Basic Defect In Cystic Fibrosis Research Articles

Alternative splicing of a previously unidentified CFTR exon introduces an in-frame stop codon 5' of the R region

The cystic fibrosis transmembrane conductance regulator (CFTR) has been extensively characterized as the carrier of the basic defect in cystic fibrosis. CFTR is part of a growing family of proteins encoded by a single gene, the variant isoforms of which are generated by alternative splicing or RNA editing. We have analyzed the CFTR mRNA in the region of exons 10–11 in T84 cells and detected an alternatively spliced exon (10b) accounting for about 5% of the CFTR mRNA. The exon lOb found in both the human and mice genomes, introduces an inframe stop codon. The resulting mRNA is translated into a truncated CFTR protein, identified in T84 cells by immunoprecipitation with the CFTR-specific monoclonal antibody MATG 1061. The insertion of a differentially spliced exon carrying an inframe stop codon is a novel cellular mechanism for the production of a protein sharing common sequences with another, but having different properties and functions.

Renal epithelial Cl‐ secretion

CONTENTS PAGE Introduction 117 A cellular model for Clsecretion in renal epithelial cells 118 The apical Clconductance(s) 120 Claccumulation at the basolateral membrane 120 Activation of basolateral K+ channels, augmentation of Clsecretion 122 Evidence for Clsecretion in intact mammalian renal tubules 124 Sites along the nephron 124 Apical Clchannels (conductance) in renal cells 124 Is Claccumulated above its electrochemical equilibrium level in renal epithelial cells? 125 Evidence from cystic fibrosis 126 The basic defect in cystic fibrosis 126 The cystic fibrosis gene product is abundantly expressed within human kidney 127 Renal defects associated with cystic fibrosis patients 130 Conclusion 131 References 131

Ion transport characteristics of the murine trachea and caecum.

1. The basic defect in cystic fibrosis relates to abnormalities of ion transport in affected tissues, such as the respiratory and gastrointestinal tracts. The identification of the cystic fibrosis gene has enabled studies on the production of a cystic fibrosis transgenic mouse to be undertaken. Knowledge of normal ion transport will be necessary for the validation of any such animal model. We have therefore characterized selected responses of the murine trachea and caecum mounted in 'mini' Ussing chambers under open-circuit conditions. 2. Basal values for the trachea were: potential difference, 1.1 mV (SEM 0.2; n = 18); equivalent short-circuit current, 20.4 microA/cm2 (3.6); conductance, 18.2 mS/cm2 (1.7). Corresponding values for the caecum were: potential difference, 0.7 mV (0.1; n = 18); equivalent short-circuit current, 11.0 microA/cm2 (1.6); conductance, 14.5 mS/cm2 (1.4). 3. Amiloride (10 mumol/l) produced a significant (P less than 0.001) fall in potential difference of 43.0% (5.7) in the trachea, but had no significant effect in the caecum. 4. Subsequently, one of three protocols was used to assess the capacity of either tissue for chloride secretion. Addition of a combination of forskolin (1 mumol/l) and zardaverine (10 mumol/l) produced rises in the potential difference of 873% (509) in the trachea and 399% (202) in the caecum. Both A23187 (10 mumol/l) and phorbol dibutyrate (10 nmol/l) increased tracheal potential difference by 350% (182) and 147% (47), respectively. Neither had a significant effect in the caecum. 5. Subsequent addition of bumetanide caused a fall in the stimulated potential difference of between 39.8% and 71.7%, depending on secretagogue and tissue type.(ABSTRACT TRUNCATED AT 250 WORDS)

Probing the basic defect in cystic fibrosis

The concurrent developments in electrophysiology studies and the identification of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has provided a unique opportunity to probe the basic cellular defect underlying cystic fibrosis. Various properties of the CFTR protein have been deduced from its primary sequence, the variety of mutations in patients and genotype-phenotype correlations, as well as the results of more recent DNA transfection studies. The most exciting observation is the fact that CFTR acts like a cAMP-regulated Cl- channel.

The basic defect in cystic fibrosis

Recent evidence strongly suggests that the cystic fibrosis gene product (CFTR) is a Cl − channel. Its properties, however, differ from those of a 30–50 pS outwardly rectifying channel previously implicated as defective in cystic fibrosis. It is still uncertain whether the pleiotropic effects of the CF defect, such as increased airway Na + absorption and mucus sulfation, are secondary to reduced Cl − conductance, or reflect additional functions of CFTR.

The cystic fibrosis defect approached from different angles — new perspectives on the gene, the chloride channel, diagnosis and therapy

The search for the basic defect in cystic fibrosis (CF) has reached a decisive stage since the recent identification of the responsible gene. Electrophysiological and biochemical research had defined the CF defect as a dysregulation of epithelial chloride channels. The putative protein product of the now identified gene shares properties with other known transport proteins, but it is not necessarily itself a chloride channel protein. Elucidation of the primary cellular defect will certainly have important aetiological and hopefully therapeutic implications. The identification of the major gene mutation already has significant consequences for genetic counselling and prenatal diagnosis. Heterozygote detection at the population level awaits identification of the probably heterogenous mutations on about 30% of the CF chromosomes. At present, about 50% of CF patients are homozygous for the recently identified major CF mutation.

Proceed With Caution: Predicting Genetic Risks in the Recombinant DNA Era

Medical genetics is in its golden age. Discoveries that elucidate a variety of genetic diseases are occurring rapidly. The definition of the basic defect in cystic fibrosis in 1989 is a spectacular example that is likely to lead to extensive genetic testing for the carrier state. A book that deals with the social and medical impact of genetic screening is therefore timely and welcome. Dr Holtzman is eminently qualified to write such a book, having spent most of his efforts over the last few years studying and pondering these issues. He starts out by providing the biologic background and explains how modern gene technology can be used in the diagnosis of genetic disease. While the molecular diagnosis of a given genetic defect is usually clear-cut, the resultant clinical manifestations often show variability in age of onset and severity. This axiom is particularly true for common diseases that depend upon interaction

Tracheal Potential Difference in the Reserpine and Isoproterenol Rat Models of Cystic Fibrosis

Basic research into cystic fibrosis (CF) has been hampered by the lack of a suitable animal model. Reserpinized or isoproterenol-treated rats have been proposed as models because they exhibit certain morphological and physiological features characteristic of CF. Recent evidence suggests that abnormal epithelial transport of Na+ [corrected] and Cl- may underlie pathogenesis, defects that may contribute to the markedly more negative transepithelial electrical potential differences (PD) recorded in CF airways compared with controls. To test the models further, we measured tracheal PD in vivo in treated rats (reserpinized - 6.9 mV, SEM 0.7 mV, n = 7; isoproterenol-treated -10.2 mV, SEM 1.5 mV, n = 12) and found it to be no different from that of controls (-8.7 mV, SEM 0.6 mV, n = 25). The animals did, however, demonstrate a reduced gain in body weight as well as increased submaxillary gland weight, which reflected an increased mucus content in the acini. These observations suggest that although the reserpinized or isoproterenol-treated rat may be useful in the study of the pathogenesis of exocrine disturbances in disease, their use as models for the effect of the basic defect of CF in the airways may be limited.

Discovery of the cystic fibrosis gene: the interface of basic science and clinical medicine.

Because the Red Journal is devoted to communication and education relating to cell and molecular biology of the lung, we would be remiss in not reviewing the recent publications that identify the gene for cystic fibrosis (CF) and further our understanding of the basic defect in CF (1-3). The CF story has been the subject of considerable publicity, in part because it is the most frequent fatal autosomal recessive disorder in Caucasians. Identification of the gene is the most recent but certainly not the only instance where investigation of a clinical pulmonary problem has resulted in major contributions to our understanding of basic biology. Of great importance to this journal is the illustration of the enormous potential that molecular biology has to impact on clinical medicine. CF was first recognized as a clinical entity in the 1930s. In the 1950s, abnormal chloride secretion in CF was recognized and established as a diagnostic test and the general approach to therapy was established (see reference 4 for review and references). The focus has been on the gene and the basic defect in the 1980s. Studies in the early 1980s identified a defect in airway epithelial chloride secretion. Subsequent work narrowed the search to some abnormality in chloride channels on the apical surface of the epithelium. It became clear that there was a defect in secretion of Clin response to beta-adrenergic agonists and that this defect was not due to altered receptor binding, to the subsequent activation of adenyl cyclase, or to an altered phosphokinase A (PKA), a kinase that phosphorylates the chloride channel. Patchclamping studies of isolated cell membranes showed that the Cl channel is normal in CF. However, there appeared to be some sort of an abnormal interaction of PKA with the apical chloride channel. Together with an energy source, ATP, PKA opened the channel and allowed Clflux to occur in normal but not in CF cells. Recent studies have shown that another kinase, phosphokinase C (PKC), activated by other receptors, also induces chloride flux in normal airway cells but fails to do so in CF cells. There may be other transport abnormalities in CF (e.g., sodium flux is excessive in cAMP-

Relation between essential fatty acid metabolism and gastrointestinal symptoms in cystic fibrosis.

Studies in our laboratory have supported the hypothesis, that the basic defect in cystic fibrosis increases the metabolism of essential fatty acids and thereby gradually gives rise to essential fatty acid deficiency, which is a well documented finding in most cases with this disease. Both the increased metabolism--giving high liberation of arachidonic acid and its metabolic products, i.e. different eicosanoids--and the subsequent essential fatty acid deficiency will cause gastrointestinal symptoms and the sequence of this development will mirror the natural history of the disease. Clinical data and results from animal research are discussed in relation to gastrointestinal symptoms and signs of cystic fibrosis.

Complement receptor expression on neutrophils at an inflammatory site, the Pseudomonas-infected lung in cystic fibrosis.

Activation of human neutrophils (PMN) is accompanied by rapid upregulation of CR1, the C3b receptor, and CR3, the iC3b receptor, which also serves as the PMN's major adherence protein. This is necessary for migration and phagocytosis, but the extent of expression of these proteins on PMN at inflammatory sites has not been determined. We used monoclonal antibodies and flow cytometry to assess CR1 and CR3 expression on PMN in bronchoalveolar lavage (BAL) fluid of cystic fibrosis (CF) patients chronically infected with pseudomonas and in sterile joint fluid of arthritis patients. Resting peripheral blood PMN from these patients and normals expressed similar low levels of CR1 and CR3, and the patients' PMN increased CR1 and CR3 expression normally when stimulated in vitro. CR3 expression on CF BAL PMN was 90 +/- 12% of that on the same patient's blood cells stimulated in vitro with FMLP. In contrast, CR1 expression on BAL PMN was only 27 +/- 8% of that on stimulated blood cells. Similar results were obtained for joint PMN. This pattern could be reproduced in vitro by treating FMLP-stimulated blood cells with BAL supernatants or with pseudomonas or PMN elastase. The serine protease inhibitors, PMSF and alpha 1-antitrypsin prevented the lavage supernatant from reducing CR1 expression, while metalloprotease inhibitors had no effect. Treatment of PMN with elastase in vitro decrease their ability to kill opsonized Pseudomonas aeruginosa. These results suggest that PMN at inflammatory sites have maximally upregulated expression of their complement receptors, but that CR1 is then cleaved by proteolysis in situ. Although not related to the basic defect in CF, this may interfere with efficient phagocytosis and contribute to the CF patient's inability to eradicate chronic lung infection.

INFLUENCE OF INTRAVENOUS FATTY ACID SUPPLEMENTATION ON NASAL TRANSEPITHELIAL POTENTIAL DIFFERENCE IN CYSTIC FIBROSIS PATIENTS

Defective regulation of arachidonic acid has been hypothesized to be a basic defect in Cystic Fibrosis(CF). In order to demonstrate the role of intravenous fatty acid supplementation on Cl− transport in CF patients, we studied the influence of intralipid 20% (Kabivitrum) infusion 12 hours 10 ml/Kg, on measurement of electrical potential difference (PD) across nasal epithelia.

The Basic Defect in Cystic Fibrosis

The Basic Defect in Cystic Fibrosis

Diet for cystic fibrosis. Nutritional requirements and prescriptions.

Diet plays no direct role in neutralizing the effects of the basic defect in cystic fibrosis, but it can prevent some of the acquired damage caused by complications. Adequate caloric intake provides the energy needed for exercise and cell metabolism, a strong diaphragm, normal cellular immunity, and a positive psychological outlook. Clinical management is aimed at achieving good nutrition. Patient education about nutrition and use of dietary supplements should be started immediately upon diagnosis. Careful clinical examinations and regular follow-up are necessary to detect complications that will interfere with good nutrition. Complications must be treated aggressively. Psychological and social stresses need to be recognized, and psychological referral may be necessary. The clinician's best tools to achieve these goals are the patient's dietary and gastrointestinal history, the anthropometric measurements taken at each office visit, regular patient assessment, and constant attention to detail in monitoring and charting the course of disease. Extraordinary measures, such as nighttime feedings by nasogastric or gastrostomy tube and intravenous hyperalimentation, may be necessary. For best results, these measures must be started before pulmonary complications threaten survival.

Biochemical and genetic exclusion of calmodulin as the site of the basic defect in cystic fibrosis.

Recent physiological studies have shown a defective beta-adrenergic regulation of chloride transport and protein secretion in tissues affected by cystic fibrosis. The exact biochemical nature of this abnormality is unknown, but an intracellular second messenger may be involved. We have tested the hypothesis that calmodulin is the site of the basic defect in CF using biochemical and molecular genetic techniques. We report here that there is no gross structural abnormality in the calmodulin protein from CF submandibular glands, and that although there are at least three distinct sequences that cross-hybridise with a calmodulin cDNA probe in the human genome, none of these can be the locus of CF. A polymorphism at the locus of a calmodulin cross-hybridising sequence at human chromosome 7p2 is described.

Establishment of a tissue culture system for epithelial cells derived from human pancreas: a model for the study of cystic fibrosis

A tissue culture system for epithelial cells derived from human foetal pancreas has been established. The cultured cells show many ultrastructural features of interlobular duct cells. Immunocytochemical and histochemical evidence is presented in support of the view that these cells are ductal in origin. They are likely to be one of the few cell types that express the basic defect of cystic fibrosis in vitro. The cells may be passaged and sufficient material obtained to permit biochemical and molecular biological analysis.

A clue to the basic defect in cystic fibrosis from cloning the CF antigen gene.

The metabolic basis of the autosomal recessive disease cystic fibrosis (CF) remains unidentified. Elevated levels of a serum protein in CF homozygotes and obligate heterozygotes have been described. As heterozygotes are clinically unaffected, any consistently observed abnormality in these individuals is a likely pointer to the aetiology of the disease. The gene for this serum protein, called cystic fibrosis (CF) antigen, has been mapped to chromosome 1. It is not the gene that is mutant in CF because this has since been assigned to chromosome 7 by cosegregation of the disease with closely linked DNA markers in CF families. CF antigen is a product of normal and leukaemic granulocytes and is inducible in the promyelocytic cell line HL60 (M.N., J.D., C. Hayward, F. Northrop, D.J.H.B., J. Walker, V. van H. and D.S.S., manuscript in preparation). We have isolated cDNA clones for this protein from a library constructed with messenger RNA from chronic myeloid leukaemia (CML) cells. The complete nucleotide sequence was obtained from the cDNA clone and by primer extension of mRNA. We have confirmed that the gene encoding CF antigen is on chromosome 1 and have localized it to a particular region. RNA blot analysis shows a 550-bp major transcript in CML cells and in induced HL60. The amino-acid sequence predicted from the nucleotide sequence shows significant homology with intestinal and brain calcium-binding proteins. Abnormal accumulation of such a protein in CF is a clue which must be pursued now that evidence is gathering that the basic defect in CF is in pathways controlling chloride channel activity.

Cystic fibrosis: A casualty of “detoxification”?

It is proposed that excessive and/or aberrant function of cytochromes P450, due to a combination of genetic and environmental influences, is the basic defect in cystic fibrosis. Organs that are involved in oxidative detoxification reactions in foetal life are thus at risk: tissue damage is initiated by excessive production of oxygen free radicals which deplete cellular antioxidants and provoke the secretion of mucus.

FETAL ABNORMALITIES IN CYSTIC FIBROSIS SUGGEST A DEFICIENCY IN PROTEOLYSIS OF CHOLECYSTOKININ

Ultrastructural and microvillous enzyme (MVE) histochemical studies of fetuses with cystic fibrosis (CF) and trisomies 13 and 18 identified features in CF which differed from the abnormalities in trisomies 13 and 18. The principal abnormalities in CF were in the tight (occluding) junctions and intracellular organelles, particularly the golgi and mitochondria, of the epithelial cells of the pancreas, respiratory system, intestine, and gallbladder. Abnormalities of amniotic fluid MVE levels in CF and trisomy 13 occur because of disruption of the pathways by which the MVE reach the amniotic fluid. Trisomy 18 shows hypoplasia and deficiency of epithelial cell microvilli. It is postulated that the basic defect in CF is due to the deficiency of an enzyme that cleaves the Arg-Asp peptide bond in cholecystokinin to produce the active octapeptide CCK-8, which normally stimulates exocrine secretion, especially in pancreas, gallbladder, and intestine, and potentiates the action of other gastrointestinal hormones.

Autonomic nervous system abnormalities in cystic fibrosis

The basic defect in cystic fibrosis, the most common lethal genetic diseases of white Americans, is unknown, but the character of the generalized exocrinopathy suggests some disorder of the regulation and control of the process of glandular secretion. Definite abnormalities in all branches of the autonomic nervous system have been demonstrated in patients with cystic fibrosis. including increased sensitivity to α-adrenergically stimulated pupillary dilation; increased responsiveness to cholinergic stimulation of pupillary constriction, parotid saliva secretion, and eccrine sweat secretion; and decreased responsiveness to β-adrenergic stimulation of the cardiovascular system as well as circulating lymphocytes and granulocytes. Since these abnormalities also occur in asymptomatic heterozygotes for cystic fibrosis (parents of patients), they are likely to be inherited characteristics and not secondarily acquired. This constellation of inherited autonomic abnormalities—α-adrenergic and cholinergic hyperresponsiveness and β-adrenergic resistance—may contribute to the pathophysiology of cystic fibrosis and may also be an important clue to the nature of the basic defect.