The cystic fibrosis J.C. Davies transmembrane conductance ... · understanding of the basics of the disease has expanded enormously. This article will review the nature of disease-causing

163Breathe | December 2008 | Volume 5 | No 2

The cystic fibrosistransmembrane conductanceregulator: state of the art

J.C. Davies

Dept of Gene TherapyImperial CollegeEmmanuel Kaye BuildingManresa RoadLondonSW3 6LRUKFax: 44 2073518340E-mail:[email protected]

ProvenanceAdapted from an ERS Schoolcourse

Educational aimsTo provide an overview of current knowledge on the structure and function of the cysticfibrosis transmembrane conductance regulator (CFTR) protein and its role in CF disease.To explore the different ways in which mutations in the CFTR gene can alter or abolish the function of the protein in vivo.

SummaryIn the 20 years since the gene responsible for cystic fibrosis (CF) was cloned, more than1,300 mutations have been documented, which interfere with the function of the CFtransmembrane conductance regulator (CFTR) protein at every stage from gene tran-scription to the working of the expressed protein. Investigation of the pathogenesis of CF has led to the development of the 'low volume'hypothesis, whereby CFTR dysfunction leads to a depletion in airway surface liquid (ASL)and a resultant reduction in mucociliary clearance (MCC). Infection begins early in lifeand elicits an exaggerated and prolonged inflammatory response, which is thought to bekey in the irreversible tissue destruction characteristic of CF.

Further material to acompanythis ERS School article can befound at www.ers-education.org

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CF is an autosomal, recessively inherited dis-ease caused by mutations in the CFTR gene.

It is common in most Caucasian populations; inthe UK, for instance, approximately one in 22people is a healthy carrier of a single mutation,giving rise to an incidence of CF of ~1:2000 inthe newborn population. In recent years, ourunderstanding of the basics of the disease hasexpanded enormously. This article will reviewthe nature of disease-causing mutations in theCFTR gene, the functions of the CFTR proteinand current hypotheses relating to diseasepathogenesis.

The CFTR geneThe CFTR gene was cloned in 1989 [1] throughpositional cloning approaches. It is located onchromosome 7q31.2, spans 250 kb of genomicDNA and contains 27 exons. The mRNA is 6.2 kband encodes a polypeptide of 1,480 amino acidswith a molecular mass of ~170,000 kDa.

Classes of disease-causing CFTR mutationsMore than 1,300 mutations have been detectedin the CFTR gene. Mutations have been found in

every exon as well as in many of the flankingintron sequences. They include mis-sense, non-sense, frameshift and splice site mutations aswell as amino acid deletions and substitutions.CFTR mutations can be divided into five classes(figure 1).

Class I: defective protein synthesisMutations in this class disrupt synthesis of theCFTR protein. They include nonsense andframeshift mutations, which lead to the creationof premature termination codons (PTCs). In somepopulations, such as Ashkenazi Jewish descen-dants, these mutations are those most com-monly seen. In addition to leading to truncatedproteins, they may also significantly reduce thehalf-life of mutant mRNA through the nonsense-mediated mRNA decay pathway. Therefore, suchmutations are expected to produce little or noprotein; this contention is supported by geno-type-phenotype studies, which usually show anassociation with severe disease. Novel forms oftreatment, directed at overcoming these PTCs,are currently in clinical trial [2].

Class II: defective protein processingNormal CFTR protein undergoes a series of pro-cesses in the endoplasmic reticulum (ER) and theGolgi, including glycosylation and folding; theseare required to enable the protein to traffic tothe apical cell membrane. Class II mutationsimpair this process, and lead to degradation ofthe protein. The commonest mutation world-wide, ΔF508, leads to a protein that is unable tofold correctly. Consequently, it is retained in theER and degraded. If the protein does succeed inreaching the apical cell membrane, it retainssome ion channel activity, although its rate ofturnover is also increased compared with wild-type protein. These observations have led to theexploration of several novel therapies aimed atcorrecting protein folding and trafficking, includ-ing the use of 4-phenylbutyrate [3]. Other com-pounds are being explored with the aid of high-throughput technology, which aims to screenlarge numbers of chemicals for the ability torestore chloride ion efflux.

Class III: defective protein regulationClass III includes mutations leading to the pro-duction of proteins that successfully reach theplasma membrane, but that cannot be activatedand therefore have a low open probability. Novelpharmacological agents directed at this class ofmutation include VX770, which has recently

164 Breathe | December 2008 | Volume 5 | No 2

The cystic fibrosis transmembrane conductance regulator: state of the art

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Figure 1The effects of the five classes ofCFTR mutation.


shown promise in clinical trials, leading tochanges in sweat electrolytes, nasal potential dif-ference and lung function [3].

Class IV: altered conductanceClass IV mutations lead to protein with alteredconductance and thus reduced chloride trans-port. Mutations in this class (and in class V) areoften associated with milder phenotypes andpancreatic sufficiency.

Class V: reduced CFTR levelClass V mutations lead to the production of nor-mal proteins, but at reduced levels, and includesplicing mutations. These can lead to variablelevels of correctly spliced transcripts among dif-ferent patients and even within organs of thesame patient.

CFTR polymorphismsIn addition to the large number of mutations,about 130 genetic polymorphisms have beenidentified in the CFTR gene. Although thesepolymorphisms are variations in the genesequence, they differ from mutations in that theyare also frequently (>1%) found in the generalpopulation. However, it has been establishedthat certain polymorphisms alter the amount offunctional CF gene product. The best-studiedexample is the thymidine polymorphism inintron 8 of the CF gene [4]. This polymorphismexists as a 5-, 7-, or 9-thymidine (T) variant. The5T variant significantly reduces the amount ofnormal CFTR transcript, because intron 8 is incor-rectly spliced, which leads to mRNA lackingexon 9.

CFTR proteinstructureThe amino acid sequence of CFTR suggests atandem repeat structure with two identicalhalves, each consisting of six putative trans-membrane α-helices and an intracellularnucleotide-binding domain, which is capable ofATP hydrolysis (main image and figure 2). Thetwo halves are linked through a highly chargedintracellular regulatory domain (R domain),which contains numerous phosphorylation sites.This protein structure indicates that CFTR is partof the ATP-binding cassette transporter proteinsuperfamily. This is a complex and rapidly evolv-ing field and several review articles have beenpublished on the subject [5].

CFTR function and thebasic defect in cysticfibrosisCFTR is expressed in the apical membrane ofepithelial cells, including airway and intes-tinal epithelium, where it functions as a cyclicAMP-regulated chloride channel. CFTR hasbeen shown to have additional functions,some of which also play a defined role in dis-ease pathogenesis, including regulation of avariety of other channels, most importantlythe epithelial sodium channel (ENaC). In CF,loss of the normal inhibition of ENaC leads tosodium hyperabsorption from the airway sur-face. Water follows down its osmotic gradi-ent, dehydrating the airway surface. Thisforms the basis of the most favoured hypoth-esis of CF disease pathogenesis, termed the"low volume" hypothesis (figure 3). Exquisiteregulation of ASL volume is required for nor-mal function of a primary innate defence sys-tem, MCC. In normal health, cilia, bathed inpericiliary liquid (PCL), beat in a coordinatedfashion to transport the overlying mucuslayer. In CF, both the PCL and the overlyingmucus layer are depleted of volume due tosodium and water hyperabsorption, leadingto failure of this mechanism [6].

Evidence in supportof the low volumehypothesisEvidence in support of this hypothesis comesboth from cell culture and in vivo experiments.Primary airway epithelial cultures were shown toregulate the volume of ASL, maintaining theheight of the PCL at ~7 μm, which correspondsto the height of an outstretched cilium. In



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Figure 2Schematic of the CFTR protein.ENaC: epithelial sodium channel;ORCC: outwardly rectifying chloride channel


contrast, over a period of 24 h, a similar volumeof fluid placed on the surface of CF culturesbecame depleted; the cilia could no longerremain upright but collapsed onto the cell sur-face, which resulted in significantly reducedmucus transport rates (figure 3) [7].Measurement of ASL height in vivo is muchmore difficult. Animal models of CF are of lim-ited value in that they do not mimic well the CFlung phenotype. In contrast, the murine nasalepithelium is much more similar, with regard toion transport abnormalities, to the human CF res-piratory epithelium. In this organ, ASL height hasbeen shown to be significantly reduced in micedeficient in CFTR [8].

MALL et al. [9] have focused on sodiumhyperabsorption, developing a transgenic mouseoverexpresses ENaC. In contrast to CFTR mutantmice, this model demonstrated reduced ASLheight and impaired mucus clearance. In addi-tion, there was a susceptibility to bacterial infec-tion and neutrophilic inflammation very similarto that observed in human CF.

Other functions ofCFTRCFTR has also been shown to regulate the activ-ity of the outwardly rectifying chloride channel,possibly through ATP transport, and probablyhelps to regulate basolateral potassium channelsand aquaporins. It is involved in the transport ofbicarbonate ions, which are likely to be importantin the composition of mucus. In addition, intra-cellular enzymatic processes are impaired in theabsence of normal CFTR; these include sulpha-tion of mucins and the sialylation of surface gly-coconjugates. The latter, in their nonsialylatedform, have been identified as receptors for bacte-ria including Staphylococcus aureus andPseudomonas aeruginosa. Although there is noevidence that direct adherence of wholepathogens to these receptors is a pathogenicmechanism in vivo, it is likely that shed bacterialproducts stimulate inflammation in this fashion.

Infection in the CFairwayIt is hypothesised that impaired MCC allowsinhaled infective organisms to gain a footholdin the CF airway. What is not clear from thelow volume hypothesis is why such a funda-mental defect in innate defence should selec-tively encourage infection with such a narrowrange of organisms, such as S. aureus,Haemophilus influenzae, P. aeruginosa, etc.Patients with a complete absence of MCC dueto genetic defects in the ciliary microstructureitself (various forms of primary ciliary dyskine-sia) appear to be less severely affected bysuch infections, although this may be becausecough clearance, which is well preserved, com-pensates for impaired MCC in a way that itdoes not in CF. Alternative hypotheses toexplain the susceptibility of CF subjects tobacterial infections include: the suggestionthat wild-type epithelial cells ingest such bac-teria; the presence of increased numbers ofreceptors on the CF cell surface; and thedecreased production of antimicrobialmolecules such as nitric oxide and glu-tathione. P. aeruginosa seems to be particu-larly well adapted to survival within the CF air-way. It secretes a complex variety ofexoproducts that incapacitate various arms ofthe host's immune defence and is capable ofrapid genetic mutations, many of which ren-der it resistant to microbial agents. It also hasan affinity for the hypoxic environment foundinside thick mucus plugs, which is one of thetriggers for biofilm formation, which furtherprotects the organism from host recognition.The chronic presence of bacteria within theairway leads to a profound neutrophilicinflammation, although whether such inflam-mation is also seen in the absence of currentor previous infection, is controversial.

ConclusionSince cloning of the CFTR gene in the late1980s, much has been learned about theeffect of the many described mutations onprotein expression, much of which has led tonovel drug targets. Disease pathogenesis isbetter understood, although there are clearlyareas where further research will offer newinsights.

166 Breathe | December 2008 | Volume 5 | No 2


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Figure 3The loss of CFTR function results inincreased sodium aborption andloss of water from the airway sur-face. Scale bar = 7 μm. Adaptedfrom [4], with permission fromthe publisher.




References1. Riordan JR, Rommens JM, Kerem B, et al. Identification of the cystic fibrosis gene: cloning and characterization of

complementary DNA. Science 1989; 245: 1066–1073.2. Zeitlin PL. Emerging drug treatments for cystic fibrosis. Expert Opin Emerg Drugs 2007; 12: 329–336.3. Cystic Fibrosis Foundation. Drug Development Pipeline. Protein Assist/Repair.

www.cff.org/research/DrugDevelopmentPipeline/#PROTEIN_ASSIST/REPAIR Date last accessed: November 11, 2008. Date lastupdated: November 11, 2008.

4. Cottin V, Thibout Y, Bey-Omar F, et al. Late CF caused by homozygous IVS8-5T CFTR polymorphism. Thorax 2005; 60: 974–975. 5. Devidas S, Guggino WB. CFTR: domains, structure, and function. J Bioenerg Biomembr 1997; 29: 443–451.6. Boucher RC. Evidence for airway surface dehydration as the initiating event in CF airway disease. J Intern Med. 2007; 261:

5–16.7. Matsui H, Grubb BR, Tarran R, Randell SH, Gatzy JT, Davis CW, Boucher RC. Evidence for periciliary liquid layer depletion, not

abnormal ion composition, in the pathogenesis of cystic fibrosis airways disease. Cell 1998; 95: 1005–1015.8. Tarran R, Grubb BR, Parsons D, et al. The CF salt controversy: in vivo observations and therapeutic approaches. Mol Cell 2001;

8: 149–158.9. Mall M, Grubb BR, Harkema JR, O'Neal WK, Boucher RC. Increased airway epithelial Na+ absorption produces cystic fibrosis-

like lung disease in mice. Nat Med 2004; 10: 487–493.

Further readingKerem E, Kerem B. Genotype-phenotype correlations in cystic fibrosis. Pediatr Pulmonol. 1996; 22: 387–395.

Tsui LC. The cystic fibrosis transmembrane conductance regulator gene. Am J Respir Crit Care Med 1995; 151: S47–S53.

Donaldson SH, Boucher RC. Sodium channels and cystic fibrosis. Chest 2007; 132: 1631–1636.

Guilbault C, Saeed Z, Downey GP, Radzioch D. Cystic fibrosis mouse models. Am J Respir Cell Mol Biol 2007; 36: 1–7.


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The cystic fibrosis J.C. Davies transmembrane conductance ... · understanding of the basics of the disease has expanded enormously. This article will review the nature of disease-causing