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www.thelancet.com Vol 373 May 30, 2009 1891

Cystic brosisBrian P OSullivan, Steven D Freedman

Cystic brosis is the most common lethal genetic disease in white populations. The outlook for patients with thedisease has improved steadily over many years, largely as a result of earlier diagnosis, more aggressive therapy, andprovision of care in specialised centres. Researchers now have a more complete understanding of themolecularbiological defect that underlies cystic brosis, which is leading to new approaches to treatment. One ofthese treatments, hypertonic saline, is already in use, whereas others are in advanced stages of development. Wereview clinical care for cystic brosis and discuss recent advances in the understanding of its pathogenesis,implementation of screening of neonates, and development of therapies aimed at treating the basic defect.

IntroductionThe outlook for people diagnosed with cystic brosisthemost common lethal genetic disease in the whitepopulationhas improved substantially in the past

1020 years. The US Cystic Fibrosis Foundationsprojected life expectancy for patients has increased from31 years to 37 years over the past decade, 1 and a UKmodel predicting that a child born with cystic brosistoday will typically live to be 50 years of age or moreseems to be realistic. 2

Cystic brosis is caused by a mutation in a gene thatencodes cystic brosis transmembrane conductanceregulator (CFTR) protein, which is expressed in manyepithelial cells and blood cells. Although CFTR functions

mainly as a chloride channel, it has many other regulatoryroles, including inhibition of sodium transport throughthe epithelial sodium channel, regulation of the outwardlyrectifying chloride channel, regulation of ATP channels,

regulation of intracellular vesicle transport, acidicationof intracellular organelles, and inhibition of endogenouscalcium-activated chloride channels. 37 CFTR is alsoinvolved in bicarbonatechloride exchange. A deciencyin bicarbonate secretion leads to poor solubility andaggregation of luminal mucins. 8

More than 1500 CFTR mutations have been identied,but only the functional importance of a small number isknown. The table shows one classication system for themost common mutations based on their functionalalterations. The absence of phenyl alanine at position 508(Phe508del, also known as F508del; see panel 1 forglossary of genetic terms), which is a class II mutation,accounts for about two-thirds of mutated alleles in

northern European and North American populations.Although CFTR mutation frequency varies frompopulation to population, world wide no other singlemutation accounts for more than approximately 5% ofCFTR mutations. 10,11

Pancreatic insuffi ciency is closely associated with classIIII mutations; however, variability in genetic back-ground (ie, all other genes in the genome) and environ-ment make genotypephenotype associations weak,especially with regard to lung disease. Mani festations of

Lancet 2009; 373: 1891904

PublishedOnlineApril 28, 2008DOI:10.1016/S0140-6736(09)60327-5

Department of Pediatrics,University of MassachusettsMedical School, Worcester, MA,USA (Prof B P OSullivan MD);and Department of Medicine,Beth Israel Deaconess MedicalCenter, Harvard MedicalSchool, Boston, MA, USA (S D Freedman MD)

Correspondence to:Prof Brian P OSullivan,Department of Pediatrics,University of MassachusettsMedical School, 55 Lake Avenue,Worcester, MA 01655, [email protected]

Search strategy and selection criteria

We searched Medline (up to September, 2008), Google

Scholar for specic topics, and the Cochrane Library forEnglish language reviews pertinent to cystic brosis.Additionally, we used systematic reviews prepared byKaren Robinson at Johns Hopkins University (Baltimore, MD,USA) for the US Cystic Fibrosis Foundations Pulmonary CareGuidelines Committee for chronic therapy, airway clearance,and exacerbation sections of the Seminar. References frompreviously published reviews of cystic brosis were reviewedwith inclusion of the most recent and relevant studies.

Effect on CFTR Functional CFTR present Sample mutations

Class I Lack of protein production No Stop codons (designation ending in X; eg, Trp1282X, Gly542X);

splicing defects with no protein production (eg, 711+1GT,17171GA)

Class II Protein traffi cking defect with ubiquitination anddegradation in endoplasmic reticulum/golgi body

No/substantially reduced Phe508del, Asn1303Lys, Gly85Glu, Leu1065Pro, Asp1507,Ser549Arg

Class III Defective regulation; CFTR not activated by ATP orcyclic AMP

No (non-functioning CFTRpresent in apical membrane)

Gly551Asp, Ser492Phe, Val520Phe, Arg553Gly, Arg560Thr,Arg560Ser

Class IV Reduced chloride transport through CFTR at theapical membrane

Yes Ala455Glu, Arg117Cys, Asp1152His, Leu227Arg, Arg334Trp,Arg117His*

Class V Splicing defect with reduced production of normalCFTR

Yes 3849+10kb CT, 1811+16kb AG, IVS8-5T, 2789+5GA

Adapted from reference 9 by permission of Edward Arnold (Publishers) Ltd. CFTR=cystic brosis transmembrane conductance regulator. *Function of Arg117His is dependentupon the length of the polythymidine track on the same chromosome in intron 8 (IVS8): 5 T, 7T, or 9T. There is more normal CFTR function with a lo nger poly-T track.

Table: Classication of CFTR mutations


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cystic brosis can be very different between patients, evensiblings, with the same CFTR genotype. Polymorphismsin non-CFTR genes might explain this discrepancy.Several studies have shown that polymorphisms intransforming growth factor 1 and mannose-bindinglectin-2 genes are associated with more severe lungdisease, with evidence of genegene interactions. 1214 Similarly, two or more modier genes seem to be themajor determinants of intestinal obstruction in newbornbabies with cystic brosis. 15 Identication of modier-genepolymorphisms could lead to more accurate prediction ofthe course of illness in an individual patient; the geneproducts could become therapeutic targets.

Cystic brosis is most common in populations ofnorthern European descent, among whom the diseaseoccurs in approximately 1 in 3000 births. 11 Birth prevalence

varies from country to country, and with ethnicbackground (gure 1). For example, the disease occurs inroughly 1 in 3000 white Americans, 1 in 400010 000 LatinAmericans, and 1 in 15 00020 000 African Americans. 11 Cystic brosis is uncommon in Africa and Asia, with areported frequency of 1 in 350 000 in Japan. 21 In Europethe Phe508del mutation predominates in the northwest,and decreases in frequency towards the southeast; themost common mutation in Israel is Trp1282X.

There are several hypotheses regarding how CFTRdysfunction leads to the phenotypic disease known ascystic brosis. Four hypotheses are outlined below; it ispossible that aspects of all four contribute to thepathogenesis of the disease.

The low-volume hypothesis postulates that the loss ofinhibition of epithelial sodium channels, because ofCFTR dysfunction, leads to excess sodium and waterreabsorption, resulting in dehydration of airway surfacematerials. 2224 Concomitant loss of chloride effl ux preventsthe epithelium from correcting the low airway surfacewater volume. The subsequent decrease in periciliarywater volume results in a reduction in the lubricatinglayer between epithelium and mucus, with compressionof cilia by mucus causing inhibition of normal ciliary andcough clearance of mucus. According to this hypothesis,mucus on the epithelium forms plaques with hypoxicniches that can harbour bacteria, particularly Pseudomonasaeruginosa.24,25

The high-salt hypothesis argues that in the absence offunctional CFTR, excess sodium and chloride are retainedin airway surface liquid. 26,27 The increased concentrationof chloride in the periciliary layer disrupts the function ofimportant innate antibiotic molecules (eg, human-defensin 1), allowing bacteria that are cleared by normalairways to persist in lungs. 28

Dysregulation of the host inammatory response hasbeen postulated as the putative basic defect in cysticbrosis. Support for this hypothesis lies in the fact thatabnormally high concentrations of inammatorymediators are seen in cystic brosis cell cultures anduninfected ex-vivo tissue samples. 2932 Furthermore,

ndings from lung lavage studies show thatinammation is present in children as young as 4 weeksof age who are apparently free of infection. 33 An increasein proinammatory molecules such as interleukin 8,interleukin 6, tumour necrosis factor , and arachidonicacid metabolites has been found in patients with cysticbrosis. 3436 Stimulation of the nuclear factor-B pathway,platelet hyper-reactivity, and abnormalities in neutrophilapoptosis have also been reported. 3739 At the same time,concentrations of native anti-inammatory substancessuch as interleukin 10, lipoxin, and docosahexaenoicacid are reduced, 31,34,40 leading to an imbalance betweenproinammatory and anti-inammatory mediators thatfavours unabated inammation.

Another hypothesis suggests that primary pre-disposition to infection is a mechanism by which CFTR

dysfunction leads to cystic brosis. In normal hosts,P aeruginosa binds to functional CFTR and initiates aninnate immune response, which is rapid and self-limiting.In patients with cystic brosis, an increase in asialo-GM1in apical cell membranes allows increased binding ofP aeruginosa and Staphylococcus aureus to airwayepithelium, without initiation of the CFTR-mediatedimmune response. 41,42 The result is that in cystic brosis,the rapid, self-limiting response that eliminatesP aeruginosa from the airways is lost at the same time asthere is enhanced attachment of bacteria to the epithelialsurface.

Diagnosis

The diagnosis of cystic brosis should be considered inany child or adult who presents with the signs orsymptoms listed in panel 2. Diagnostic algorithms forclassic and non-classic cystic brosis have beenpublished by the European Union Cystic FibrosisDiagnostic Working Group and the US Cystic FibrosisFoundation. 43,44 These guidelines concur that thediagnosis of cystic brosis consists of nding specicclinical (phenotypic) characteristics in combination withbiochemical or genetic markers of CFTR dysfunction.In general, a diagnosis of cystic brosis can be made ina patient with clinical features of the disease if theconcentration of chloride in sweat is greater than60 mmol/L or if it is in the intermediate range

(3059 mmol/L for infants less than 6 months of age,4059 mmol/L for older individuals), and twodisease-causing CFTR mutations are identied. 44

The sweat test remains the most readily available andclinically useful way of making the diagnosis of cysticbrosis, provided it is done according to strict guidelines,with pilocarpine iontophoresis and a quantitativedetermination of chloride concentration. 45 Sweat chlorideconcentration increases with age in people without cysticbrosis; however, a concentration greater than 60 mmol/Lis still diagnostic of the disease. 46 Panel 3 lists some of thecauses of false-negative and false-positive sweat testresults.

Panel : Glossary ofterminology usedto identifycommon CFTR genemutations

Ala455Glu: A455EAla559Thr: A559TAla561Glu: A561EArg1162X: R1162XArg117Cys: R117CArg117His: R117HArg334Trp: R334WArg347Pro: R347PArg553Gly: R553GArg533X: R533X

Arg560Thr: R560TArg1066Cys: R1066CArg560Ser: R560SAsn1303Lys: N1303KAsp1152His: D1152HAsp1507: D1507Gly85Glu: G85EGly542X: G542XGly551Asp: G551DIle507del: I507delLeu227Arg: L227RLeu346Pro: L346PLeu1065Pro: L1065PPhe508del: F508del

Ser492Phe: S492FSer549Arg: S549RSer549Asn: S549NTrp846X: W846XTrp1282X: W1282XVal520Phe: V520F

CFTR=cystic brosistransmembrane conductanceregulator. Left-hand termsuse three-letter aminoacidcodes; right-hand terms useone-letter aminoacid code.


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Several methods of CFTR mutation detection arecommercially available. In general, use of a discretegroup of mutation probes is faster and less costly thanexpanded mutation analysis, and incorporation of the40 most frequent disease-associated mutations will detectover 90% of affected individuals in most populations. Aconcern with limited analysis is that the diagnosis will bemissed if the patient is affected by a mutation that is notscreened. Full sequence analysis will detect most CFTRmutations; however, it might reveal polymorphisms andnovel mutations of unknown importance.

A helpful method in assessing individuals who might

have cystic brosis, but who do not meet classic diagnosticcriteria, is measurement of nasal transepithelial potentialdifference (NPD). 43,44 Unfortunately, measurement ofNPD is labour intensive, technically diffi cult, and notavailable at all cystic brosis centres. NPD might beuseful in the research setting as a marker of the ability ofpharmaceutical agents to alter chloride channelfunction.

The number of people recognised as having milderproblems possibly associated with CFTR dysfunction isgrowing. These problems include male infertility,recurrent pancreatitis, chronic sinusitis, and primarysclerosing cholangitis. 47,48 Sweat testing is rarely helpful,

since these patients will frequently have borderlinechloride concentrations; however, a sweat chlorideconcentration more than 60 mmol/L will conrm thediagnosis of classic cystic brosis. CFTR mutationanalysis, if done, should be interpreted by someone withgood knowledge of cystic brosis genetics, since variantsthat are not diagnostic might be found. For practical andpsychological reasons, it is best to use the termCFTR-related diseases for this group of disorders,rather than saying that the patient has cystic brosis.

Newborn screeningNewborn screening is done by the measurement ofimmunoreactive trypsinogen (IRT) in blood spots takenfrom newborn infants. A very high IRT concentrationsuggests pancreatic injury consistent with (but not specicfor) cystic brosis. This marker is increased even in infantswith class IV or V mutations that are associated withpancreatic suffi ciency. Infants who have a high IRTconcentration on initial testing undergo further assess-ment via a repeat IRT 13 weeks later (IRT/IRT), or byanalysis of the initial blood spot for a specied group ofCFTR mutations (IRT/DNA). 49,50 The advantages ofIRT/IRT screening are that it avoids the problemsassociated with detecting mutations of uncertain clinical

Figure : Approximate cystic brosis birth prevalence and common mutations for selected countriesBirth prevalence is reported as number of live births per case of cystic brosis. Common/important mutations in each region are listed below the prevalence. The birth

prevalence can vary greatly between ethnic groups in a country. Data from references 10, 11, and 1620.

Low birth prevalenceIntermediate birth prevalenceHigh birth prevalence

1:3500Gly542XCFTRdele2,3

1:3200Gly551AspGly542XAsn1303Lys

1:2900

Gly551AspGly542XArg553X

1:2500Asn1303LysGly551Asp

1:2000Gly542XTrp1282X1:900

Ala455GluGly542X621+1GT

1:3000Trp1282X3849+10 kb CT

1:3500Gly542XLeu346Pro

1:4200Asn1303LysArg1162X

1:25000394delTTGly542X

1:5600394delTT

3659delC

1:550017171GAArg553X

1:5500Gly542X3849+10 kb CT

1:6000Ala561Glu

Arg1066Cys

1:2800CFTRdele2,3

Gly551Asp

1:2850Gly542X

Asn1303Lys

1:4500Ala455Glu

17171GA

1:3300Arg553X

Asn1303LysGly542X

1:4700Gly542X

711+1GT

1:3700Gly542X

1811+16kb AG1:2700

(Brittany)1078delT

Gly551AspTrp846X

1:1400Gly551AspArg117His

1:2500Gly551Asp

Gly542X621+1GT

1:8500Gly542X

Ile507delSer549Asn

1:2400Gly551AspArg117His

1:4700394delTT

Asn1303Lys

1:3200(USA white)

Gly542XGly551AspTrp1282X

1:15000(African American)

Ala559Thr3120+1GA

1:10000(Latin American)

Gly542X4061GA

1811+16kb AG1:10500

(Native American)Arg1162X

Leu1093Pro


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signicance, and does not require immediate discussionof carrier status or non-paternity. The IRT/IRT algorithmneeds a second blood spot to be taken 13 weeks after therst one, which could be a major logistical problem insome populations, whereas the IRT/DNA method allowscomplete testing on one blood specimen. Mutationsincluded in the DNA analysis should reect the frequencyof specic cystic brosis mutations in the local population. 50 A positive screening result by either method only indicates

that a child is at increased risk for cystic brosis; a sweattest must still be done to conrm the diagnosis.

In regions that undertake newborn screening, it hasbecome highly unusual to see infants present with cysticbrosis at an advanced age, or with the classic symptomsof respiratory disease and emaciation. Several studieshave shown that newborn screening for cystic brosisleads to improved nutritional outcomes. 51,52 Other studieshave shown that weight for age in infancy correlatespositively with lung function at 6 years of age. 53,54 Thereare data supporting the hypothesis that improved growthin infancy as a result of newborn screening leads toimproved pulmonary function later in life. 55 Two UKCystic Fibrosis Trust registry-based studies have shownthe clinical benet and cost-effectiveness of newbornscreening; 56,57 the US Centers for Disease Control and

Prevention supports use of cystic brosis newbornscreening throughout the USA. 58

Clinical manifestationsCystic brosis-related symptoms appear throughout life,with great overlap and variability of symptoms andtiming from patient to patient. Figure 2 shows theapproximate age of onset of some of the major clinicalcomplications of the disease.

Gastrointestinal symptomsAround 15% of infants with cystic brosis are born withmeconium ileus, an obstructive condition secondary toinspissated material in the small and large bowels. 8590%

of infants with cystic brosis develop pancreatic insuf-ciency, which can be present at birth or evolve over therst year of life. Typical signs of pancreatic insuffi ciencyare greasy stools, atulence, abdominal bloating, and poorweight gain. Pancreatic insuffi ciency leads to steatorrhoea,fat-soluble-vitamin deciency, and malnutrition. At thetime cystic brosis was rst recognised in 1938, the lifeexpectancy of patients was only months; death was causedby malnutrition. 59 With the introduction of pancreaticenzyme replacement therapy, malnutrition becamemanageable; however, adequate caloric intake andcorrection of fat-soluble-vitamin deciencies remaincrucial components of disease control. 60 Thickenedintestinal secretions, malabsorption, and decreased gut

motility can lead to distal intestinal obstruction or chronicconstipation in older patients. 61 Poor absorption of fatsoluble vitamins (A, D, E, and K) can lead to acro derma-titis, anaemia, neuropathy, night blindness, osteoporosis,and bleeding disorders.

Patients with cystic brosis are at risk for focal biliarycirrhosis caused by obstruction of intrahepatic bile ducts,but clinically apparent cirrhosis occurs in only about 5%of patients, and usually presents by 15 years of age. 61 Bleeding from oesophageal varices is life-threatening forpatients who have portal hypertension; intensiveintervention by gastroenterologists and surgeons isneeded to control it.

Panel : Signs and symptoms of cystic brosis

General (any age) Family history of cystic brosis Salty-tasting skin Clubbing of ngers and toes Cough with sputum production MucoidPseudomonas aeruginosa isolated from airway

secretions Hypochloraemic metabolic alkalosis

Neonatal Meconium ileus Protracted jaundice Abdominal or scrotal calcications Intestinal atresia

Infancy Persistent inltrates on chest radiographs Failure to thrive Anasarca or hypoproteinaemia Chronic diarrhoea Abdominal distention Cholestasis Staphylococcus aureus pneumonia Idiopathic intracranial hypertension (vitamin A deciency) Haemolytic anaemia (vitamin E deciency causes

anaemia by increasing fragility and reducing lifespan ofred blood cells)

Childhood Chronic pansinusitis or nasal polyposis Steatorrhoea Rectal prolapse Distal intestinal obstruction syndrome or intussusception Idiopathic recurrent or chronic pancreatitis Liver disease

Adolescence and adulthood Allergic bronchopulmonary aspergillosis Chronic pansinusitis or nasal polyposis Bronchiectasis Haemoptysis Idiopathic recurrent pancreatitis Portal hypertension Delayed puberty

Azoospermia secondary to congenital bilateral absence ofthe vas deferens


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Pulmonary diseaseThe lungs of children with cystic brosis are normal inappearance at birth, but quickly become infected andinamed, with polymorphonuclear cells present inbronchoalveolar lavage uid obtained from evenhealthy-looking infants. 33 Chronic airway infection,progressing to bronchiectasis, gas trapping, hypoxaemia,and hypercarbia is the hallmark of cystic brosis lungdisease; pulmonary insuffi ciency is responsible for atleast 80% of cystic brosis-related deaths. 1

Typically, infants with cystic brosis are rapidlycolonised by Haemophilus inuenzae or S aureus, or both.Within a short time, P aeruginosa becomes thepredominant organism found in the airways. 62 One groupof investigators showed that 39 (98%) of a cohort of40 cystic brosis infants had serological or culture

evidence of P aeruginosa infection by 3 years of age.63

Persistent infection leads to generation and secretion ofchemotactic cytokines, which recruit large numbers ofpolymorphonuclear cells into the airways. P aeruginosa amplies the cycle of infection and inammation byreleasing toxins and elastases that cleave crucial surfacemarkers on polymorphonuclear cells. These cells thenrelease their own proteases and elastases that exacerbateinjury to any viable polymorphonuclear cells in theregion. 64 Thereafter, bacterial exotoxins and products ofthe damaged neutrophils spur further polymorphonuclearcell recruitment, more inammation, and increasedtissue damage. Release of DNA from senescentpolymorphonuclear cells leads to increased sputum

viscosity.65

The airways of cystic brosis patients are conducive tothe growth of P aeruginosa for several reasons: permissivemicroenvironments within the hypoxic niches of adherentmucous plaques, increased bacterial binding to theepithelium, and decreased bacterial clearance via innateimmune mechanisms. 23,25,41,42 Initially, P aeruginosa growsas a non-mucoid strain that can be cleared by the host, oreradicated with aggressive antibiotic treatment. 66,67 Overtime, P aeruginosa colonies synthesise an alginate coat andform biolms. 42 These biolms, once established, arediffi cult if not impossible to clear with standard antibiotictreatment. There is a pronounced survival benet forthose patients who remain free of pseudomonas

infection.68,69

For this reason, heightened surveillance forP aeruginosa has become commonplace, with strategies toeradicate early infection by use of inhaled antibiotics withor without oral quinolones under investigation. 66,70,71

Cystic brosis airways can be infected with otherpathogens, such as Burkholderia cepacia (a complex ofat least nine different species), Stenotrophomonasmaltophilia, meticillin-resistant S aureus (MRSA), andatypical mycobacteria. 72 Many Burkholderia species haveinnate antibiotic resistance, are transmissible fromperson to person, and are highly virulent. Infection withB cepacia complex can cause a rapid decline in pulmonaryfunction, and increased mortality in patients with cystic

brosis. 73 Occasionally, infection with the complex cancause an invasive, fatal bacteraemiathe so-calledcepacia syndrome. Burkholderia cenocepacia, one of thespecies in the complex, is highly transmissible: infectionwith it is associated with a striking deterioration in

Panel : Causes of false-positive or false-negative sweattest results

False-positive result Atopic dermatitis (eczema) Malnutrition Congenital adrenal hyperplasia Mauriac syndrome Fucosidosis Ectodermal dysplasia Klinefelters syndrome Nephrogenic diabetes insipidus Adrenal insuffi ciency Hypothyroidism Autonomic dysfunction Environmental deprivation

Munchausen syndrome by proxyFalse-negative result Dilution of sample Malnutrition Peripheral oedema Low sweat rate (quantity not suffi cient) Hypoproteinaemia Dehydration CFTR mutations with preserved sweat duct function

(eg, 3849+10kb CT; Arg117His7T)

Figure : Approximate age of onset of clinical manifestations of cystic brosisABPA=allergic bronchopulmonary aspergillosis. CBAVD=congenital bilateral absence of the vas deferens.CFRD=cystic brosis-related diabetes mellitus. DIOS=distal intestinal obstruction syndrome. HPOA=hypertrophicpulmonary osteoarthritis.

Infection

Fetal echogenic bowel Meconium ileus Pancreatic insufficiency Rectal prolapse

Dehydration Hyponatraemic hypochloraemicmetabolic alkalosis

Renal calculi Hyponatraemic hypochloraemic

metabolic alkalosis

Delayed puberty, osteoporosis, CFRD Renal calculi, renal failure CBAVD, HPOA Arthritis, vasculitis Hyponatraemic hypochloraemic

metabolic alkalosis

DIOS Intussuception Hepatic steatosis, biliary brosis Rectal prolapse

DIOS Intussuception Biliary brosis, cirrhosis Digestive tract cancer (adenocarcinoma)

ABPA

Sinusitis Polyposis

ABPA

Haemoptysis, pneumothorax Respiratory failure Sinusitis, polyposis, anosmia

Sinopulmonary

Gastrointestinal

Renal, endocrine, other

Infancy Childhood Adolescence/adulthood


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health, perhaps because of the organisms ability to elicita more robust inammatory response from host cellsthan other B cepacia species. 74,75 Other B cepacia complexspecies can also cause acute deterioration, highlightingthe need for effective infection control at all cystic brosiscentres. 76

Approximately 1520% of patients with cystic brosiscarry MRSA in their airways; this colonisation isassociated with poorer lung function. 77 S maltophilia hasbeen found in increasing numbers in patients withcystic brosis, but so far has not been shown to beassociated with more rapid decline in pulmonaryfunction or wellbeing. 78 Atypical mycobacteria aresometimes found in airway secretions from patientswith cystic brosis; it remains unclear whether thisnding represents true infection in all cases, or is

only saprophagous colonisation in some patients.Mycobacterium avium complex (72%) and Mycobacteriumabscessus (16%) were the most common atypical mycobacteria found in a survey of US cystic brosiscentres. 79 A cross-sectional study from Israel showed anassociation between non-tuberculous mycobacterialinfection and more severe underlying disease, a resultnot found by the US study. 80 The clinical signicance ofrecovering atypical mycobacteria in sputa from cysticbrosis patients is not fully understood, but patientswith persistently positive sputum smears or culturesshould be monitored closely for development ofworsening disease.

Another organism that can cause colonisation without

invasive infection is Aspergillus fumigatus. An intenseallergic response to this fungusknown as allergicbronchopulmonary aspergillosis (ABPA)is seen in115% of patients with cystic brosis, with a frequencythat varies geographically. 81,82 Clinical manifestations ofABPA are wheezing, pulmonary inltrates, and centralbronchiectasis. The minimum diagnostic criteria forABPA are acute or subacute clinical deterioration, totalserum IgE concentration more than 500 IU/mL(1200 ng/mL), immediate cutaneous reactivity toaspergillus, and one of the following: (1) precipitins toA fumigatus or IgG antibody to A fumigatus, or(2) abnormalities on chest radiograph or CT scan thathave not cleared with standard antibiotic treatment. 81

Endocrine disordersPancreatic dysfunction is caused by obstruction ofintrapancreatic ducts with thickened secretions. Withtime, the pancreas undergoes autolysis with replacementof the body of the pancreas with fat. When a certainproportion of islet cells are no longer functional, thepatient will develop insulin insuffi ciency and carbohydrateintolerance, possibly coexisting with insulin resistance. 83,84 Cystic brosis-related diabetes mellitus (CFRD) is notthe same as typical type I or type II diabetes mellitus.Several factors unique to cystic brosis affect glucosemetabolism, including raised energy expenditure, acute

and chronic infection, glucagon deciency, liver dys-function, decreased intestinal transit time, and increasedwork of breathing. 83 As cystic brosis patients get older,clinically apparent CFRD is more likely to develop; up to30% of patients aged over 25 years are reported to havethe condition. 1 However, in one study, nearly 40% ofadolescent cystic brosis patients not previouslydiagnosed with CFRD had abnormal ndings on oralglucose tolerance testing. 84 Notably, female patients withdiabetes have poorer survival than male patients. 85 Because of the association between CFRD and moresevere pulmonary disease, more frequent pulmonaryexacerbations, and poorer nutritional status, any patientirrespective of age who has unexplained weight loss or adecrease in pulmonary function should be assessed forCFRD. Periodic screening with a random glucose

concentration should be done in all cystic brosispatients; a yearly oral glucose tolerance test is thought tobe the best screening method for those 10 years of ageand older. 86

Osteoporosis secondary to vitamin D deciency,chronic systemic inammation, and intermittent cortico-steroid use is increasingly being recognised as acomplication of cystic brosis. Osteopenia starts inchildhood, but generally manifests in adulthood. Boneresorption exceeds bone formation even in wellnourished, clinically stable patients. 87

ReproductionThe vas deferens is very sensitive to CFTR dysfunction.

Virtually all men with classic cystic brosis haveazoospermia and are infertile because of congenitalbilateral absence of the vas deferens, which can also beseen in men with only one CFTR mutation and no othermanifestations of cystic brosis. 47,88 Women with cysticbrosis are fertile. Although there is some controversyabout the effects of pregnancy in cystic brosis, theconsensus is that a woman who has adequate nutritionaland pulmonary reserve can successfully complete theterm of pregnancy. 89

TreatmentChronic pulmonary treatmentAn aggressive approach to cystic brosis care is supported

by two epidemiological studies showing that cysticbrosis centres with high median pulmonary functiontest results see patients more frequently, obtain morefrequent respiratory-tract cultures, and use more oraland intravenous antibiotics than do centres with lowermedian lung function results. 54,90 In an evidence-basedreview, the US Cystic Fibrosis Foundation used the USPreventive Services Task Force recommendation gradesto assess chronic pulmonary therapies. 91 The highestrecommendations were given to inhaled dornase alfa(recombinant human deoxyribonuclease; given daily),and inhaled tobramycin (300 mg twice daily, given in28-day on-off cycles for use in patients with moderate to


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severe disease with P aeruginosa in their airways). 91 Thesetwo agents were also recommended for use in patientswith mild lung disease, although the evidence for benetis not as robust. The Cystic Fibrosis Foundationguidelines also support the use of inhaled hypertonicsaline, chronic azithromycin, ibuprofen, and inhaled agonists in specic patient populations. 91

Despite the inammatory nature of the disease, neitheroral nor inhaled corticosteroids were recommended forroutine use in patients with cystic brosis, because of anunacceptable adverse event prole of oral corticosteroids,and absence of proof of effi cacy for the inhaledmedication. 9193 Intravenous colistin has been found to bebenecial in the acute setting when combined withanother antipseudomonal antibiotic. 94 Inhaled colistinhas been used widely in Europe as a chronic, suppressive

treatment, but only two trials met the Cystic FibrosisFoundation Pulmonary Guidelines Committees eligibilitycriteria for inclusion in their report. Neither of thesestudies showed a benet from inhaled colistin. 95,96

Hypertonic saline, macrolide antibiotics, and ibuprofendeserve special note. These are inexpensive, readilyavailable drugs, which are easy to administer and havevery few side-effects, and thus could be successfully usedin worldwide cystic brosis care. Inhaled hypertonicsaline represents an exciting change to the notion ofcystic brosis care that treats only symptoms to one thatcorrects the underlying defect. 97 By acting as a hyper-osmolar agent, hypertonic saline presumably drawswater into the airways even when CFTR dysfunction is

present. Rehydration of the periciliary layer then allowsimproved mucociliary clearance. Elkins and colleagues 98 found that patients with cystic brosis who receivedhypertonic saline (4 mL of 7% hypertonic saline twice aday via nebulisation for 48 weeks) had a larger increasein forced expiratory volume in 1 s (FEV 1) and fewerpulmonary exacerbations than patients who received09% saline. Similarly, in a study of 24 patients whoreceived 7% hypertonic saline four times a day for 14 dayswith or without amiloride, Donaldson and co-workers 99 showed that hypertonic saline improved mucociliaryclearance and FEV 1 more when given alone than whengiven with amiloride. In-vitro data further suggested thatsustained hydration of airway surfaces was the factor that

caused the improved mucociliary clearance.99

If hyper-tonic saline can correct the basic hydration defect inairways of cystic brosis patients, as the results fromthese studies suggest, it would be most effective usedearly in life, before pulmonary disease becomes estab-lished. In a pilot study, inhalation of 7% hypertonic salinewas well tolerated in a cohort of 13 infants between6 months and 3 years of age. 100 Larger studies of hypertonicsaline in infants are underway.

Macrolide antibiotics have been used for many years totreat patients with diffuse panbronchiolitis, a disease thatshares many features with cystic brosis. 101 Four studieshave addressed the chronic use of macrolides in cystic

brosis. 102105 The largest of these showed that improve-ment in FEV 1 and reduction in pulmonary exacerbationswere higher after treatment with thrice weeklyazithromycin than after placebo in P aeruginosa-positivepatients. 105 The precise mechanisms of action of macrolideantibiotics remain unclear, but azithromycin reducesvirulence factor production, decreases biolm production,and has bactericidal effects on P aeruginosa when it isgrowing in its stationary (biolm) phase. 106 Furthermore,macrolides can affect cytokine production by many celltypes and alter polymorphonuclear cell function, makingthem effective as both antibiotic and anti-inammatoryagents. 107

High-dose oral ibuprofen has been studied in two large,long-term, placebo-controlled trials. 108,109 In a single-centrestudy, Konstan and colleagues 108 showed a decrease in the

rate of loss of lung function over 4 years after ibuprofentreatment compared with placebo, with the largest benetseen in younger patients (513 years). A multicentre trialin Canada enrolled patients 618 years of age with mildlung disease. 109 In this study there was no signicanteffect of ibuprofen treatment on the primary endpoint,FEV1, compared with placebo, although the ibuprofen-treated group spent fewer days in the hospital thanpatients in the placebo group (18 days vs 41 days peryear). No signicant adverse events were reported ineither of these studies; however, a retrospective reportfrom another institution showed that many patientstreated with high-dose ibuprofen chose to discontinuetreatment, often because of gastrointestinal side-effects. 110

Ibuprofen treatment, if used, seems to be most benecialwhen started before the development of severeinammation and pathological changes in the lung. 108

Frequent use of oral antibiotics to reduce symptomssuch as cough and sputum production is warranted inthe treatment of symptomatic cystic brosis patients.Whether prophylactic antibiotics should be used in theasymptomatic infant who is colonised or infected withS aureus, however, is less clear. In a randomised,placebo-controlled trial, Stutman and colleagues 111 showed that use of prophylactic cefalexin for up to 6 yearsdecreased rates of positive S aureus airway cultures, butat the cost of increased rates of P aeruginosa infection.UK studies of the use of prophylactic ucloxacillin in

younger children, for a shorter duration, showedreduction of clinical symptoms without an increased rateof pseudomonas infections. 112,113 In view of the severeconsequences of P aeruginosa infection in patients withcystic brosis, the US Cystic Fibrosis Foundation recom-mends against the use of prophylactic antistaphylococcalagents, 91 but universal agreement about this issue hasnot been reached. 114

Pulmonary exacerbationsTreating ares of cystic brosis lung disease aggressively,especially with intravenous antibiotics, improves pul-monary outcomes, and presumably extends life


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expectancy. 90,115,116 Unfortunately, what constitutes a pul-monary exacerbation of cystic brosis is not clearlydened, as highlighted in a series of reviews onpulmonary exacerbation epidemiology, prevention, andtreatment. 117120 Increased cough, change in sputum colouror quantity, decreased appetite or weight, and change inrespiratory rate and examination (ie, presence of newcrackles or wheezes on auscultation of the chest) areparticularly important features (panel 4). The importance

of a unied approach to the denition of exacerbationsand then treating them appropriately has been shown byKraynack and colleagues 122 who reported that the medianFEV1 of the patients at their centre rose substantially in avery short time after uniform, aggressive standards wereadopted.

Once identied, treatment for a pulmonary exacerbationof cystic brosis generally includes antibiotics (oral,inhaled, or intravenous), increased use of airwayclearance techniques, and improved nutrition. Intra-venous antibiotic treatment has been shown to reducesputum Pseudomonas spp density, and improvepulmonary function. 116 Combination antibiotic treatmentwith agents that have different modes of action is

preferred to single agent treatment to avoid emergenceof resistant strains, with treatment lasting about 14 days. 123 Since most patients with exacerbations will haveP aeruginosa in their airways, the usual in-hospitaltreatment is a combination of a lactam, which interfereswith cell wall biosynthesis, and an aminoglycoside, whichbinds bacterial ribosome subunits and inhibits proteinproduction; 123 however, addition or substitution of otherantibiotics specic for S aureus, H inuenzae , or MRSAmight be necessary. Home-based treatment withintravenous antibiotics is feasible, 124 but might not be aseffective as hospital-based treatment. 125,126 Use of com-bined oral and inhaled antibiotics without hospital

admission might be suffi cient for milder exacerbationand allows the patients daily life to continueunimpeded.

Airway clearance techniquesThere are many techniques used by patients with cysticbrosis to augment clearance of tenacious airway secre-tions. These methods include percussion and post uraldrainage, positive expiratory pressure (PEP) devices, highpressure PEP devices, active cycle of breathing tech-niques, airway-oscillating devices, high-frequency chestwall oscillation devices, and autogenic drainage (ie, chestphysiotherapy in which the patient does a series ofrespiratory huffs and coughs designed to move mucusfrom distal to proximal airways so it can be coughed out).Despite a paucity of well designed, controlled studies,

nearly all cystic brosis caregivers believe in the benet ofairway clearance techniques as a part of the therapeuticregimen. 127 Bradley and colleagues, 128 summarising severalCochrane systematic reviews, and McCool and Rosen, 129 in guidelines written for the American College of ChestPhysicians, describe evidence to support the inclusion ofchest physical treatment in the care of patients with cysticbrosis, but could not nd evidence that one form ofairway clearance was better than another. Thus,convenience, ease of administration, and patient satis-faction are the main driving forces for the choice ofairway clearance method. Notably, percussion andpostural drainage done early in life should avoid thehead-down position, which could increase the risk of

gastroesophageal reux and aspiration.130

Exercise has many benecial effects on cardiovasculartness and sense of wellbeing. Several studies show thataerobic and anaerobic exercise improve quality of life inpatients with cystic brosis and might stabilise lungfunction to some degree, but there is no evidence thatexercise alone should be used as an alternative to airwayclearance. 131

Lung transplantationLung transplantation is the nal therapeutic option forpatients with endstage lung disease. Transplantation hasthe potential to extend and substantially improve qualityof life in properly selected patients. How best to select

patients, especially children, for this high-risk procedureis the subject of vigorous debate. 132134 In Europe, it isunusual for children with cystic brosis to be consideredfor transplantation unless they have a projected lifeexpectancy of less than 2 years despite maximum medicaltherapy. This cautious approach improves the riskbenetratio. 134 5-year survival post transplant for children is lessthan 50%, with slightly better outcomes in adults (50% ofrecipients are alive 6 years post transplant). 135,136 For adults,referral for transplantation generally occurs when apatients FEV 1 plateaus at less than 30% of that predicted.However, age, sex, lung infection and colonisation, andrate of decline of FEV 1 all affect the decision. The presence

Panel : Signs and symptoms of an acute pulmonaryexacerbation in cystic brosis

This mnemonic is based on Dorothy Andersons originaldescription of cystic brosis as cystic brosis of thepancreas.59 Adapted with permission from reference 121(copyright Elsevier, 1994).

C=Cough: increase or change in characterF=Fever: low-grade rise in body temperature

P=Pulmonary function tests (decrease in FEV1)A=Appetite: decrease in appetiteN=Nutrition: weight lossC=Complete blood count: increase in white blood cell countR=Radiograph: new ndings on chest radiographE=Examination: new crackles or wheezesA=Activity: reduction in activity levelS=Sputum: increase in quantity or change in quality

FEV1=forced expiratory volume in 1 s.


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or absence of certain species of B cepacia can affectoutcome and should be taken into account when assessinglung transplant candidates. 137 Use of nocturnal non-invasiveventilation can improve chest symptoms, sleep-associatedhypoventilation, and quality of life in patients with awakehypercapnia who are awaiting transplantation. 138

NutritionThe benets of maintaining good nutrition in regard tolong-term survival and lung health cannot be overstated.In a classic study from 1988, Corey and co-workers 139 reported a clear-cut survival advantage for well nourishedcystic brosis patients compared with less well nourishedpatients. Peterson and colleagues 140 reported improvedFEV1 trajectory in children who gained weight at anappropriate and uninterrupted rate.

Supplementation with pancreatic enzymes should beused in patients who present with pancreatic insuffi ciencyeither on clinical grounds (steatorrhoea, failure to thrive),or as shown by low human faecal elastase-1 con-centration. 60,141 Fat-soluble-vitamin supplementation ismandatory in all patients with pancreatic insuffi ciency.Infants with cystic brosis can be safely breastfed, andthis form of feeding might confer lifelong benets. 142 Patients height and weight should be measured andtheir body-mass index (BMI) calculated at every cysticbrosis clinic visit; those showing a decrease in BMI (orBMI percentage in children) or stunting should receivenutritional counselling. In view of the strong correlationbetween nutritional status and pulmonary function,

attention to nutritional wellbeing should be regarded asone of the cornerstones of good lung health in cysticbrosis; supplements (given orally or via gastrostomytube) should be strongly considered in any patient withless than optimum growth.

New horizonsThe pronounced improvement over the past two decadesin life expectancy for patients with cystic brosis islargely the result of centralisation of care at cystic brosiscentres and aggressive treatment of symptoms. Largepatient registries have been used to examine treatmentoutcomes, and to implement quality improvement pro-gram mes. 54,57,90,143,144 Recent advances in the understanding

of cystic brosis pathophysiology have not yet had timeto result in substantial improvements in clinical care.The great hope for the future is that therapies that treatthe basic defect will normalise life expectancy for thoseborn with CFTR mutations.

Animal modelsA frustrating aspect of cystic brosis research has beenthe lack of a good animal model of the disease. Thewidely used mouse models do not have pronounced lungdisease, making them poor surrogates for the study ofpulmonary treatments. Recently, both cystic brosisheterozygote ferrets and pigs have been developed, 145,146 as

has a litter of CFTR-decient piglets that showsphenotypic similarities to human infants with thedisease. 147 It is hoped that these models will lead toimproved understanding and treatment of cysticbrosis.

Mutation-specic therapiesClass I nonsense mutations are single base substitutionsthat lead to premature termination of mRNA transcriptsand result in a loss of production of full length CFTR.About 10% of patients with cystic brosis carry in-framenonsense mutations, with Trp1282X and Gly542X beingthe most common. 10 Welch and co-workers 148 have reporteda moleculePTC124that has been shown to allowread-through of premature stop codons without disruptionof normal termination signals. Administration of the

compound to a mouse model expressing the CFTR-Gly542Xmutation suppressed the mutation and restored CFTRprotein and function. 149 These promising results led to aphase II human trial, in which an improvement in NPDmeasurements was seen in many, but not all, of thepatients who received the test material. 150

Class II mutations result in faulty processing of nascentCFTR protein. In the most common example, Phe508del,mRNA is translated into a protein that has a foldingdefect. This defect is recognised by the quality controlmechanisms in the endoplasmic reticulum where themisfolded protein forms a stable conformer with achaperone, is ubiquinated, and marked for degradationbefore it can leave the endoplasmic reticulum. 151,152 CFTR

proteins with class III and IV mutations reach the apicalcell membrane, but do not function properly. 153 Hypo-thetical agents that correct the localisation of Phe508delfrom the endoplasmic reticulum to the cell membranehave been called correctors; drugs that increase functionof CFTR that is correctly located at the cell membrane aretermed potentiators.

If a misfolded protein does reach the cell surface, itmight retain a degree of its normal function. This is truefor the Phe508del-CFTR protein. Discovery of a drug thatcould overcome the endoplasmic reticulum qualitycontrol mechanism and allow mutant protein to leaveintracellular organelles and proceed to the cell membranewould have important implications not only for cystic

brosis, but also for many other diseases that are causedby altered protein folding. 151 Several chemicals and smallmolecules that either act as chaperones or that couldallow passage of altered CFTR to the cell membrane areunder investigation. 154158 Chaperones are small intra-cellular molecules that regulate protein traffi cking andhelp nascent proteins to achieve their native structure.With normal folding, chaperones engage and disassociatefrom proteins as the folding process proceeds. If anaminoacid deletion or substitution interferes with normalprotein folding, chaperones fail to disengage and insteadmark the nascent protein for degradation. Modiers ofchaperone function, including phenylbutyrate, have


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shown promise in vitro, but no derivatives have reachedclinical trials. A nutritional supplement, curcumin,generated a great deal of interest when it was rstproposed as a corrector of CFTR misfolding. 156 Un-fortunately, other investigators have not been able toreplicate the initial ndings.

Potentiators of CFTR channel activity could benetpatients who are affected by mutations in which CFTRreaches the apical cell membrane but does not respondappropriately to cAMP-mediated phosphorylation(class III), or does not have normal chloride conductance(class IV). Examples of potentiators include genistein,VX-770 (Vertex Pharmaceuticals, Cambridge, MA, USA),alkylxanthines (eg, CPX [SciClone Pharmaceuticals,Foster City, CA, USA]), and phosphodiesterase-5inhibitors. 155,158162 In a phase IIa clinical trial, patients with

at least one copy of the Gly551Asp mutation showedgreater improvement in chloride channel function, asindicated by decreased sweat chloride concentration andimproved NPD results, after treatment with VX-770 for28 days than did patients given placebo. 161 These ndingswith VX-770 and PTC124 pave the way for targeted therapyof the molecular defect in the near future.

A cocktail of a corrector and a potentiator might be theultimate treatment for most patients with cystic brosis,since Phe508del-CFTR protein that is delivered to theapical cell membrane via a corrector will still havefunctional abnormalities because of loss of thephenylalanine residue in the rst nucleotide bindingdomain. 158,159 However, even before a corrector is available,

Phe508del-homozygous patients might benet frompotentiators, since some of their defective protein reachesthe apical membrane despite the endoplasmic reticulumsquality control mechanisms.

Ways of correcting the biochemical aberrations of cysticbrosis without affecting CFTR function directly includeupregulation of non-CFTR-associated chloride channels(eg, with Moli1901)162 or reduction of epithelial sodiumreabsorption through epithelial sodium channels (eg,with denufosol). 163

Gene therapyIt should be possible to treat an autosomal recessivedisease such as cystic brosis with insertion of one copy

of normally functioning DNA into the affected cells,independent of the class of mutation the recipient hadbefore gene therapy. Although easy in concept (and invitro), in practice gene therapy has proven to be quitediffi cult. Initial gene therapy trials with adenovirusvectors proved to be impractical because of immuno-genicity and low effi ciency of viral vectors to insert DNAinto epithelial cells. 164166 Recently, attention has turned toadeno-associated viruses (AAV) and liposomes aspotential vectors. Flotte and colleagues 167 have shown aphysiological correction of chloride movement in nasalepithelial cells from recombinant AAV-serotype-2 CFTRgene therapy recipients, even in those with low CFTR

mRNA expression. Unfortunately, a phase IIb trial ofrepeated doses of aerosolised AAV CFTR treatment didnot result in signicant improvement in spirometricvalues.168 Additionally, concerns remain about toxicity,and immunological responses to repeated administrationof this vector. 169

The UK Cystic Fibrosis Gene Therapy Consortium hasworked to develop non-viral vectors for gene transfer. 170 So far, their best results in animal models have been witha cationic lipid vector. In their 2006 review, theyanticipated that repeat doses of non-viral vectors and useof new plasmidsand new methods of delivering thesevectorswould be developed in the next few years. 170 It ishoped that next-generation vectors will lead to effectivegene transfer and sustained cure of the pulmonarydisease associated with cystic brosis. For now, however,

the prospect of gene therapy remains a hope more than areality.

ConclusionsCystic brosis is a multifaceted disease that requiresclose attention to pulmonary and nutritional variables.Patients should be seen in centres that have experienceof caring for individuals with the disease and that canoffer expertise in a broad range of areas. Physicians alonecannot provide adequate care; a team consisting ofnurses, nutritionists, respiratory therapists, socialworkers, and others is necessary to achieve the bestoutcomes. The goal in 2009 is to preserve lung functionby maximising current treatment regimens, so that

patients can benet fully from future therapies that couldcorrect the basic defect and turn cystic brosis into amanageable disease.ContributorsBPOS and SDF both participated in the writing of this Seminar. Bothauthors saw and approved the nal version.

Conicts of interestBPOS declares that he has no conict of interest. SDF is a co-inventoron a patent application through Beth Israel Deaconess Medical for theuse of docosahexaenoic acid for the treatment of conditions related tocystic brosis gene mutations. BPOS and SDF do not hold equity inany company producing a cystic brosis-related project. BPOS andSDF have both served on committees for the US Cystic FibrosisFoundation.

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