Patients with CF have abnormal transport of chloride and sodium across the respiratory epithelium, resulting in thickened, viscous airway secretions. Patients develop chronic infection of the respiratory tract with a characteristic array of bacterial flora, leading to progressive respiratory insufficiency and eventual respiratory failure.
CF is caused by mutations in a single large gene on chromosome 7 that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) protein. CFTR belongs to the ABC (ATP-Binding Cassette) family of proteins, a large group of related proteins that share transmembrane transport functions. ABC proteins include bacterial transporters for amino acids and other nutrients, as well as the mammalian multidrug resistance (MDR) protein (or P-glycoprotein). CFTR has been shown to function as a regulated chloride channel, which in turn may regulate the activity of other chloride and sodium channels at the cell surface. The CFTR gene spans 250 kilobases on chromosome 7, encoding 1480 amino acids in the mature protein.
The protein has two groups of six membrane-spanning regions, two intracellular nucleotide-binding folds (NBFs), and a highly charged "R domain" containing multiple phosphorylation sites. Activation of the chloride channel requires phosphokinase A-mediated phosphorylation of the R domain, and the continuous presence of ATP in the NBFs.
Genetic changes in CFTR
The phenotypic expression of disease varies widely, primarily as a function of the specific mutation (or mutations) present. More than 1200 distinct sequence changes in the CFTR gene are associated with clinical disease . The severity of clinical disease appears to vary as a function of the specific genetic mutations present in a patient with CF.
Mutations of the CFTR gene have been divided into four different classes:
Class I - Defective protein production — This defect is usually caused by nonsense, frameshift, or splice-site mutations, leading to premature termination of the mRNA and complete absence of CFTR protein.
Class II - Defective protein processing — With class II defects, the mutations in the CFTR sequence prevent the protein from trafficking to the correct cellular location. The most common CFTR mutation, delta F508 (deletion of a single phenylalanine residue in the first NBF), belongs to this category.
Class III - Defective regulation — These mutations lead to diminished channel activity in response to ATP. Many involve alterations of the NBF regions, NBO1 and NBO2, which may retain varying degrees of sensitivity to nucleotide binding (show figure 2). Other CF mutations, mapped to the R domain, may also fall into this category.
Class IV - Defective conduction — With class IV mutations, the protein is produced and correctly localized to the cell surface. However, although chloride currents are generated in response to cAMP stimulation, the rate of ion flow and the duration of channel opening are reduced when compared to normal CFTR function.
The pathogenesis of the organ dysfunction seen in CF has been studied in humans and CFTR-knockout mice, but remains incompletely understood. It appears that the physical and chemical abnormalities of CF airway secretions result in chronic infection with phenotypically unique bacteria, particularly Pseudomonas species. Other genetic factors, including polymorphisms of the tumor necrosis alpha (TNF-a) gene, may increase susceptibility to Pseudomonas aeruginosa infection and contribute to the clinical manifestations of CF.
Primary abnormalities in fatty acid metabolism have been noted in biopsies of CFTR-expressing tissue from patients with CF. These changes, which result in increased tissue levels of arachidonic acid, are also present in the mouse model of CF, but are not seen in tissue from patients with inflammatory bowel disease. Thus, increased tissue expression of arachidonic acid and its metabolites may contribute to the abnormal inflammation characteristic of CF.
Abnormal secretions — CFTR malfunction leads to defective cAMP-dependent chloride secretion from the respiratory epithelium. In addition, sodium absorption from the airway lumen. is increased, possibly due to a failure of CFTR-mediated regulation of sodium channel activity An associated finding is an increased concentration of chloride in sweat secretions, which constitutes one of the methods of diagnosis of CF. The high chloride concentration of epithelial secretions may contribute to the increased risk of pulmonary infection, because bacterial killing via neutrophils and beta-defensins within the airways appears to require a normal chloride concentration. The net result of these changes is an alteration in the rheology of airway secretions, which become thick and difficult to clear.
Chronic infection — The chronic airway obstruction caused by viscous secretions is followed by progressive colonization with pathogenic bacteria, including Haemophilus influenzae, Staphylococcus aureus, and eventually Pseudomonas aeruginosa. P. aeruginosa and some other Gram negative bacteria often acquire a nonmotile mucoid phenotype, which is rare in other disease states. Once infection is established, neutrophils are unable to control the bacteria, even though there is massive infiltration of these inflammatory cells into the lung tissue. Recruited neutrophils subsequently release elastase, which overwhelms the antiproteases of the lung and contributes to tissue destruction. In addition, large amounts of DNA and cytosol matrix proteins are released by degranulating neutrophils, contributing to the increased viscosity of the airway mucus.
While some degree of inflammation has been noted prior to the development of bacterial colonization, chronic infection appears to be the major stimulus for an exuberant inflammatory response that ultimately results in bronchiectasis.
Pseudomonas aeruginosa — Chronic infection with P. aeruginosa is due in part to increased oxygen utilization by epithelial cells in patients with CF, which results in an abnormally decreased oxygen tension within the hyperviscous mucous layer. This local hypoxia may induce the characteristic phenotypic changes in P. aeruginosa, including alginate production and loss of motility, noted in patients with CF and chronic Pseudomonas infection. In addition, P. aeruginosa is able to multiply and form a reservoir of macrocolonies within the hypoxic regions of the mucous layer.
The organism may also acquire the ability to persist in the environment of the CF lung. One study found that 36 percent of CF patients were colonized with a hypermutable strain of P. aeruginosa that often persisted for years; such mutator strains were not isolated from patients with acute P. aeruginosa infection who did not have CF.
The frequent colonization and persistent infection caused by P. aeruginosa in CF patients is also related to the defective CFTR protein itself. CFTR-knockout mice have an impaired ability to control Pseudomonas lung infections. Normal CFTR protein serves as the receptor for binding of P. aeruginosa lipopolysaccharide (LPS) in vitro, and extracts LPS from the surface of the organism for endocytosis into epithelial cells.
This results in increased intranuclear translocation of the nuclear transcription factor NF kappa B and subsequent immunoactivation. This process does not occur in the presence of abnormal CFTR or in CFTR-knockout mice, which may partially explain the inability of CF patients to control these infections.
Respiratory tract involvement
Respiratory manifestations include a persistent, productive cough, hyperinflation of the lung fields on chest radiograph, and pulmonary function tests that are consistent with obstructive airway disease. As the disease progresses, chronic bronchitis with or without bronchiectasis develops and is accompanied by acute exacerbations characterized by increased cough, tachypnea, dyspnea, increased sputum production, and weight loss. Digital clubbing is often seen in patients with moderate to advanced disease. Colonization of the airway with pathogenic bacteria often occurs early in life. Staphylococcus aureus and Haemophilus influenzae are common pathogens during early childhood, but Pseudomonas aeruginosa is ultimately isolated from the respiratory secretions of most patients. This predisposition to infection may be in part because of impaired clearance directly induced by a defect in CFTR. In support of this hypothesis is the observation that CFTR-knockout mice have an impaired ability to control Pseudomonas lung infections, and that CFTR gene transfer to respiratory epithelial cells from patients with CF decreases binding of P. aeruginosa to the epithelium
The majority of CF patients develop sinus disease, and radiographs reveal panopacification of the paranasal sinuses in 90 to 100 percent of patients older than 8 months of age. Nasal polyposis is seen in 10 to 32 percent of patients. Some cases of chronic rhinosinusitis in the general population may in fact be due to mutations in the CFTR gene. One case-control study of 147 consecutive patients with chronic rhinosinusitis and 123 controls found a significantly higher proportion of CFTR mutations among patients with chronic rhinosinusitis (7 versus 2 percent) . Nine of 10 CFTR mutations detected in this study had the polymorphism M470V.
Failure to thrive is a presenting sign in many infants and children; less commonly, infants may present with hypoproteinemia and edema. These problems can often be reversed with oral supplementation of pancreatic enzyme extracts . In severe cases, the fat malabsorption can lead to deficiencies of the fat-soluble vitamins A, D, E, and K; however, routine vitamin supplementation has made clinical evidence of vitamin deficiency uncommon.
An increased prevalence of CFTR mutations has been observed in patients with idiopathic chronic pancreatitis, suggesting that certain CF genotypes (perhaps in combination with environmental or genetic cofactors) predispose to this complication in patients who lack chronic sinopulmonary disease, or abnormal sweat test or nasal potential difference measurements.
Pancreatic insufficiency is present from birth in most patients with CF. Insufficient secretion of enzymes leads to malabsorption of fat (with steatorrhea) and protein. In addition, primary abnormalities in fatty acid metabolism have been noted in biopsies of CFTR-expressing tissue from patients with CF. These changes, which result in increased tissue levels of arachidonic acid, are also present in the mouse model of CF, but are not seen in tissue from patients with inflammatory bowel disease.
Glucose metabolism in patients with CF and pancreatic disease is variable. Those with no pancreatic exocrine deficiency have normal insulin secretion and responsiveness. In comparison, patients with exocrine deficiency have decreased insulin secretion, but normal glucose tolerance resulting from the unusual combination of increased hepatic glucose production and increased peripheral glucose utilization. How the latter adaptation occurs is unclear, but it is initially able to counteract the effects of insulin deficiency. Patients with exocrine deficiency and either impaired glucose tolerance or overt diabetes have reductions in both peripheral glucose utilization and hepatic insulin sensitivity.
Meconium ileus and distal ileal obstruction
Meconium ileus is the presenting problem in 10 to 20 percent of newborns with CF, and is virtually pathognomonic of the disease. No specific CFTR mutations predispose to the development of meconium ileus. A familial recurrence rate of nearly 30 percent suggests that other genetic factors are involved, and a cystic fibrosis modifier locus for the development of meconium ileus has been mapped to chromosome 19. Episodes of small bowel obstruction may also occur in older children and adults, and have been variously termed "meconium ileus equivalent" or "distal ileal obstructive syndrome" (DIOS). DIOS occurs in up to 15 percent of adult patients with CF, and is more common in patients with severe CFTR genotype and advanced lung disease. Pancreatic insufficiency is present in many, but not all, affected patients When identified early, DIOS can usually be controlled medically. Surgical intervention is sometimes required to alleviate severe obstruction, and may lead to recurrent problems from adhesions.
Focal biliary cirrhosis caused by inspissated bile produces symptomatic portal hypertension in 2 to 5 percent of patients with CF. Asymptomatic liver disease is a common finding at autopsy; antemortem elevations of serum alkaline phosphatase and lobular hepatomegaly may be found incidentally.
Cholelithiasis has been reported in up to 12 percent of patients, and may result from excessive loss of bile acids in the stool with consequent production of lithogenic bile . Asymptomatic cholelithiasis generally does not require treatment, although prophylactic cholecystectomy is performed in such patients prior to lung transplantation in some centers.
Several observational studies have suggested that administration of ursodeoxycholic acid may arrest the progression of liver disease related to CF, but an unequivocal effect of this therapy has not been confirmed in randomized trials.
Despite adequate spermatogenesis, defects in sperm transport render more than 95 percent of men with CF infertile . Incompletely developed Wolffian structures, most commonly absent vas deferens, are almost universally found in these men. These anomalies probably reflect a critical role for CFTR in the organogenesis of these structures. Further evidence is based on the observation that nearly half of all men with congenital bilateral absence of the vas deferens and normal lung function have two CFTR mutations. Microsurgical epididymal sperm aspiration and intracytoplasmic sperm injection can permit affected men to become biological fathers. The incidence of female infertility may be as high as 20 percent. It is related to secondary amenorrhea (induced primarily by malnutrition) and the production of abnormally tenacious cervical mucus. When patients with CF become pregnant, maternal and fetal outcomes are generally favorable if the prepregnancy FEV1 exceeds 50 to 60 percent of the predicted value. Careful genetic counseling for prospective CF parents is essential, since all offspring of such individuals will be carriers of CF mutations, and the risk of children affected with CF is high.
Mineralization of bone appears to be lower in patients with CF, averaging 20 percent less than age- and gender-matched controls. Reduced rates of bone accretion and accelerated rates of bone loss explain the high prevalence of low bone mineral density in adults with cystic fibrosis. Clinically significant reductions in bone density are present in up to 30 percent of patients with CF. These changes may result in a higher risk of fracture. However, in one questionnaire study, the risk of fracture, adjusted for age and sex, was similar among patients (aged 6 to 25 years) with CF, pancreatic insufficiency, and mild to moderate lung disease and control subjects. Limited evidence suggests that patients homozygous for the delta F508 mutation are at independently increased risk of decreased bone mineral density. The possible contribution of this mutation in the pathogenesis of bone loss remains speculative.
Calcium supplementation alone appears to be insufficient to prevent significant bone loss, and the ability of bisphosphonate therapy to increase bone density in patients with CF has been assessed in small trials. This promising therapy remains under investigation; possible limitations include bone pain during intravenous infusion and poor oral absorption of standard doses of these agents.
Hypertrophic osteoarthropathy is a syndrome characterized by abnormal proliferation of the skin and osseous tissue at the distal parts of the extremities, occurring in association with radiographically confirmed periosteal new bone formation. Clubbing and hypertrophic osteoarthropathy appear to be different manifestations of the same disease process (show figure 1). Although clubbed fingers and toes are common in patients with long-standing CF, hypertrophic osteoarthropathy is much less frequently observed (5 percent of patients).
A CF-associated arthropathy, which occurs in 2 to 9 percent of patients, is characterized by relatively short episodes of pain and swelling of joints; these features are occasionally accompanied by painful nodular skin lesions and purpura.
CF appears to be a risk factor for recurrent venous thrombosis. In a review of 120 children and young adults with acute venous thromboembolism, recurrent thrombosis occurred in 19. Among these, six had CF (compared to none of the patients without recurrence) and five of the six were colonized with Burkholderia cepacia. Most, but not all, of the recurrent thrombosis events occurred in the presence of a central venous catheter.
More than 1000 new cases of cystic fibrosis were diagnosed in 200. More than 40 percent of these patients initially presented with respiratory symptoms; other common early manifestations included failure to thrive (29 percent), steatorrhea (24 percent), and meconium ileus (19 percent). Less than 10 percent of cases were diagnosed on the basis of newborn screening programs; this statistic may be expected to change as more states institute screening programs. The indications for genetic screening and prenatal testing in the diagnosis of CF are discussed elsewhere.
The diagnosis of CF is based upon compatible clinical findings with biochemical or genetic confirmation. The sweat chloride test is the mainstay of laboratory confirmation, although tests for specific mutations, nasal potential difference, immunoreactive trypsin, stool fecal fat, or pancreatic enzyme secretion may also be useful in some cases.
Although CF is generally diagnosed in infants and children who present with recurrent pulmonary infections, patients with unrecognized disease may present later in life with atypical symptoms. One large retrospective cohort study of more than 1000 patients with CF found that 7 percent were diagnosed at age 18 years. Patients diagnosed in adulthood were more likely than children to present with gastrointestinal symptoms, diabetes mellitus, and infertility. In addition, adults presenting with CF were more likely than children to have unusual genetic mutations, normal pancreatic function, and equivocal results on sweat chloride tests.
The sweat chloride test remains the gold standard for the diagnosis of CF, since less than 90 percent of CF mutations can be identified by typical screening for 20 to 30 of the most prevalent mutations. The test is performed by the collection of sweat with pilocarpine iontophoresis and by chemical determination of the chloride concentration.
A sweat chloride value greater than 60 meq/L distinguishes most patients with CF from those with other forms of chronic pulmonary disease. In 252 CF patients tested by Shwachman, sweat sodium and chloride levels were uniformly elevated when compared to normal controls . However, a normal sweat chloride concentration may be observed in approximately 1 percent of patients with CF, who have unusual genotypes such as the 3849 + 10kb C-T or poly-T defects.
The sweat chloride test must be carried out with extreme care, and should be obtained by a laboratory that performs the assay regularly. Patients are frequently referred to CF centers with a misdiagnosis of CF, based upon factitious elevations in the sweat chloride level. At least 50 mg of sweat must be collected within a 45-minute period for the test to be valid, and collecting sufficient sweat is frequently problematic in young infants. Another problem is that hypoproteinemic edema and concurrent administration of steroids can lead to a false decrease in the sweat chloride concentration. A variety of other clinical conditions may be associated with elevated sweat chloride levels, but none of them are readily confused with CF.
Molecular diagnosis is usually carried out by direct mutation analysis. This may be accomplished by a variety of techniques for identification of specific known mutations in the nucleotide sequence of the CFTR gene. These techniques include: Allele–specific oligonucleotide hybridization Allele–specific amplification Ligase amplification Direct sequencing Restriction enzyme analysis.
The Cystic Fibrosis Mutation Data Base lists more than 1250 different mutations in the CFTR gene. Most diagnostic laboratories in the United States screen for 20 to 30 of the most common mutations, which identifies approximately 90 percent of CF chromosomes (and 97 percent of chromosomes in families of Ashkenazic Jewish ancestry). The remaining 10 percent of affected chromosomes are comprised of more than 1000 different pathologic mutations of CFTR, making more extensive screening impractical. Rare mutations can be identified, but the size of the CFTR gene makes this a time-consuming and expensive task.
Linkage analysis can be performed for prenatal diagnosis or carrier detection in CF families carrying unidentified mutations . A list of testing laboratories is available through the GeneTests website (www.genetests.org). Additional information can be obtained from the Cystic Fibrosis Foundation (www.cff.org).
Nasal potential difference measurements
Abnormalities in epithelial chloride secretion can be demonstrated in most CF patients by evaluating the nasal transepithelial potential difference in the basal state, after nasal perfusion with amiloride, and after nasal perfusion with a chloride-free solution. This test should be performed in experienced centers because standardization of both solution preparation and potential difference measurement is necessary. The presence of nasal polyps or inflammation may result in false negative results.
The degree of abnormality in nasal transepithelial potential difference is not correlated with the severity of CF lung disease. However, specific abnormalities in nasal transepithelial potential difference are associated with distinct phenotypes of CF. In general, abnormal chloride secretion is associated with pancreatic insufficiency, while sodium hyperabsorption is more common among patients with severe of lung disease.
Newborn screening — Most infants with CF have elevated blood levels of immunoreactive trypsin (IRT), which can be quantified by radioimmunoassay or by an enzyme–linked immunoassay. The test can be performed on the dried blood sample obtained for newborn screening purposes, and allows detection of at least 95 percent of newborns with CF. IRT levels fall rapidly during infancy, and a negative result is not informative after 8 weeks of age. In addition, the rates of false positive and false negative results are relatively high in many series. The test is primarily used for neonatal screening, but also may be useful for small or malnourished infants, in whom the sweat chloride test cannot be successfully performed.
The rationale for newborn screening is that early detection of CF may lead to earlier intervention and improved outcomes, as demonstrated by the following: A meta-analysis of randomized trials examining newborn screening in Europe and Australia demonstrated modest improvements in survival by 10 years of age. In this study, newborn screening reduced mortality by 5 to 10 per 100 children with CF without meconium ileus. Several observational studies have shown that children with CF detected via screening demonstrate better lung function and improved growth parameters with less intensive treatment than children diagnosed with CF on clinical grounds, although these findings have not yet been confirmed in randomized trials. Early nutritional intervention appears to improve neurocognitive outcome in infants and young children with CF. Neonatal identification of CF patients allows early referral to centers specializing in the management of CF, and identifies a population for studying the mechanism of early (preclinical) lung injury. In the future, the effectiveness of novel therapies to delay or prevent the onset of lung disease could be assessed in these patients with preclinical disease.
In the United States, the Centers for Disease Control and Prevention (CDC) concluded in 2004 that CF screening programs were justified on the basis of moderate benefit and low risk of harm, but noted that the decision to implement these programs was dependent upon the resources and priorities of individual states . Statewide testing programs are in place in 26 states in the US, and regional or elective programs are available in several other areas. An updated list of state screening programs is available on the cystic fibrosis foundation website: http://www.cff.org/legislative_action/state_issues/StatesNewbornScreening/.
One to 2 percent of patients with clinical features compatible with CF are found to have normal sweat chloride levels on repeated testing. For these individuals, a variety of other techniques may help clarify the diagnosis if genetic screening is not conclusive. As an example, the presence of azoospermia in sexually mature males may provide supportive evidence for a CF diagnosis.
Pancreatic secretion may be evaluated by collecting duodenal fluid after stimulation with secretin and cholecystokinin. Decreased levels of pancreatic enzymes can be indicative of CF, but the technique is time–consuming, technically demanding, and uncomfortable for the patient. Similar information can be gathered by calculating the percentage of ingested fat in a 72-hour stool collection; the value is elevated in individuals with pancreatic insufficiency.