Chloride conductance and genetic background modulate the cystic fibrosis phenotype of Delta F508 homozygous twins and siblings.

To investigate the impact of chloride (Cl(-)) permeability, mediated by residual activity of the cystic fibrosis transmembrane conductance regulator (CFTR) or by other Cl(-) channels, on the manifestations of cystic fibrosis (CF), we determined Cl(-) transport properties of the respiratory and intestinal tracts in Delta F508 homozygous twins and siblings. In the majority of patients, cAMP and/or Ca(2+)-regulated Cl(-) conductance was detected in the airways and intestine. Our finding of cAMP-mediated Cl(-) conductance suggests that, in vivo, at least some Delta F508 CFTR can reach the plasma membrane and affect Cl(-) permeability. In respiratory tissue, the expression of basal CFTR-mediated Cl(-) conductance, demonstrated by 30% of Delta F508 homozygotes, was identified as a positive predictor of milder CF disease. In intestinal tissue, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid-insensitive (DIDS-insensitive) Cl(-) secretion, which is indicative of functional CFTR channels, correlated with a milder phenotype, whereas DIDS-sensitive Cl(-) secretion was observed mainly in more severely affected patients. The more concordant Cl(-) secretory patterns within monozygous twins compared with dizygous pairs imply that genes other than CFTR significantly influence the manifestation of the basic defect.

Residual chloride secretion in intestinal tissue of deltaF508 homozygous twins and siblings with cystic fibrosis. The European CF Twin and Sibling Study Consortium.

BACKGROUND & AIMS: Cholinergic stimulation of chloride secretion is impaired in the intestines of patients with cystic fibrosis (CF). However, intestinal chloride secretion has been observed in patients with mild CF mutations. The aim of this study was to investigate residual Cl(-) secretion in the intestine of DeltaF508 homozygous CF patients, and examine the contribution of cystic fibrosis transmembrane conductance regulator (CFTR) and alternative Cl(-) conductances. Twins and siblings with identical CFTR genotypes were investigated to determine the impact of factors other than CFTR on chloride secretion. METHODS: Chloride secretion in rectal tissue was investigated by applying Ca(2+) and adenosine 3',5'-cyclic monophosphate (cAMP)-linked agonists before and after the inhibition of alternative Cl(-) conductances with 4,4'-diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS). RESULTS: cAMP-mediated Cl(-) secretion was observed in 73% of patients, and 20% showed DIDS-sensitive Ca(2+)-activated Cl(-) secretion. This DIDS-sensitive alternative chloride conductance was seen only in CF patients who also responded to cAMP agonists. Chloride secretion was more concordant within monozygous twins than within dizygous pairs. CONCLUSIONS: These results suggest the presence of CFTR-mediated Cl(-) secretion in a subgroup of patients, implying that a portion of deltaF508 CFTR can be processed in vivo and function as a chloride channel in the apical membrane of intestinal cells. Moreover, a considerable number of deltaF508 homozygous patients express chloride conductances other than CFTR in their intestinal epithelia.

Characterization of a novel 21-kb deletion, CFTRdele2,3(21 kb), in the CFTR gene: a cystic fibrosis mutation of Slavic origin common in Central and East Europe.

We report a large genomic deletion of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, viz., a deletion that is frequently observed in Central and Eastern Europe. The mutation, termed CFTRdele2,3(21 kb), deletes 21,080 bp spanning introns 1-3 of the CFTR gene. Transcript analyses have revealed that this deletion results in the loss of exons 2 and 3 in epithelial CFTR mRNA, thereby producing a premature termination signal within exon 4. In order to develop a simple polymerase chain reaction assay for this allele, we defined the end-points of the deletion at the DNA sequence level. We next screened for this mutation in a representative set of European and European-derived populations. Some 197 CF patients, including seven homozygotes, bearing this mutation have been identified during the course of our study. Clinical evaluation of CFTRdele2,3(21 kb) homozygotes and a comparison of compound heterozygotes for deltaF508/CFTRdele2,3(21 kb) with pairwise-matched deltaF508 homozygotes indicate that this deletion represents a severe mutation associated with pancreatic insufficiency and early age at diagnosis. Current data show that the mutation is particularly common in Czech (6.4% of all CF chromosomes), Russian (5.2%), Belorussian (3.3%), Austrian (2.6%), German (1.5%), Polish (1.5%), Slovenian (1.5%), Ukrainian (1.2%), and Slovak patients (1.1%). It has also been found in Lithuania, Latvia, Macedonia and Greece and has sporadically been observed in Canada, USA, France, Spain, Turkey, and UK, but not in CF patients from Bulgaria, Croatia, Romania or Serbia. Haplotype analysis has identified the same extragenic CF-haplotype XV-2c/KM. 19 "A" and the same infrequent intragenic microsatellite haplotype 16-33-13 (IVS8CA-IVS 17bTA-IVS 17bCA) in all examined CFTRdele2,3(21 kb) chromosomes, suggesting a common origin for this deletion. We conclude that the 21-kb deletion is a frequent and severe CF mutation in populations of Eastern- and Western-Slavic descent.

Categories of deltaF508 homozygous cystic fibrosis twin and sibling pairs with distinct phenotypic characteristics.

Cystic fibrosis (CF), the most common severe autosomal recessive trait among Caucasians, is caused by molecular lesions in the cystic fibrosis transmembrane conductance regulator gene (CFTR). The course of the multi-organ disease CF is highly variable, suggesting the influence of environmental factors and/or modulating genes other than CFTR on the disease phenotype. To evaluate the cause of CF disease variability, the European CF Twin and Sibling Study collected data on two clinical parameters most sensitive for the course and prognosis of CF, ie weight predicted for height (wfh)% (representative for the nutritional status) and FEVPerc (representative for the pulmonary status) for a cohort of 277 sibling pairs, 12 pairs of dizygous twins and 29 pairs of monozygous twins. Of these 318 CF twin and sib pairs, 114 were reported to be homozygous for the most frequent CF disease-causing lesion, deltaF508. Intra-pair discordance was assessed by the intra-pair differences with wfh% and FEVPerc and by DELTA, a composite parameter defined by linear combination of wfh% and FEVPerc in order to describe discordance with respect to the overall disease severity. Monozygous twins had a significantly lower DELTA than dizygous twins (P = 0.05) indicating that CF disease severity is modulated by an inherited component in addition to the CFTR gene itself. Extreme phenotypes are considered to be more informative for the analysis of any quantitative trait. Thus, we aimed to quantify disease severity and intra-pair discordance in order to select pairs with the extreme phenotypes DIS (discordant patient pairs), CON+ (concordant and mildy affected patient pairs) and CON- (concordant and severely affected patient pairs). The algorithm reliably discriminated between pairs DIS, CON+ and CON- among the cohort of deltaF508 homozygotes. The selected pairs from these categories demonstrated non-overlapping properties for wfh%, FEVPerc and the intra-pair difference of both parameters.

Clinical presentation of exclusive cystic fibrosis lung disease.

The diagnosis of cystic fibrosis (CF) is based on the occurrence of two mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and on assays that measure the basic defect of abnormal chloride transport in the affected organs. However, in cases of atypical CF not all diagnostic tests may be positive. We present a patient with an atypical CF phenotype in whom the only presenting symptom was severe CF-like lung disease substantiated by an abnormal nasal potential difference. Genetic analysis showed that the patient was a symptomatic heterozygote, which suggests that one lesion in the CFTR gene may be sufficient to cause CF-like lung disease.

Cystic-fibrosis-like disease unrelated to the cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis (CF) is considered to be a monogenic disease caused by molecular lesions within the cystic fibrosis transmembrane conductance regulator (CFTR) gene and is diagnosed by elevated sweat electrolytes. We have investigated the clinical manifestations of cystic fibrosis, CFTR genetics and electrophysiology in a sibpair in which the brother is being treated as having CF, whereas his sister is asymptomatic. The diagnosis of CF in the index patient is based on highly elevated sweat electrolytes in the presence of CF-related pulmonary symptoms. The investigation of chloride conductance in respiratory and intestinal tissue by nasal potential difference and intestinal current measurements, respectively, provides no evidence for CFTR dysfunction in the siblings who share the same CFTR alleles. No molecular lesion has been identified in the CFTR gene of the brother. Findings in the investigated sibpair point to the existence of a CF-like disease with a positive sweat test without CFTR being affected. Other factors influencing sodium or chloride transport are likely to be the cause of the symptoms in the patient described.

Infections with Pseudomonas aeruginosa in patients with cystic fibrosis.

The lung infection with Pseudomonas aeruginosa is regarded as one of the major causes of health decline in patients with cystic fibrosis (CF). The CF host response to the persistent bacterial antigen load in the endobronchiolar lumen is characterized by a pronounced humoral response, local production of cytokines, influx of neutrophils into the lung and a protease-protease inhibitor imbalance predominantly sustained by released neutrophil elastase. CF is an autosomal recessive disease, and we could demonstrate for our local patient population that the age-dependent risk to become chronically colonized with P. aeruginosa can be differentiated by the disease-causing CFTR mutation genotype. The age-specific colonisation rates were significantly lower in pancreas sufficient than in pancreas insufficient patients. P. aeruginosa is occasionally detected in throat swabs already in infancy or early childhood in most patients although there is a lapse of several years amenable to preventive measures such as vaccination until onset of persistent colonization. The epidemiology of the infection with P. aeruginosa was investigated by quantitative macrorestriction fragment pattern analysis. The distribution and frequency of clones found in CF patients match that found in other clinical and environmental aquatic habitats, but the over-representation of specific clones at a CF clinic indicates a significant impact of nosocomial transmission for the prevalence of P. aeruginosa-positive patients at a particular center. Most patients remain colonized with the initially acquired P. aeruginosa clone. According to direct sputum analysis the majority of patients is carrying a single clonal variant at a concentration of 10(7)-10(9) CFU. Co-colonization with other species or other clones is infrequent. Independent of the underlying genotype, the CF lung habitat triggers a uniform, genetically fixed conversion of bacterial phenotype. Most CFP, aeruginosa strains become non-motile, mucoid, LPS-, pyocin- and phage-deficient, secrete less virulence determinants and shift the production of cytokines evoked in neutrophils. On the other hand, other properties such as antimicrobial susceptibility or adherence to bronchial mucins remain highly variable reflecting the capacity of P. aeruginosa to adapt to ongoing changes in the CF lung habitat.

Analysis of microsatellites by direct blotting electrophoresis and chemiluminescence detection.

We describe a fast and reliable method for the nonradioactive analysis of microsatellites. For three dinucleotide repeats within the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the separation of polymerase chain reaction (PCR) products generated with biotinylated primers on a direct blotting electrophoresis system and subsequent chemiluminescence detection is shown. In direct blotting electrophoresis, the separation of DNA fragments depended linearly on size. The reproducible resolution allowed reliable assignment of allele lengths to a given signal. The nonradioactive detection protocol was advantageous compared to radioactive methods: samples could be analyzed within one day due to the fast signal development by 3-(4-methoxyspiro[1,2-dioxetane-3,2'-(5'- chloro)tricyclo[3.3.1.1.3,7]decan]-4-yl)phenylphosphate disodium salt (CSPD). Variation of exposure times enabled differentiation between major bands and byproducts of comparable intensity that are due to the slippage of the Taq polymerase during PCR amplification.

Detection of more than 50 different CFTR mutations in a large group of German cystic fibrosis patients.

We have conducted a comprehensive study of the molecular basis of cystic fibrosis (CF) in 350 German CF patients. A screening approach based on single-strand conformation analysis and direct sequencing of genomic polymerase chain reaction products has allowed us to detect the molecular defects on 95.4% of the CF chromosomes within the coding region and splice sites of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The spectrum of sequence changes comprises 54 different mutations, including 17 missense mutations, 14 nonsense mutations, 11 frameshift mutations, 10 splice site variants and two amino acid deletions. Eleven of these mutations have not previously been described. Our results reflect the marked mutational heterogeneity of CF in a large sample of patients from a non-isolated population.