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SCNN1B encodes the beta subunit of the epithelial sodium channel ENaC. Previously, we reported an association between SNP markers of SCNN1B gene and disease severity in cystic fibrosis-affected sibling pairs. We hypothesized that factors interacting with the SCNN1B genomic sequence are responsible for intrapair discordance. Concordant and discordant pairs differed at six SCNN1B markers (Praw = 0.0075, Pcorr = 0.0397 corrected for multiple testing). To identify the factors binding to these six SCNN1B SNPs, we performed an electrophoretic mobility shift assay and captured the DNA–protein complexes. Based on protein mass spectrometry data, the epithelial splicing regulatory protein ESRP2 was identified when using SCNN1B-derived probes and the ESRP2-SCNN1B interaction was independently confirmed by coimmunoprecipitation assays. We observed an alternative SCNN1B transcript and demonstrated in 16HBE14o− cells that levels of this transcript are decreased upon ESRP2 silencing by siRNA. Furthermore, we confirmed that mildly and severely affected siblings have different ESPR2 genetic backgrounds and that ESRP2 markers are linked to the response of CF patients’ nasal epithelium to amiloride, indicating ENaC involvement (Pbest = 0.0131, Pcorr = 0.068 for multiple testing). Our findings demonstrate that sibling pairs clinically discordant for CF can be used to identify meaningful DNA regulatory elements and interacting factors.

Cystic fibrosis is a genetic disease caused by mutation of the CFTR gene, which encodes a chloride and bicarbonate transporter in epithelial cells. Due to the vast range of geno- and phenotypes, it is difficult to find causative treatments; however, small-molecule therapeutics have been clinically approved in the last decade. Still, the search for novel therapeutics is ongoing, and thousands of compounds are being tested in different assays, often leaving their mechanism of action unknown. Here, we bring together a CFTR-specific compound database (CandActCFTR) and systems biology model (CFTR Lifecycle Map) to identify the targets of the most promising compounds. We use a dual inverse screening approach, where we employ target- and ligand-based methods to suggest targets of 309 active compounds in the database amongst 90 protein targets from the systems biology model. Overall, we identified 1038 potential target–compound pairings and were able to suggest targets for all 309 active compounds in the database.

The Transmembrane member 16A (TMEM16A), also known as anoctamin-1 (ANO1), is a calcium-activated chloride channel present in the airway epithelium. It is known to be involved in the apical chloride secretion indicating that TMEM16A could be addressed for the treatment of chloride secretion defects like in Cystic- Fibrosis (CF). In this paper we generated knockout cell lines using CRISPR/Cas9-mediated ablation in a healthy human iPSC line (MHHi001-A), in a CF patient iPSC line (MHHi002-A) and in its corrected counterpart (MHHi002-A-1). These lines can be used for gaining information about the role of TMEM16A for mucus secretion and/or production and evaluating its therapeutic potential.

Different causative therapeutics for CF patients have been developed. There are still no mutation-specific therapeutics for some patients, especially those with rare CFTR mutations. For this purpose, high-throughput screens have been performed which result in various candidate compounds, with mostly unclear modes of action. In order to elucidate the mechanism of action for promising candidate substances and to be able to predict possible synergistic effects of substance combinations, we used a systems biology approach to create a model of the CFTR maturation pathway in cells in a standardized, human- and machine-readable format. It is composed of a core map, manually curated from small-scale experiments in human cells, and a coarse map including interactors identified in large-scale efforts. The manually curated core map includes 170 different molecular entities and 156 reactions from 221 publications. The coarse map encompasses 1384 unique proteins from four publications. The overlap between the two data sources amounts to 46 proteins. The CFTR Lifecycle Map can be used to support the identification of potential targets inside the cell and elucidate the mode of action for candidate substances. It thereby provides a backbone to structure available data as well as a tool to develop hypotheses regarding novel therapeutics.

The cystic fibrosis transmembrane conductance regulator (CFTR) gene is influenced by the fundamental cellular processes like epithelial differentiation/polarization, regeneration and epithelial–mesenchymal transition. Defects in CFTR protein levels and/or function lead to decreased airway surface liquid layer facilitating microbial colonization and inflammation. The SERPINA1 gene, encoding alpha1-antitrypsin (AAT) protein, is one of the genes implicated in CF, however it remains unknown whether AAT has any influence on CFTR levels. In this study we assessed CFTR protein levels in primary human lung epithelial cells grown at the air-liquid-interface (ALI) alone or pre-incubated with AAT by Western blots and immunohistochemistry. Histological analysis of ALI inserts revealed CFTR- and AAT-positive cells but no AAT-CFTR co-localization. When 0.5 mg/mL of AAT was added to apical or basolateral compartments of pro-inflammatory activated ALI cultures, CFTR levels increased relative to activated ALIs. This finding suggests that AAT is CFTR-modulating protein, albeit its effects may depend on the concentration and the route of administration. Human lung epithelial ALI cultures provide a useful tool for studies in detail how AAT or other pharmaceuticals affect the levels and activity of CFTR.

BackgroundNasal potential difference (NPD) and intestinal current measurements (ICM) are cystic fibrosis transmembrane conductance regulator (CFTR) biomarkers recommended to make a diagnosis in individuals with inconclusive sweat test and CFTR genetics and a clinical suspicion for cystic fibrosis (CF) or CFTR-related disorder (CFTR-RD).MethodsNPD and ICM were measured according to standard operating procedures of the European Cystic Fibrosis Society Diagnostic Network Working Group.ResultsWe assessed 219 individuals by NPD or ICM who had been referred to our laboratory due to clinical symptoms suggestive of CF, but inconclusive sweat test and CFTR genetics (median age: 16.3 years, range 0.4 to 76 years). CF or CFTR-related disorder was diagnosed in 22 of 29 patients (76%) with a CFTR genotype of unknown or variable clinical significance and in 51 of 190 carriers (27%) of one (35/42) or no (16/148) identified CFTR mutation. If two CFTR sequence variants had been identified, the outcome of NPD and ICM was consistent with the classification of the CFTR2 database. Moreover, a suspected false-positive diagnosis of CF was confirmed in seven and withdrawn in eight patients. Of 26 individuals assessed by both NPD and ICM, eleven individuals exhibited discordant tracings of ICM and NPD, with one measurement being in the CF range and the other in the normal range.ConclusionThe majority of patients whom we diagnosed with CF or CFTR-RD by extended electrophysiology are carriers of the wild-type CFTR coding sequence on at least one of their CF alleles. The disease-causing genetic lesions should reside in the non-coding region of CFTR or elsewhere in the genome, affecting the regulation of CFTR expression in a tissue-depending fashion which may explain the large within-group variability of CFTR activity in the respiratory and intestinal epithelium seen in this group.

Background The impact of complex alleles on CFTR processing and function has yet not been investigated in native human tissue. Methods Intestinal current measurements (ICM) followed by CFTR immunoblot were performed on rectal biopsies taken from two siblings who are compound heterozygous for the CFTR mutations p.Phe508del and the complex allele p.[Arg74Trp;Val201Met;Asp1270Asn]. Results Normal and subnormal chloride secretory responses in the ICM were associated with normal and fourfold reduced amounts of the mature glycoform band C CFTR, respectively, consistent with the unequal clinical phenotype of the siblings. Conclusion The combined use of bioassay and protein analysis is particularly meaningful to resolve the CFTR phenotype of “indeterminate” borderline CFTR genotypes on a case‐to‐case basis.

The three-base-pair deletion c.1521_1523delCTT (p.Phe508del, F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) is the most frequent disease-causing lesion in cystic fibrosis (CF). The CFTR gene encodes a chloride and bicarbonate channel at the apical membrane of epithelial cells. Altered ion transport of CFTR-expressing epithelia can be used to differentiate manifestations of the so-called CF basic defect. Recently, an 11p13 region has been described as a CF modifier by the North American CF Genetic Modifier Study Consortium. Selecting the epithelial-specific transcription factor EHF (ets homologous factor) as the likely candidate gene on 11p13, we have genotyped two intragenic microsatellites in EHF to replicate the 11p13 finding in the patient cohort of the European CF Twin and Sibling Study. We could observe an association of rare EHF haplotypes among homozygotes for c.1521_1523delCTT in CFTR, which exhibit a CF-untypical manifestation of the CF basic defect such as CFTR-mediated residual chloride secretion and low response to amiloride. We have reviewed transcriptome data obtained from intestinal epithelial samples of homozygotes for c.1521_1523delCTT in CFTR, which were stratified for their EHF genetic background. Transcripts that were upregulated among homozygotes for c.1521_1523delCTT in CFTR, who carry two rare EHF alleles, were enriched for genes that alter protein glycosylation and trafficking, both mechanisms being pivotal for the effective targeting of fully functional p.Phe508del-CFTR to the apical membrane of epithelial cells. We conclude that EHF modifies the CF phenotype by altering capabilities of the epithelial cell to correctly process the folding and trafficking of mutant p.Phe508del-CFTR.

The manifestation of the monogenic disease cystic fibrosis results from the cystic fibrosis transmembrane conductance regulator (CFTR)-mediated basic defect defined as an altered chloride transport. An association study using contrasting endophenotypes was conducted with 17 markers to allow fine-mapping of a previously reported association signal within the CLCA gene cluster. Markers were analyzed for association with the manifestation of the basic defect in the patient population of the European CF Twin and Sibling Study composed of 101 families with a total of 171 patients. The manifestation of the basic defect was associated with markers rs11807298-rs6684219, encompassing the CLCA4 promoter (Praw=0.0013; Pcorr=0.0157). Refined analysis of the CLCA4 association signal among F508del homozygous CF patients who exhibit either no, CFTR-mediated or Ca(2+)-mediated residual chloride conductance revealed that allele distributions for markers rs11807298-rs113894048-rs6684219 differed significantly among these three patient groups. Our data strongly argue that CLCA4 modulates the capability to express residual chloride secretion in colonic tissue. The latter finding is in consistency with the now favored role of the CLCA proteins in signal transduction in epithelial cells.

The major cystic fibrosis mutation F508del has been classified by experiments in animal and cell culture models as a temperature-sensitive mutant defective in protein folding, processing and trafficking, but literature data on F508del CFTR maturation and function in human tissue are inconsistent. In the present study the molecular pathology of F508del CFTR was characterized in freshly excised rectal mucosa by bioelectric measurement of the basic defect and CFTR protein analysis by metabolic labelling or immunoblot. The majority of investigated F508del homozygous subjects expressed low amounts of complex-glycosylated mature F508del CFTR and low residual F508del CFTR-mediated chloride secretory activity in the rectal mucosa. The finding that some F508del CFTR escapes the ER quality control in vivo substantiates the hope that the defective processing and trafficking of F508del CFTR can be corrected by pharmacological agents.

Background The cystic fibrosis (CF) basic defect, caused by dysfunction of the apical chloride channel CFTR in the gastrointestinal and respiratory tract epithelia, has not been employed so far to support the role of CF modifier genes. Methods Patients were selected from 101 families with a total of 171 F508del-CFTR homozygous CF patients to identify CF modifying genes. A candidate gene based association study of 52 genes on 16 different chromosomes with a total of 182 genetic markers was performed. Differences in haplotype and/or diplotype distribution between case and reference CF subpopulations were analysed. Results Variants at immunologically relevant genes were associated with the manifestation of the CF basic defect (0.01

Cystic fibrosis (CF) is a monogenic disease characterized by a high variability of disease severity and outcome that points to the role of environmental factors and modulating genes that shape the course of this multiorgan disease. We genotyped families of cystic fibrosis sib pairs homozygous for F508del-CFTR who represent extreme clinical phenotypes at informative microsatellite markers spanning a 38 Mb region between CFTR and 7qtel. Recombination events on both parental chromosomes were compared between siblings with concordant clinical phenotypes and siblings with discordant clinical phenotypes. Monitoring parent-of-origin-specific decay of genomic sharing delineated a 2.9-Mb segment on 7q34 in which excess of recombination on paternal chromosomes in discordant pairs was observed compared with phenotypically concordant sibs. This 2.9-Mb core candidate region was enriched in imprinting-related elements such as predicted CCCTC-binding factor consensus sites and CpG islands dense in repetitive elements. Moreover, allele frequencies at a microsatellite marker within the core candidate region differed significantly comparing mildly and severely affected cystic fibrosis sib pairs. The identification of this paternally imprinted locus on 7q34 as a modulator of cystic fibrosis disease severity shows that imprinted elements can be identified by straightforward fine mapping of break points in sib pairs with informative contrasting phenotypes.

On 19q13, TGFB1 and the cystic fibrosis modifier 1 locus (CFM1) have been identified as modifiers of the course of the monogenic disease cystic fibrosis (CF). Recently, we have described a transmission disequilibrium at the microsatellite D19S197, localized between TGFB1 and CFM1. To map the corresponding molecular variants, we have selected informative SNP markers within a 600-kb area and compared two-marker-haplotype-distributions between phenotypically contrasting sib pair groups, intending to type only phylogenetically old markers by aiming for close-to-maximal polymorphism information content of the SNPs. Starting with a seed set of five SNPs that cover intermarker distances of up to 50 kb, we have iteratively added more SNPs to the map, until we could identify two genomic fragments of 3,289 and 2,052 bp for which pairs with contrasting phenotypes showed different haplotype distributions on the final 17-SNP-map (P raw = 0.0002, P corr17SNPs = 0.0106 and P raw = 0.0008, P corr17SNPs = 0.0469, respectively). Resequencing of these fragments of four unrelated individuals for each element showed that the mildly and severely affected pairs differ in seven SNPs and concordant pairs differ from discordant pairs in five SNPs. Annotation of these variants indicate that CEACAM6 and a regulatory element near the 3′ end of CEACAM3 are associated with CF disease severity and intrapair discordance, respectively. While our approach was only guided by the markers’ position, the involvement of genes from the CEACAM family in host defense and innate immunity designates these proteins as likely modifiers of the multi-organ disease cystic fibrosis which is known for its cytokine imbalance and pro-inflammatory phenotype.

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.

We have analyzed frequent naturally occurring variants in the autogene FAS in two independent cystic fibrosis (CF) patient populations. Analysis of FAS expression levels from intestinal epithelial biopsies from 16 unrelated F508del-CFTR homozygotes showed a correlation between FAS intron 2 SNP rs7901656 and signals for Affymetrix GeneChip U133 Plus 2.0 probeset 204781_s_at consistent with a dominant model (P=0.0009). Genotype and haplotype analysis at six informative SNPs spanning the FAS gene locus was carried out on 37 nuclear families representing extreme clinical phenotypes that were selected from the European CF Twin and Sibling Study population of more than 300 affected sibling pairs. Case-control comparison of the haplotype composed of rs2296603-rs7901656-rs1571019 encompassing intron 2 of FAS reached significance (P=0.0246). Comparative phylogenetic analysis and functional annotation of the FAS intron 2 sequence revealed a conserved non-coding sequence surrounding rs7901656 and predicted binding sites for four transcription factors whereby the binding site of c-Rel is altered by rs7901656. Taken together, these findings from two independent CF patient cohorts indicate that allelic variants within FAS intron 2 alter FAS gene expression and that these functional variants modulate the manifestation of CF disease.

Two entities localised within in a 5 Mb interval on 19q13, that is the transforming growth factor beta 1 (TGFbeta1) and the cystic fibrosis modifier 1, have been reported to modulate disease severity of cystic fibrosis (CF), albeit the designation of the risk allele for TGFbeta1 differs between studies. We have analysed genotyping data at seven microsatellite loci and four single nucleotide polymorphisms targeting the 19q13 area from 37 nuclear CF families with two affected offspring exhibiting extreme clinical phenotypes for indicators of transmission-ration distortion, maternal genetic or maternal non-genetic effects. Evidence for a transmission-ratio distortion was obtained at D19S112 (P=0.0304) near the recently characterised myotonic dystrophy locus myotonic dystrophy protein kinase (DMPK). Maternal and paternal genotype distributions were significantly different at rs1982073 (Leu10Pro at TGFbeta1) whereby all CF sibs heterozygous at rs1982073 inherited the Leu10 allele from their mother (P=0.000132) in our sibling panel. To ask whether the improved survival in CF over the last decades has any influence on TGFbeta1 allele frequencies, we analysed unrelated F508del homozygotes who were stratified by birth cohort. Sensitivity with respect to the survivor bias was reflected by significantly higher incidence of mild cystic fibrosis transmembrane conductance regulator mutation genotypes in the early born patient cohort (P=0.0169), and an allelic imbalance was also observed at TGFbeta1 (P=0.0664). In conclusion, the role of TGFbeta1 as a CF modulator, suggested from studies with a case-control setting, needs to be interpreted with caution unless family-based analysis is carried out to identify parental genetic and non-genetic effects.

In the context of cystic fibrosis, the epithelial cell has been characterized in terms of its ion transport capabilities. The ability of an epithelial cell to initiate CFTR-mediated chloride and bicarbonate transport has been recognized early as a means to regulate the thickness of the epithelial lining fluid and recently as a means to regulate the pH, thereby determining critically whether or not host defense proteins such as mucins are able to fold appropriately. This review describes how the epithelial cell senses the presence of pathogens and inflammatory conditions, which, in turn, facilitates the activation of CFTR and thus directly promotes pathogens clearance and innate immune defense on the surface of the epithelial cell. This paper summarizes functional data that describes the effect of cytokines, chemokines, infectious agents, and inflammatory conditions on the ion transport properties of the epithelial cell and relates these key properties to the molecular pathology of cystic fibrosis. Recent findings on the role of cystic fibrosis modifier genes that underscore the role of the epithelial ion transport in host defense and inflammation are discussed.

This report describes the first biosynthetic analysis of the cystic fibrosis transmembrane conductance regulator (CFTR) in freshly excised human rectal biopsies. Expression of functional CFTR was assessed by intestinal current measurement (ICM) prior to biosynthetic studies. Several structural features of CFTR are found to be comparable to those established in CFTR-expressing cell lines. Interestingly, maturation of CFTR increases substantially in tissue incubated at 26 °C. Our data provide a solid basis for future studies on the characterisation of CFTR in pathological cases.

IL1R modifies disease severity in CF patients, emphasizing the significance of IL-1 signalling for CF pathogenesis http://ow.ly/pIN730gL10G

Ex vivo biochemical analysis of rectal biopsies of a carrier of the mild 2789+5 G-A CFTR frameshift splice site mutation revealed mutant truncated CFTR of expected size and an imbalance of more core-glycosylated and less mature full-length CFTR. This first immunoblot analysis of a non-F508del CFTR mutant protein derived from human tissue demonstrates that splice site mutations should not only be investigated at the mRNA, but also at the protein level to properly interpret the associations between genotype, molecular pathology and disease.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator gene (CFTR). Disease severity in CF varies greatly, and sibling studies strongly indicate that genes other than CFTR modify disease outcome. Syntaxin 1A (STX1A) has been reported as a negative regulator of CFTR and other ion channels. We hypothesized that STX1A variants act as a CF modifier by influencing the remaining function of mutated CFTR. We identified STX1A variants by genomic resequencing patients from the Bernese CF Patient Data Registry and applied linear mixed model analysis to establish genotype-phenotype correlations, revealing STX1A rs4363087 (c.467-38A>G) to significantly influence lung function. The same STX1A risk allele was recognized in the European CF Twin and Sibling Study (P=0.0027), demonstrating that the genotype-phenotype association of STX1A to CF disease severity is robust enough to allow replication in two independent CF populations. rs4363087 is in linkage disequilibrium to the exonic variant rs2228607 (c.204C>T). Considering that neither rs4363087 nor rs2228607 changes the amino-acid sequence of STX1A, we investigated their effects on mRNA level. We show that rs2228607 reinforces aberrant splicing of STX1A mRNA, leading to nonsense-mediated mRNA decay. In conclusion, we demonstrate the clinical relevance of STX1A variants in CF, and evidence the functional relevance of STX1A variant rs2228607 at molecular level. Our findings show that genes interacting with CFTR can modify CF disease progression.European Journal of Human Genetics advance online publication, 10 April 2013; doi:10.1038/ejhg.2013.57.

Background F508del-CFTR, the most frequent disease-causing mutation among Caucasian cystic fibrosis (CF) patients, has been characterised as a mutant defective in protein folding, processing and trafficking. We have investigated the two neighbouring cytokeratin genes KRT8 and KRT18 in a candidate gene approach to ask whether variants in KRT8 and/or KRT18 modify the impaired ion conductance known as the CF basic defect, and whether they are associated with correct trafficking of mutant CFTR and disease severity of CF. Methods We have selected contrasting F508del-CFTR homozygous patient subpopulations stratified for disease severity, comparing 13 concordant mildly affected sib pairs vs. 12 concordant severely affected sib pairs, or manifestation of the CF basic defect in intestinal epithelium, comparing 22 individuals who exhibit CFTR-mediated residual chloride secretion vs. 14 individuals who do not express any chloride secretion, for an association. The KRT8/KRT18 locus was initially interrogated with one informative microsatellite marker. Subsequently, a low density SNP map with four SNPs in KRT8 and two SNPs in KRT18, each selected for high polymorphism content, was used to localize the association signal. Results KRT8, but not KRT18, showed an association with CF disease severity (Pbest = 0.00131; Pcorr = 0.0185) and CFTR mediated residual chloride secretion (Pbest = 0.0004; Pcorr = 0.0069). Two major four-marker-haplotypes spanning 13 kb including the entire KRT8 gene accounted for 90% of chromosomes, demonstrating strong linkage disequilibrium at that locus. Absence of chloride secretion was associated with the recessive haplotype 1122 at rs1907671, rs4300473, rs2035878 and rs2035875. The contrasting haplotype 2211 was dominant for the presence of CFTR mediated residual chloride secretion. In consistency, the KRT8 haplotype 2211 was associated with mild CF disease while 1122 was observed as risk haplotype. Analysis of microsatellite allele distributions on the SNP background suggests that the mild KRT8 haplotype 2211 is phylogenetically older than its severe counterpart. Conclusions The two opposing KRT8 alleles which have been identified as a benign and as a risk allele in this work are likely effective in the context of epithelial cell differentiation. As the mild KRT8 allele is associated with CFTR mediated residual chloride secretion among F508del-CFTR homozygotes, the KRT8/KRT18 heterodimeric intermediary filaments of the cytoskeleton apparently are an essential component for the proper targeting of CFTR to the apical membrane in epithelial cells.

The molecular pathology of mutant F508del CFTR is partially corrected in vitro by the secondary amino acid substitution R553Q in the ABC signature motif. An individual with the CFTR genotype R553X/F508del-R553Q showed the typical symptoms and electrophysiological anomalies of cystic fibrosis in the airways and intestine. Sweat chloride concentrations were normal early in life, but were later raised into the range that is diagnostic for cystic fibrosis, suggesting that R553Q could temporarily correct the basic defect in sweat glands. R553Q caused a delay in diagnosis because of false negative sweat tests but was not a disease reverting suppressor mutation as had been inferred from cellular models.

Background A major boost to cystic fibrosis disease research was given by the generation of various mouse models using gene targeting in embryonal stem cells. Moreover, the introduction of the same mutation on different inbred strains generating congenic strains facilitated the search for modifier genes. From the original Cftr TgH(neoim)Hgu mouse model with a divergent genetic background (129/Sv, C57BL/6, HsdOla:MF1) two inbred mutant mouse strains CF/1-Cftr TgH(neoim)Hgu and CF/3-Cftr TgH(neoim)Hgu had been generated using strict brother × sister mating. CF/1-Cftr TgH(neoim)Hgu and CF/3-Cftr TgH(neoim)Hgu mice were fertile and showed normal growth and lifespan. In this work the Cftr TgH(neoim)Hgu insertional mutation was backcrossed from CF/3-Cftr TgH(neoim)Hgu onto the inbred backgrounds C57BL/6J and DBA/2J generating congenic animals in order to clarify the differential impact of the Cftr mutation and the genetic background on the disease phenotype of the cystic fibrosis mutant mice. Clinical and electrophysiological features of the two congenic strains were compared with those of CF/1-Cftr TgH(neoim)Hgu and CF/3-Cftr TgH(neoim)Hgu and wild type controls. Results Under the standardized housing conditions of the animal facility, the four mouse strains CF/1-Cftr TgH(neoim)Hgu , CF/3-Cftr TgH(neoim)Hgu , D2.129P2(CF/3)-Cftr TgH(neoim)Hgu and B6.129P2(CF/3)-Cftr TgH(neoim)Hgu exhibited normal life expectancy. Growth of congenic cystic fibrosis mice was comparable with that of wild type controls. All mice but D2.129P2(CF/3)-Cftr TgH(neoim)Hgu females were fertile. Short circuit current measurements revealed characteristic response profiles of the HsdOla:MF1, DBA/2J and C57BL/6J backgrounds in nose, ileum and colon. All cystic fibrosis mouse lines showed the disease-typical hyperresponsiveness to amiloride in the respiratory epithelium. The mean chloride secretory responses to carbachol or forskolin were 15–100% of those of the cognate wild type control animals. Conclusion The amelioration of the clinical features and of the basic defect that had emerged during the generation of CF/3-Cftr TgH(neoim)Hgu mice was retained in the congenic mice indicating that the Cftr linkage group or other loci shared between the inbred strains contain(s) the major modifier(s) of attenuation of cystic fibrosis symptoms.

Background: Knowledge of how CFTR mutations other than F508del translate into the basic defect in cystic fibrosis (CF) is scarce due to the low incidence of homozygous index cases. Methods: 17 individuals who are homozygous for deletions, missense, stop or splice site mutations in the CFTR gene were investigated for clinical symptoms of CF and assessed in CFTR function by sweat test, nasal potential difference and intestinal current measurement. Results: CFTR activity in sweat gland, upper airways and distal intestine was normal for homozygous carriers of G314E or L997F and in the range of F508del homozygotes for homozygous carriers of E92K, W1098L, R553X, R1162X, CFTRdele2(ins186) or CFTRdele2,3(21 kb). Homozygotes for M1101K, 1898+3 A-G or 3849+10 kb C-T were not consistent CF or non-CF in the three bioassays. 14 individuals exhibited some chloride conductance in the airways and/or in the intestine which was identified by the differential response to cAMP and DIDS as being caused by CFTR or at least two other chloride conductances. Discussion: CFTR mutations may lead to unusual electrophysiological or clinical manifestations. In vivo and ex vivo functional assessment of CFTR function and indepth clinical examination of the index cases are indicated to classify yet uncharacterised CFTR mutations as either disease-causing lesions, risk factors, modifiers or neutral variants.

Background From the original Cftr TgH(neoim)Hgu mutant mouse model with a divergent genetic background (129P2, C57BL/6, MF1) we have generated two inbred Cftr TgH(neoim)Hgu mutant strains named CF/1-Cftr TgH(neoim)Hgu and CF/3-Cftr TgH(neoim)Hgu , which are fertile and show normal growth and lifespan. Initial genome wide scan analysis with microsatellite markers indicated that the two inbred strains differed on the genetic level. In order to further investigate whether these genetic differences have an impact on the disease phenotype of cystic fibrosis we characterised the phenotype of the two inbred strains. Results Reduced amounts, compared to wild type control animals, of correctly spliced Cftr mRNA were detected in the nasal epithelia, lungs and the intestine of both inbred Cftr TgH(neoim)Hgu strains, with higher residual amount observed for CF/1-Cftr TgH(neoim)Hgu than CF/3-Cftr TgH(neoim)Hgu for every investigated tissue. Accordingly the amounts of wild type Cftr protein in the intestine were 9% for CF/1-Cftr TgH(neoim)Hgu and 4% for CF/3-Cftr TgH(neoim)Hgu . Unlike the apparent strain and/or tissue specific regulation of Cftr mRNA splicing, short circuit current measurements in the respiratory and intestinal epithelium revealed that both strains have ameliorated the basic defect of cystic fibrosis with a presentation of a normal electrophysiology in both tissues. Conclusion Unlike the outbred Cftr TgH(neoim)Hgu insertional mouse model, which displayed the electrophysiological defect in the gastrointestinal and respiratory tracts characteristic of cystic fibrosis, both inbred Cftr TgH(neoim)Hgu strains have ameliorated the electrophysiological defect. On the basis of these findings both CF/1-Cftr TgH(neoim)Hgu and CF/3-Cftr TgH(neoim)Hgu offer an excellent model whereby determination of the minimal levels of protein required for the restoration of the basic defect of cystic fibrosis can be studied, along with the modulating factors which may affect this outcome.

Transepithelial nasal potential difference (NPD) measurements assess ion conductance in the upper respiratory epithelium. NPD is useful in assisting in the diagnosis of classical and atypical cystic fibrosis (CF) and of cystic fibrosis transmembrane regulator (CFTR)-related disorders, as well as for monitoring the effect of pharmacological agents and gene transfer approaches to correct the abnormalities of ion transport in CF. The article summarizes the objectives and the principle of NPD measurements, describes a hands-on protocol of the procedure and provides quality control measures, practical hints and troubleshooting.

Intestinal current measurements (ICM) on rectal suction biopsies are a tool for the ex vivo diagnosis of classical and atypical cystic fibrosis (CF). We present the basic ICM protocol, typical tracings and their interpretation. The ICM technique allows the registration of CF-induced changes in electrogenic transepithelial ion transport (Cl−, HCO3−, K+) in a Cl− secretory epithelium, and on the basis of pharmacological criteria, is able to discriminate between CFTR-mediated Cl− secretion and secretion through alternative anion channels. ICM is particularly useful for the classification of individuals with CF-like clinical features with equivocal sweat test values and/or no or one identifiable CFTR mutation.