Modifying Factors of Cystic Fibrosis Disease: Residual Chloride Secrefion, Genefic Background and Epigenetics

Cystic fibrosis (CF) is an autosomal recessive disease caused by genetic lesions in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. This CFTR gene was cloned in 1989,1-3 and located to the long arm of chromosome 7 (7q3L2). lt encodes the CFTR protein that functions as a adenosine 3',5'-cyclic monophosphate (cAMP)-regulated chloride channe1 in the apical rnembrane of exocrine epithelia, 4,5 like the sweat gland, subrnandibular glands, and the pulmonary, gastrointestinal, hepatobiliaty, and urogenital tracts. In individuals with CF the defective chloride transport leads to abnormal ion and water transport,6 whieh causes dehydration of secretions and malfunctioning of the obstructed exocrine glands, whieh typically results in chronic airway obsttuction, pancreatie insufficiency (PI), and intestinal malabsorption. The sunrival of CF patients has immensely improved throughout the last century: while in 1938, 70% of babies died within the first year of life, 7 the median survival is now reported to be towards 30 years of age,8,9 most probably due to the introduction of new therapeutic regimes, like physiotherapy, aggressive antibiotic treatment, pancreatic enzyme replacement, and proper nutrition. CF is the most common, lethal, inherited disease in the Caucasian population. lO There have been many reports on the incidence in Europe vmying from 1 in 2000 live bitths in Ireland 11 to 1 in 40000 live bit-ths in Finland. 12 In the Netherlands the incidence was estimated around 1 in 3600 life births,13 CF is found to be rare in persons from non-Caucasian origin. The most common CFTR gene mutation in the Caucasian population is the ó.F508 rnutation, a deletion of the amino acid phenylalanine at position 508, which occurs in approximately 70% of CF al1eles and 90% of CF patients. Vet, presently over 870 different CFTR mutations have been identified,14 which give rise to the cystic fibrosis phenotype

An association study on contrasting cystic fibrosis endophenotypes recognizes KRT8 but not KRT18 as a modifier of cystic fibrosis disease severity and CFTR mediated residual chloride secretion

<p>Abstract</p> <p>Background</p> <p>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 <it>KRT8 </it>and <it>KRT18 </it>in a candidate gene approach to ask whether variants in <it>KRT8 </it>and/or <it>KRT18 </it>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.</p> <p>Methods</p> <p>We have selected contrasting F508del-<it>CFTR </it>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 <it>KRT8</it>/<it>KRT18 </it>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.</p> <p>Results</p> <p><it>KRT8</it>, but not <it>KRT18</it>, showed an association with CF disease severity (P_best= 0.00131; P_corr= 0.0185) and CFTR mediated residual chloride secretion (P_best= 0.0004; P_corr= 0.0069). Two major four-marker-haplotypes spanning 13 kb including the entire <it>KRT8 </it>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 <it>KRT8 </it>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 <it>KRT8 </it>haplotype 2211 is phylogenetically older than its severe counterpart.</p> <p>Conclusions</p> <p>The two opposing <it>KRT8 </it>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 <it>KRT8 </it>allele is associated with CFTR mediated residual chloride secretion among F508del-<it>CFTR </it>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.</p

Challenging the diagnosis of Cystic Fibrosis in a patient carrying the 186-8T/C allelic variant in the CF Transmembrane Conductance Regulator gene

BACKGROUND: This report describe for the first time a clinical case with a CFTR allelic variant 186-8T/C (c.54-8 T/C) in intron 1 of CFTR and underline the importance of applying a combination of genetic and functional tests to establish or exclude a diagnosis of Cystic Fibrosis. In this case the diagnostic algorithm proposed for CF has been successfully applied at our Center and previous CF diagnosis assigned in a different Center was not confirmed.Case report: A 38 year-old Italian woman had been treated as affected by CF since 2010, following diagnosis based on sweat tests (reported values of 73 and 57 mEq/L) and a clinical history consistent with CF. No mutations were identified by first level of genetic analysis. Afterwards the patient referred to our center for assessing the relevance of these findings. The genetic variant 186-8T/C (c.54-8 T/C) in intron 1 of the CFTR gene was detected by sequencing. Low-level interstitial-alveolar infiltration was recorded by high-resolution computerized tomography. Lung function was normal and sputum and Broncho Alveolar Lavage cultures resulted bacteriologically negative. Sweat chloride levels was re-assessed and resulted with values of 57 and 35 mEq/L, with a borderline range between 40 and 60 mEq/L. Nasal Potential Difference measurements resulted in three reliable measurements consistent with a non-CF phenotype. Differential diagnosis with ciliary dyskinesia was excluded, as was exon 2 skipping of CFTR gene that might have caused a CFTR functional defect. Furthermore, single cell fluorescence analysis in response to cAMP agonists performed in patient's monocytes overlapped those obtained in healthy donors. CONCLUSION: We concluded that this patient was not affected by CF. This case highlights the need for referrals to highly specialized centers and the importance of combined functional and genetic tests in making a correct diagnosis. Moreover, we confirmed a correlation between NPD tracings and cell depolarization in monocytes providing a rationale for proposing the use of leukocytes as a potential support for CF diagnosis