the diagnostic value of parameters of this mucociliary cascade, the results of ciliary investigations in over 500 individuals (controls, acquired, and inherited abnormalities) were reviewed. Ciliary beat frequency, ciliary coordination and ultrastructural abnormalities were measured and evaluated in biopsies and after ciliogenesis in culture. There is a considerable overlap for all investigated parameters in biopsy material of controls, secondary, and primary ciliary dyskinesia. There is not one parameter that is diagnostic for primary ciliary dyskinesia or can be used as an exclusion criterion. After ciliogenesis in culture, cilia are always completely normal except for the inherited abnormalities. Absence of coordinated ciliary activity after ciliogenesis in culture is 100% sensitive and specific for the diagnosis of primary ciliary dyskinesia.

Several diseases have been clinically or genetically related to cystic fibrosis (CF), but a consensus definition is lacking. Here, we present a proposal for consensus guidelines on cystic fibrosis transmembrane conductance regulator (CFTR)-related disorders (CFTR-RDs), reached after expert discussion and two dedicated workshops.A CFTR-RD may be defined as "a clinical entity associated with CFTR dysfunction that does not fulfil diagnostic criteria for CF".The utility of sweat testing, mutation analysis, nasal potential difference, and/or intestinal current measurement for the differential diagnosis of CF and CFTR-RD is discussed. Algorithms which use genetic and functional diagnostic tests to distinguish CF and CFTR-RDs are presented.According to present knowledge, congenital bilateral absence of vas deferens (CBAVD), acute recurrent or chronic pancreatitis and disseminated bronchiectasis, all with CFTR dysfunction, are CFTR-RDs.

Introduction Inhaled mannitol has beneficial effects on lung function, mucociliary clearance, quality of life and sputum properties. This trial examined the efficacy of inhaled mannitol in children with cystic fibrosis (CF). Methods The efficacy of inhaled mannitol in children with CF aged 6–17years was assessed in a phase 2, randomised, placebo-controlled crossover study. Subjects were randomly assigned to mannitol 400mg every 12h or matching placebo for 8weeks, followed by an 8week washout and an 8week period with the alternate treatment. The primary endpoint was the absolute change from baseline in ppFEV1 (percent predicted FEV1). Results A total of 92 subjects were studied, with a mean age of 12years and mean baseline ppFEV1 of 72.2%. During mannitol treatment ppFEV1 was 3.42% (p=0.004) higher compared to placebo or a 4.97% (p=0.005) relative difference; relative change from baseline FEF25-75 was 10.52% (p=0.013). During mannitol treatment, acute post-treatment sputum weight was higher (p=0.012). In pre-specified subgroups (rhDNase use, age, and disease severity), the treatment differences consistently favoured mannitol. The most common AEs were cough and pulmonary exacerbations. Pulmonary exacerbation AEs were approximately 30% lower in the mannitol group. Conclusions In children with CF, inhaled mannitol was associated with significant improvements in lung function and sputum weight, irrespective of rhDNase use, age or disease severity. Inhaled mannitol was well tolerated and was associated with a reduced incidence of pulmonary exacerbation AEs. (Clinical Trials.Gov: NCT 01883531)

Human β defensins contribute to the first line of defense against infection of the lung. Polymorphisms in these genes are therefore potential modifiers of the severity of lung disease in cystic fibrosis. Polymorphisms were sought in the human β-defensin genes DEFB1, DEFB4, DEFB103A , and DEFB104 in healthy individuals and cystic fibrosis (CF) patients living in various European countries. DEFB1, DEFB4 , and DEFB104 were very polymorphic, but DEFB103A was not. Within Europe, differences between control populations were found for some of the frequent polymorphisms in DEFB1 , with significant differences between South-Italian and Czech populations. Moreover, frequent polymorphisms located in DEFB4 and DEFB104 were not in Hardy Weinberg equilibrium in all populations studied, while those in DEFB1 were in Hardy Weinberg equilibrium. Sequencing of a monochromosomal chromosome 8 mouse–human hybrid cell line revealed signals for multiple alleles for some loci in DEFB4 and DEFB104 , but not for DEFB1 . This indicated that more than one DEFB4 and DEFB104 gene was present on this chromosome 8, in agreement with recent findings that DEFB4 and DEFB104 are part of a repeat region. Individual DEFB4 and DEFB104 PCR amplification products of various samples were cloned and sequenced. The results showed that one DNA sample could contain more than two haplotypes, indicating that the various repeats on one chromosome were not identical. Given the higher complexity found in the genomic organization of the DEFB4 and DEFB104 genes, association studies with CF lung disease severity were performed only for frequent polymorphisms located in DEFB1 . No association with the age of first infection by Pseudomonas aeruginosa or with the FEV1 percentage at the age of 11–13 years could be found.