BackgroundThe 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.MethodsPatients 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.ResultsVariants at immunologically relevant genes were associated with the manifestation of the CF basic defect (0.01

Chronic airway infections determine most morbidity in people with cystic fibrosis (CF). Herein, we present unbiased quantitative data about the frequency and abundance of DNA viruses, archaea, bacteria, moulds and fungi in CF lower airways.Induced sputa were collected on several occasions from children, adolescents and adults with CF. Deep sputum metagenome sequencing identified, on average, approximately 10 DNA viruses or fungi and several hundred bacterial taxa.The metagenome of a CF patient was typically found to be made up of an individual signature of multiple, lowly abundant species superimposed by few disease-associated pathogens, such as Pseudomonas aeruginosa and Staphylococcus aureus, as major components. The host-associated signatures ranged from inconspicuous polymicrobial communities in healthy subjects to low-complexity microbiomes dominated by the typical CF pathogens in patients with advanced lung disease. The DNA virus community in CF lungs mainly consisted of phages and occasionally of human pathogens, such as adeno- and herpesviruses. The S. aureus and P. aeruginosa populations were composed of one major and numerous minor clone types.The rare clones constitute a low copy genetic resource that could rapidly expand as a response to habitat alterations, such as antimicrobial chemotherapy or invasion of novel microbes.

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We have used a stepwise approach consisting of initial interrogation, confirmation and fine mapping to analyze STAT3, IL1B and IFNGR1 as modifiers of cystic fibrosis disease building upon the data and sample collection of the European Cystic Fibrosis Twin and Sibling Study. We have observed direct correlation between the length of the intronic microsatellite STAT3Sat to STAT3 expression levels among F508del-CFTR homozygous patients (P¼0.0075), and an association of longer STAT3Sat-alleles with the presence of CFTR-mediated residual chloride secretion (P¼0.0031), measured as the manifestation of the CF basic defect in intestinal tissue. Both, family-based analysis by TDT and case-reference comparison identified consistently the same intragenic IL1B haplotype as a risk variant (P raw ¼0.055 for TDT, P raw o0.3 for case-reference comparison). Using haplotypeguided hierarchical fine mapping, we have identified two single nucleotide exchanges for which concordant and discordant sibling pairs differ at a 7 kb -spanning core haplotype in IFNGR1 (P raw ¼0.0113). Taken together, our findings imply that immunorelevant pathways and ion secretion, dominated by CFTR in intestinal and respiratory epithelium, merge at the level of the epithelial cell to integrate the signaling of cytokines due to innate and acquired immune defense.

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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.

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.

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