Prenatal diagnosis of cystic fibrosis by trehalase enzyme assay in amniotic fluid

Amniocentesis and amniotic fluid trehalase enzyme assay were offered to 14 pregnant women at a 1 in 4 risk for a child with cystic fibrosis. Twelve of these pregnancies were screened at the 18th week of gestation; ten proceeded to term, seven following the finding of a normal trehalase activity and three despite the low enzyme level in amniotic fluid. In all ten cases prenatal diagnosis proved to be correct. In two cases with low enzyme activity parents opted for termination at the 19th week, and with PAS-Alcian Blue staining some slight histochemical lesions characteristic of cystic fibrosis were seen in the exocrine glands, including the pancreas and intestinal mucosa, of both fetuses. The total protein content in the meconium of these fetuses was significantly higher than in the controls. Results suggest that trehalase assay in the amniotic fluid is a potential prenatal test for cystic fibrosis and it appears that in fetuses with cystic fibrosis some histochemical and biochemical abnormalities can be observed as early as the 19th week of gestation. The role of ultrasound examination as an additional procedure for the prenatal diagnosis of cystic fibrosis is also discussed.     

An abnormal pattern of amniotic fluid microvillar enzymes signalling fetal cystic fibrosis

We have observed seven pregnancies at risk for fetal cystic fibrosis where second-trimester amniotic fluid microvillar enzyme activities presented an unusual pattern. Low gamma-glutamyltranspeptidase and borderline alpha-glucosidase values were associated with normal aminopeptidase M and intestinal alkaline phosphatase values. All seven pregnancies went to term; five of the seven infants were affected with cystic fibrosis.     

Affinity for glycoproteins of bacteria found in the respiratory tract in cystic fibrosis

The attraction of bacteria commonly found in the respiratory tract in cystic fibrosis to glycoprotein at pH 7 has been studied. The effect of washing on the removal of bacteria from the glycoprotein has also been investigated, and the value of doing both washed and unwashed sputum cultures is discussed.     

Pancreatic fluid secretion and protein hyperconcentration in cystic fibrosis

To study pancreatic protein and water secretion in 28 patients with cystic fibrosis and 21 controls matched for pancreatic acinar function as defined by trypsin secretion, we used a quantitative-marker perfusion technique and continuous intravenous secretin-pancreozymin stimulation. Regardless of the level of pancreatic acinar function, secretions from the patients contained significantly higher concentrations of protein than those from the controls. Total protein output and albumin:protein ratios were not increased in secretions from the patients, but their fluid secretion was significantly decreased at any level of pancreatic function. A significant linear correlation was found between protein and volume secretion in the patients (r = 0.86, P less than 0.001), most of whom had a fluid output of less than 4.2 ml per kilogram of body weight per hour. No such relation was found in the control subjects, whose flow was always above 4.2 ml per kilogram per hour. We conclude that fluid secretion in patients with cystic fibrosis may be a rate-limiting factor in protein output and that a limited flow of hyperconcentrated protein secretions may predispose to protein precipitation and ductal obstruction in the pancreas.     

Salt-independent abnormality of antimicrobial activity in cystic fibrosis airway surface fluid

The link between the genetic defect in cystic fibrosis (CF) and the recently described breach in pulmonary host defense has focused on the role of salt and water metabolism in the airways. Using a human bronchial xenograft model we demonstrate a salt-independent abnormality in bacterial killing in CF airway surface fluid (ASF). Biochemical characterization implicates the absence or dysfunction of a molecule critical to the constitution of normal bacterial killing. Our study suggests that CF transmembrane conductance regulator (CFTR) deficiency causes a primary abnormality in the composition of ASF that leads to a salt-independent defect in host defense. Importantly, this defect is corrected by adenovirus-mediated gene transfer of CFTR.     

IL-8 and IFN-gamma in tear fluid of patients with cystic fibrosis.

Cystic fibrosis (CF) is inherited as an autosomal recessive disorder. It is caused by mutations in the protein-coding gene of chromosome 7, resulting in chronic pulmonary disease and pancreatic insufficiency. The disease affects all secretory epithelia, including the eye. The pathogenesis of ocular changes in CF is still unknown, but the involvement of immunologic processes in patients with CF has been studied in recent years. We measured interleukin-8 (IL-8) and interferon-gamma (IFN-gamma) levels in tears in a group of patients and a group of normal controls to determine if the levels of these cytokines are elevated in CF. The levels of these cytokines in tears and the clinical severity of CF and eye disease were compared. Tear samples were collected from 24 patients with CF at the department of pediatric diseases, Medical University of Bialystok, Poland. Cytokine levels were determined by ELISA. Ophthalmic examinations, including tests for keratoconjunctivitis sicca (dry eye), were used to study the ocular surface. The tear levels of IL-8 and IFN-gamma in the CF patients were significantly higher than those in controls. The clinical severity of CF correlated significantly with the IL-8 and IFN-gamma levels. We found positive correlation between the tear levels of IFN-gamma and dry eye findings in CF patients. Our results suggest that the inflammatory cytokines IL-8 and IFN-gamma may play key roles in the regulation of ocular surface inflammation and the immunologic reaction in patients with CF. The tear levels of IL-8 and IFN-gamma may be candidate markers for evaluation of the clinical status of CF and eye disease. These findings help to provide a new insight into the pathogenesis of dry eye in patients with CF and provide potential targets for therapy.     

Co-ordinate regulation of the cystic fibrosis and multidrug resistance genes in cystic fibrosis knockout mice

The cystic fibrosis (Cftr and multidrug resistance (Mdr1) genes encode structurally similar proteins which are members of the ABC transporter superfamily. These genes exhibit complementary patterns of expression in vivo, suggesting that the regulation of their expression may be co- ordinated. We have tested this hypothesis in vivo by examining Cftr and Mdr1 expression in cystic fibrosis knockout transgenic mice (Cftr(tm1CAM)). Cftr mRNA expression in Cftr(tm1CAM)/Cftr(tm1CAM) mice was 4-fold reduced in the intestine, as compared with littermate wild- type mice. All other Cftr(tm1CAM)/Cftr(tm1CAM) mouse tissues examined showed similar reductions in Cftr expression. In contrast, we observed a 4-fold increase in Mdr1 mRNA expression in the intestines of neonatal and 3- to 4-week-old Cftr(tm1CAM)/Cftr(tm1CAM) mice, as compared with age-matched +/+ mice, and an intermediate level of Mdr1 mRNA in heterozygous Cftr(tm1CAM) mice. In 10-week-old, Cftr(tm1CAM)/Cftr(tm1CAM) mice and in contrast to the younger mice, Mdr1 mRNA expression was reduced, by 3-fold. The expression of two control genes, Pgk-1 and Mdr2, was similar in all genotypes, suggesting that the changes in Mdr1 mRNA levels observed in the Cftr(tm1CAM)/Cftr(tm1CAM) mice are specific to the loss of Cftr expression and/or function. These data provide further evidence supporting the hypothesis that the regulation Cftr and Mdr1 expression is co-ordinated in vivo, and that this co-ordinate regulation is influenced by temporal factors.      

First report of cystic fibrosis mutations in Libyan cystic fibrosis patients

Background: There are few data on the molecular basis of Cystic Fibrosis (CF) in North Africa, probably due to under-diagnosis.

Aim: This is the first study of cystic fibrosis transmembrane conductance regulator (CFTR) mutations in the Libyan population.

Subjects and methods: This study analysed the complete coding region and flanking intronic sequences of the CFTR gene in 10 unrelated Libyan CF patients.

Results: This study identified four mutations (F508del, c.1670delC, N1303K and E1104X), with a high frequency of the latter.

Conclusion: Identification of CF mutations facilitates molecular investigation of cystic fibrosis in the Libyan population and helps to provide effective genetic counselling among CF families.     

Autoantibodies in cystic fibrosis

Autoantibodies to smooth muscle were found more frequently (21%) in the sera of patients with cystic fibrosis than in age- and sex-matched controls. This appears to be associated with liver involvement. There was no increase in the frequency of occurrence of antibodies to ‘reticulin’ or cell nuclei in the sera of patients with cystic fibrosis.     

Pregnancy and cystic fibrosis

A 20-year old parturient with moderate to severe cystic fibrosis presented for an Elective Caesarean Delivery at 31 weeks gestation due to intra-uterine growth retardation and persistent maternal weight loss. The procedure was successfully performed using a combined spinal-epidural anaesthetic technique. A review of the literature is presented and the anaesthetic implications of pregnancy in cystic fibrosis patients are discussed.     

Differential localization of the cystic fibrosis transmembrane conductance regulator in normal and cystic fibrosis airway epithelium.

Deletion of the amino acid residue Phe 508 of the cystic fibrosis transmembrane conductance regulator (CFTR) protein represents the most common mutation identified in cystic fibrosis (CF) patients. A monoclonal and a polyclonal antibody directed against different regions of CFTR were used to localize the CFTR protein in normal and CF airway epithelium derived from polyps of non-CF and CF subjects homozygous for the delta Phe 508 CFTR mutation. To identify the cellular and subcellular localization of CFTR, immunofluorescent light microscopy, confocal scanning microscopy, and immunogold transmission electron microscopy were performed on cryofixed tissue. A markedly different subcellular distribution was identified between normal and CF airway epithelial cells. In normal epithelium, labeling was restricted to the surface apical compartment of the ciliated cells. In contrast, in the epithelium from homozygous delta Phe 508 CF patients, CFTR markedly accumulated in the cytosol of all the epithelial cells. These findings are consistent with the concept that the CFTR delta Phe 508 mutation modifies the intracellular maturation and trafficking of the protein, leading to an altered subcellular distribution of the delta Phe 508 mutant CFTR.     

Prenatal detection of intestinal obstructions, aneuploidy syndromes, and cystic fibrosis by microvillar enzyme assays (disaccharidases, alkaline phosphatase, and glutamyltransferase) in amniotic fluid

Microvillar enzymes (disaccharidases, alkaline phosphatase, and gamma-glutamyltransferase) were assayed in amniotic fluid from pregnancies with normal and abnormal fetuses to determine their specificity and reliability for the prenatal detection of intestinal obstructions and cystic fibrosis. All fetuses with imperforate anus, duodenal atresia, jejuno-ileal atresia, multiple intestinal atresia, or other forms of intestinal obstructions, with or without associated ventral wall defect or aneuploidy syndrome, showed diminished microvillar enzyme activities below the normal range of control amniotic fluid samples. The exclusively intestinal hydrolases maltase, sucrase, palatinase, and alkaline phosphatase were the most reliable and sensitive markers to detect intestinal obstructions whereas more widely distributed trehalase and gamma-glutamyltransferase activities were less sensitive. The combination of intestinal disaccharidase maltase, sucrase or palatinase and ALP assays is more accurate for prenatal diagnosis of CF than a combination of intestinal ALP and GGTF assays.     

Identification of SNPs in the cystic fibrosis interactome influencing pulmonary progression in cystic fibrosis

There is growing evidence that the great phenotypic variability in patients with cysticfibrosis (CF) not only depends on the genotype, but apart from a combination ofenvironmental and stochastic factors predominantly also on modifier gene effects. It hasbeen proposed that genes interacting with CF transmembrane conductance regulator (CFTR)and epithelial sodium channel (ENaC) are potential modifiers. Therefore, we assessed theimpact of single-nucleotide polymorphisms (SNPs) of several of these interacters on CFdisease outcome. SNPs that potentially alter gene function were genotyped in 95well-characterized p.Phe508del homozygous CF patients. Linear mixed-effect model analysiswas used to assess the relationship between sequence variants and the repeatedmeasurements of lung function parameters. In total, we genotyped 72 SNPs in 10 genes.Twenty-five SNPs were used for statistical analysis, where we found strong associationsfor one SNP in PPP2R4 with the lung clearance index (P≤0.01), thespecific effective airway resistance (P≤0.005) and the forced expiratoryvolume in 1 s (P≤0.005). In addition, we identified one SNP inSNAP23 to be significantly associated with three lung function parameters aswell as one SNP in PPP2R1A and three in KRT19 to show a significantinfluence on one lung function parameter each. Our findings indicate that directinteracters with CFTR, such as SNAP23, PPP2R4 and PPP2R1A, may modify the residualfunction of p.Phe508del-CFTR while variants in KRT19 may modulate the amount ofp.Phe508del-CFTR at the apical membrane and consequently modify CF disease.     

CFTR与囊性纤维化

囊性纤维化跨膜传导调节因子（CFTR）是一种cAMP激活的ATP门控性氯离子通道,表达于气道,消化道和生殖道上皮细胞的顶部质膜中。囊性纤维化(CF)是白人中最常见的遗传性疾病之一,由CFTR基因突变造成。对CFTR基因的破译使人们进一步了解CF的发病机制,并为该疾病的诊断提供了新的线索。