Cystic fibrosis (CF) is a genetic disease that causes a dysfunctional cystic fibrosis transmembrane conductance regulator protein (CFTR) to be produced. This essay firstly will focus on the mutations of this defective protein and the intracellular effects. It will then consider the symptoms of the disease that can be observed including pulmonary, gastrointestinal, endocrine and reproductive problems. Then focus will be on the current treatment methods which target the consequences of the CFTR dysfunction such as phlegm retention and infection and the new treatment methods which treat the underlying CFTR defect such as targeting the trafficking of the protein.
Cystic fibrosis is an autosomal recessive inherited disease caused by a gene defect on chromosome seven that is responsible for the cystic fibrosis transmembrane conductance regulator protein (CFTR). This is found in the apical plasma membrane of epithelial cells in the lungs, sweat glands, pancreas amongst other tissues. This causes dysfunctional CFTR to be produced leading to a thick sticky mucus causing a recurrent cough, frequent lung infections by bacteria such as Psuedomonas aeruginosa and digestive problems. More than 1,500 mutations have been found including DF508 which is the most common, caused by a deletion of phenylalanine. The mutant allele was first isolated in 1989 and since then life expectancy has improved greatly to between 31 and 37 years old and is still increasing today. Numerous mutations have been identified which are classed differently (class I – VI) depending on how the dysfunctional protein is handled within the cell.
Cystic fibrosis is an autosomal recessive disease which means both parents must be heterozygous carriers of the CF allele in order for the offspring to have a 25% chance of inheriting the disease, or 50% of being a carrier (figure 1). There are over 1,500 observed mutations of the CFTR protein but the majority of these are rare. The most common mutation is caused by a deletion of phenyl-alanine in position 508 (DF508) which accounts for 66% of CF cases. The CF mutations are grouped into 6 classes depending on their functional consequences within the cell (table 1)and the DF508 belongs to class II. Classes I
III are more common and often have associated pancreatic insufficiency though class IV-VI are more rare and these patients are normally pancreatic sufficient. The DF508 for example produces a misfolded CFTR and is recognised within the cell endoplasmic reticulum as an abnormal protein, leading to it being proteolytically degraded in the proteasome. This results in only small amounts of CFTR reaching the plasma membrane but this has a short half life leading to an insufficiency of chloride transport. The misfolded CFTR leads to a protein trafficking problem, hence new drugs that aim to rescue the protein from ER degradation could be therapeutic strategies to re-develop intracellular protein movement. Since different mutations lead to different problems with the CTFR protein, certain treatment strategies may only work on a small proportion of patients.
Table 1: Different classes of CFTR mutations and the effects of each intracellularly – adapted from O’Sullivan, B.P. & Freedman, S.D. (2009) Cystic Fibrosis. Lancet 373: 1891-904
There are several hypotheses as to how this CFTR mutation causes the disease known as cystic fibrosis. The first is the low-volume hypothesis. The loss of inhibition of sodium channels causes excess sodium and water reabsorption causing dehydration of airway surface materials and lack of a compensatory mechanism. This lower water volume causes inhibition of normal ciliary and cough clearance of the mucus and plaques form that harbour bacteria. Secondly, the salt hypothesis believes excess sodium and chloride are retained in airway surface liquid and the increased concentration of chloride disrupts the innate antibiotic molecules so bacteria persist. Thirdly, it is hypothesised disease is due to the dysregulation of host inflammatory response which is backed up by the abnormally high concentration of inflammatory mediators found in children as young as 4 weeks who appear disease free. Finally, the increased presence of asialo-GM1 receptors in apical membranes allow increased binding of P. aeruginosa and S. aureus without the rapid self-limiting innate immune response since in normal patients it is believed the binding of bacteria to functioning CFTR generates an innate immune response which would not function in CF patients. This is made worse by the combination of increased bacterial binding. The CFTR gene defect causes absent or malfunctioning CTFR protein causing abnormal chloride conductance on apical membrane of epithelial cells in the lungs. 
CFTR belongs to a family of transmembrane proteins called adenosine triphosphate binding cassette transporters and is a chloride channel. It also has several other functions such as inhibition of sodium transport through sodium channels in the epithelium, regulation of ATP channels, regulation of intracellular vesicle transport, acidification of intracellular organelles and inhibition of endogenous calcium activated chloride channels. In the lungs, this dysfunctional CFTR causes airway surface liquid depletion leading to decreased ciliary stability and ciliary collapse with decreased mucociliary transport causing phlegm retention, infection and inflammation of the airways.
Increased cAMP levels leads to phosphorylation of CFTR causing chloride transport but since this is not functioning in CF patients the chloride channel fails to open and respond to cAMP (a second messenger). This causes a decreased secretion of Cl? into the lumen airway so excessive water and sodium is absorbed. This cannot cross the epithelial membrane due to the osmotic gradient created leading to increased viscosity of mucus. Local mediators that are secreted onto airway surface liquid help regulate the surface liquid volume as they induce CFTR dependent and independent chloride secretion.
The alternative chloride channel mediates chloride secretion since the P2Y receptor is activated by ATP in both CF and non CF epithelium which is triggered by movement. Respiratory syncytical viruses that may infect the airways have increased ATPase activity so more ATP is broken down; the loss of this compensatory mechanism that would activate the alternative chloride channel has a negative effect on airway clearance becoming a problem in CF patients.
Cystic fibrosis can be diagnosed at different stages of a child’s life; newborn testing occurs as standard since all babies are tested by a heel-prick blood sample as part of the Guthrie test and antenatal testing is carried out on women considered to be high risk of having a child with CF. Carrier testing is a mouthwash test to establish if each parent is a carrier and a genetic test via a swab on the inside of the cheek probes for 40 of the most common CF mutations which correctly diagnoses 90% of cases. One further test is to test the sweat on the skin of babies or children since patients with CF have a high salt concentration in the sweat and CF can be diagnosed if the salt concentration is above 60 mmol/L – this is because CFTR resorbes chlorine into cells of sweat glands and if this is dysfunctional this cannot occur.
General symptoms that lead to a diagnosis include a family history, salty-skin, clubbing of the toes and fingers, a cough with sputum production, mucoid Pseudomonas aeruginosa isolated – repeated chest infections, diarrhoea and poor weight gain. The further symptoms can be grouped into the organ they affect from pulmonary to gastrointestinal, digestive system, endocrine and reproductive symptoms.
Pulmonary symptoms are perhaps the most obvious and commonly associated with the disease. A thick secretion of high levels of mucus into the lungs occurs which leads to frequent bronchial infections and a recurrent cough. Pseudomonas aeruginosa and Staphylococcus aureus are the most commonly isolated bacteria and can be found at high affinities in CF lungs. It is the failure of the mucosal defence system to clear these organisms that is the issue. Early studies suggested P. aeruginosa binds to CF epithelial cells at higher density than normal individuals due to more asialo-GM1 receptors in CF patients, however other theories hypothesised CFTR itself is a receptor for the bacteria that mediates intracellular uptake of the bacteria and killing of it that would be absent in patients with defective CFTR protein. Current studies however suggest the bacteria are present on the mucus layer on respiratory epithelial cells rather than the cell membranes making it unlikely this is the case. It was hypothesised salt-sensitive cationic antimicrobial peptides called defensins could not function in CF patients if the luminal side of the epithelium has an increased sodium chloride concentration. This seems unlikely though as not all defensins are salt sensitive. It is now thought dehydration of the airway surface liquid impairs cilia functioning and mucociliary clearance so inhaled bacteria colonise. Furthermore CF sputum has below normal oxygen levels that switch P. aeruginosa from non-mucoid to mucoid form that is resistant to host defences. “The persistence of chronic P. aeruginosa infections in cystic fibrosis patients is due to biofilm growing mucoid strains.”  These biofilms exhibit increased tolerance to antibiotics and resist phagocytosis as well as parts of the innate and adaptive immune system. This leads to complex-mediated chronic inflammation which can cause lung damage. The bacteria are also so persistent as the mutate and have low metabolic rates and increased doubling times.
In the gastrointestinal tract, several problems occur throughout life. At the newborn stage, some babies may need an operation to remove mucus that is obstructing the bowel – a condition known as meconium ileus. Pancreatic insufficiency is also seen causing symptoms such as greasy stools, flatulence, abdominal bloating, poor weight gain and fat soluble vitamin deficiency with malnutrition. Since it is hard to digest food, malnutrition can occur which causes poor growth, physical weakness and delayed puberty. This requires a pancreatic enzyme therapy with high calorie intake to manage. Older patients’ may develop an intestinal obstruction and the lack of absorption of vitamins A, D, E and K can lead to conditions such as anaemia, neuropathy and osteoporosis.
The endocrine system can sometimes be affected in later life due to obstruction of the pancreatic ducts due to thickened secretions. As pancreatic disease develops the proportion of islet cells declines leading to a lack of insulin production where the blood sugar cannot be controlled which is then diagnosed as CF related diabetes mellitus, with symptoms such as constant thirst, hunger, weight loss and urination, however CF diabetes is not the same as type 1 and 2 diabetes. The reproductive system in women patients does not seem to be affected and they still produce healthy eggs, in men however the sperm ducts are blocked leading to male infertility.
Some other symptoms include frequent sinusitis and hay fever that requires nasal spray or antibiotics and adults may develop nasal polyps. Incontinence can sometimes develop and in some patients bile ducts in the liver become blocked by mucus and the patient may require a liver transplant.
Treatment of cystic fibrosis currently focuses on the consequences of the CFTR dysfunction such as phlegm retention, infection and inflammation though new strategies target the underlying gene defect. Currently, physiotherapy is one main treatment strategy used in combination with other management techniques. The thick sticky mucus secretions that block the airway in CF patients causing infections and coughing can be dislodged either by mechanical chest thumps or autogenic drainage and positive expiratory pressure. Physiotherapy is needed every day from between 15 and 50 minutes depending on the level of mucus present. Physical activity is also important as it prevents deterioration of the lungs and increases bulk and strength.
Medication is used to treat cystic fibrosis such as lung medication including bronchiodilator drugs to open airways by relaxing the surrounding muscles, relieving tightness and shortening of breath and can be taken by being inhaled in nebulisers, taken orally or intravenously. Other medication includes antibiotics to treat persistent pulmonary infections, steroids to reduce inflammation of the airways and DNase to break down the mucus making it easier for the body to digest. Repeated pulmonary infections and thick mucus secretions can become so severe that the patient may need a lung transplant and possibly a heart or liver transplant also.
Due to the nutritional problems associated with the disease, enzyme pills are taken with every meal and snack to replace pancreatic enzymes so more energy is gained from the food since there is a lack of digestive enzymes hence less nutrients can be absorbed from the food. These problems occur due to blocking of the small channels carrying digestive juices by mucus causing enzymes to build up in the pancreas that damages it over time. Nutritional supplements may also be given such as high energy drinks, and insulin may be necessary if the patient develops CF related diabetes mellitus. A suitable diet that is high in calories is also required to ensure adequate energy is gained. The lack of mineral absorption can lead to osteoporosis – weakening of the bones – which can be treated with bisphosphonates.
There are a variety of new treatment possibilities targeting the underlying gene defect in the transmembrane receptor rather than downstream effects. Anti inflammatory drugs are one option due to persistent endobronchial inflammation in patients. The first main possibility is CF transmembrane regulator replacement therapy. This has already been tested using a variety of vectors such as adneoviruses, adeno-associated viruses and cationic lipids to transfect the functioning gene into epithelial cells. Some successful gene transfer has been seen into airway epithelial cells however it was short-lived CFTR expression and was hard to prove the link between improvement in CFTR function and clinical manifestations. The issue is it is yet unknown how much improvement in CTFR function is needed in order to make a big difference. The current research now focuses on the correct vector to use to minimise adverse effects and increase expression time – this is difficult as viral vectors have good transfection rates but more adverse effects and as multi dose therapy would be needed, virus-specific immune responses would devleop whereas liposomal vectors have less negative effects but worse transfection rates. 
A second option being researched currently is CFTR pharmacotherapy involving drugs with affect intracellular trafficking of CFTR. This would not work for all patients due to the specific classes of mutations so it is of limited benefit. Class I mutations are stop mutations that decrease or eliminate production of CFTR. Aminoglycosides induce read through of premature stop codons so would produce a full length functioning CFTR protein, these can be topically applied and an improvement in CFTR functioning has been seen however the concentration needed is high and adverse effects mean they are not clinically suitable. An alternative to this includes PTC 124 – premature termination codon – which acts in a similar way but lacks toxicity. Class II mutations have misfolded CFTR and the trafficking of these is impaired due to proteosomic degradation; this CFTR does have chloride transport function however it is prematurely degraded and most does not reach the membrane. This gives a new target – drugs which reduce degradation of the misfolded protein and increase trafficking to the membrane – and libraries of chemical agents are being screened for applicants. Class III mutations have a reduced probability of opening the CFTR channel but these are rarer. Compounds which activate CFTR would aid class III mutations such as VX770 (a potentiator) that is being used in trials for patients with the G551D mutation that could show improvements in function of the CFTR as well as reduced sweat chloride concentration. However effects in class II may also be seen if used in combination with a corrector compound that brings CFTR to the surface and then the potentiator can activate it. 
Option three involves opening alternative chloride channels to compensate for the lack of function of the CFTR channel. CFTR is not the only chloride transport channel in a membrane, a calcium-dependent chloride channel also secretes chloride in epithelial cells and increasing the activity of this may be an option so enough chloride transport occurs in the cell. Two drugs have shown to have an ability to do this via the P2Y receptor. First of these is denufusol, which bypasses the defective channel and activates the alternative chloride transporter – “This activation results in an increase in airway surface epithelial hydration, and through these actions and effects on cilia beat frequency, increases mucociliary clearance” and has been shown to be an early intervention strategy when inhaled. The second of these drugs is lacovutide (Moli990) increases intracellular calcium level and activates alternative chloride channels, it does not bind with receptors but instead interacts with phospholipids on the plasma membrane.
The CFTR protein has several functions – chloride transport, inhibiting sodium transport as well as regulation of ATP channels. Inhibition of sodium absorption was hypothesised as a treatment option however amiloride (an epithelial sodium channel blocker with a short half life) was shown to have no clinical benefit and a tendency to decreased lung function. Studies on mice have shown when given as an early intervention the disease progression was prevented, however there is little evidence to show this in humans. An improvement may be seen in a blocker with a longer half life.
Finally, airway surface liquid rehydration could improve the inadequate water content of the surface liquid by increasing the airway fluid layer with an inhaled osmotic agent. Hypertonic saline was found to have a positive effect on mucociliary transport and lung function due to induction of coughing and hydrating the mucus and new evidence has shown it also increased depth of the airway surface liquid. Inhaled powdered mannitol is an alternative. Effectiveness is limited to those with established lung disease but again, early intervention may prove more effective. 
Cystic fibrosis is a lifelong eventually fatal disease caused by a genetic defect in the CFTR protein. How this protein functions and which factor is responsible for all the symptoms seen in CF patients is not yet confirmed though it is clear the dehydration if airway surface liquid causing the thick mucus that is hard to dislodge and harbours biofilms of bacteria leading to frequent infection is a major factor. Current treatment strategies target the downstream effects of CF such as the phlegm retention and make the disease manageable. The new development of drugs targeting the underlying defect is occurring with some in clinical trials though the benefit to each patient is unknown. This is because of the diversity of mutations and varying symptoms within each patient making this a difficult disease to treat.
O’Sullivan, B.P. & Freedman, S.D. (2009) Cystic Fibrosis. Lancet 373: 1891-904
Ratjen, F. (2009) Cystic Fibrosis: Pathogenesis and Future Treatment Strategies. Respiratory Care 54: 595-605
Kellerman D, Rossi Mop A, Engels J, Schaberg A, Gorden J, Smiley L, Denufosol: a review of studies with inhaled P2Y(2) agonists that led to Phase 3.( Pulm Pharmacol Therapeutics. 2008 Aug;21(4):600-7. Epub 2007 Dec 31)
Development, Inspire Pharmaceuticals, Inc., 4222 Emperor Blvd, Suite 200, Durham, NC, USA. [email protected]
Hoiby N, Ciofu O, Bjarnsholt T, Pseudomonas aeruginosa biofilms in cystic fibrosis, (Future Microbiology 2010 Nov;5(11):1663-74)
Department of Clinical Microbiology 9301, Rigshospitalet, University of Copenhagen, Juliane Maries Vej 22, Copenhagen, Denmark. [email protected]
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Griffiths, Wessler, Lewonitin, Carroll, Introduction to Genetic Analysis, 9th edition, Freeman
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