Genetics

Cystic fibrosis is caused by mutations in a gene on chromosome 7 encoding the protein subsequently termed the CFTR gene. More than 1800 mutations have been reported to the Cystic Fibrosis Genetic Analysis Consortium. Most of these mutations are rare, and only four mutations occur in a frequency of more than 1%. CFTR mutations can be grouped into five classes: CFTR is not synthesized (I), is inadequately processed (II), is not regulated (III), shows abnormal conductance (IV), or has partially defective production or processing (V). Class I, II, and III mutations are more common and associated with pancreatic insufficiency, whereas patients with the less common class IV and V mutations often are “pancreatic sufficient” (Figure 44-1).

Figure 44-1 Major classes (I-V) and molecular consequences of cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations.

The most common mutation worldwide, found in approximately 66% of patients with CF, is a class II mutation caused by a deletion of phenylalanine in position 508 (F508del) of CFTR. F508del CFTR is misfolded and trapped in the endoplasmic reticulum (ER) and subsequently proteolytically degraded. However, small amounts of F508del CFTR reach the plasma membrane of epithelial cells and have some functional activity. These findings suggest that F508del CFTR rescue from ER degradation may be a potential therapeutic intervention.

The CFTR gene belongs to a family of transmembrane proteins called adenosine triphosphate (ATP)–binding cassette (ABC) transporters and functions as a chloride (Cl⁻) channel in apical membranes. However, CFTR possesses other functions in addition to being a chloride channel. CFTR has been described as a regulator of other membrane channels, including the epithelial sodium channel (eNaC) and the outwardly rectifying chloride channel (ORCC). CFTR also transports or regulates bicarbonate (HCO₃⁻) transport through epithelial cell membranes and may act as a transporter for other proteins, such as glutathione.

A relationship exists between CFTR genotype and clinical phenotype in CF. Patients who carry two “severe” mutations (classes I, II, and III) that cause loss of function in CFTR have classic CF, characterized by pancreatic insufficiency, early age of diagnosis, and elevated sweat chloride. In contrast, patients who have at least one “mild” mutation with partial function in CFTR are typically diagnosed at an older age, have sweat chloride values closer to normal, and are pancreatic sufficient.

Whereas classes IV and V CFTR mutations are linked with pancreatic sufficiency, attempts to link specific mutations to the severity of lung disease have shown large phenotypic variability. This is best documented for patients homozygous for the F508del mutation who exhibit a wide spectrum in lung disease severity. This wide phenotypic variation suggests that environmental factors and genes other than CFTR influence the development, progression, and disease severity of CF (Figure 44-2).

Figure 44-2 Spectrum of lung disease with chest radiograph, FEV₁, and age. FEV₁, forced expiratory volume in 1 second. pred, of predicted value.

Pathophysiology

Although there is ongoing debate on how CFTR mutations cause disease, some of the fundamental questions have been clarified in recent years. CFTR is expressed in higher quantities in tissues clinically affected by CF, such as sinuses, lungs, pancreas, liver, gastrointestinal (GI) tract, and reproductive tract, although low levels also occur elsewhere. Because lung disease is the most pertinent clinical feature of CF, the focus here is on its pathophysiology in the respiratory tract.

Airway epithelial cells secrete chloride and absorb sodium chloride (NaCl), the balance of which is regulated through apical channels, including CFTR (Figure 44-3). Ion secretion and absorption affect water transport, and a balance between secretion and absorption is thought to be important to maintain an adequate layer of airway surface liquid (ASL). The ASL supports the thin mucous layer on top of epithelial cells, which is constantly transported out of the lungs through ciliary movement. Lack or dysfunction of CFTR leads to reduced chloride secretion and NaCl hyperabsorption with depletion of ASL. In the absence of adequate ASL, respiratory cilia collapse, leading to breakdown of mucociliary transport. Mucus accumulates in the lower airways, and inhaled bacteria are trapped in this viscous mucous layer on top of respiratory epithelial cells.

Figure 44-3 Restoring airway surface liquid in cystic fibrosis.

(From Ratjen F: Restoring airway surface liquid in cystic fibrosis, N Engl J Med 354:291–293, 2006.)

The spectrum of bacteria that are relevant for CF lung disease is relatively limited. Overall, Pseudomonas aeruginosa is the most common isolate, followed by Staphylococcus aureus and Haemophilus influenzae. Later in the course of disease, multiresistant organisms such as Stenotrophomonas maltophilia, Achromobacter (Alcaligenes) xylosoxidans, and Burkholderia cepacia complex may be isolated. As in other chronic pulmonary diseases, nontuberculous mycobacteria (usually Mycobacterium avium-intracellulare or M. abscessus) may be isolated. It is challenging to prove whether these organisms are causing ongoing disease requiring treatment, or if they are colonizing only the damaged lung. For a more detailed discussion of nontuberculous mycobacteria (NTM) infections, see Chapter 31.

Stenotrophomonas maltophilia, Alcaligenes xylosoxidans, and Burkholderia cepacia complex are isolated in less than 10% of patients with CF. B. cepacia complex is an unusual organism that is found in the environment (soil and water) and causes chronic infection in only CF and chronic granulomatous disease. It is inherently multiresistant and difficult to treat and in CF is associated with a significantly worse prognosis. There is evidence for person-to-person spread in patients with CF. Approximately 15% to 20% of patients with CF who are infected with B. cepacia complex will have rapidly progressive deterioration, so-called cepacia syndrome, with necrotizing pneumonia, greatly elevated white blood cell (WBC) counts, bacteremia, and almost 100% mortality.

The mucus in CF lacks oxygen, leading to anaerobic growth conditions for bacteria. Anaerobic bacteria exist in high numbers in CF airways, but their clinical significance is uncertain. The anaerobic growth conditions trigger a switch of S. aureus and P. aeruginosa from nonmucoid to mucoid cell types, the predominant phenotype in CF lungs. These mucoid strains form biofilms in CF airways that are resistant to killing by the host defense system, resulting in chronic infection. Inflammatory products (e.g., elastase) released by neutrophils stimulate mucus secretion, perpetuating the cycle of mucus retention, infection, and inflammation.

Evidence indicates that inflammation is dysregulated in CF airways. Neutrophilic airway inflammation has been detected in infants with CF in the first months of life, as well as in CF fetal lung tissue. Whether or not inflammation is directly related to the CFTR defect is still disputed. However, an exaggerated, sustained, and prolonged inflammatory response to bacterial and viral pathogens is an accepted feature of CF lung disease. The persistent endobronchial inflammation is deleterious for the course of lung disease (Figure 44-4).

Figure 44-4 Sequence of cystic fibrosis pathophysiology.

Exocrine pancreatic insufficiency is present in approximately 85% to 90% of patients with CF, generally in those patients who carry two copies of the class I, II, or III CFTR mutations. The exocrine pancreas has great functional reserve, and 98% to 99% of its function must be lost before malabsorption will occur. Patients who are pancreatic sufficient do not have normal pancreatic exocrine function but have sufficient function to prevent fat malabsorption. Pancreatic disease begins in utero and is thought to result from decreased volume of pancreatic secretions with decreased concentrations of HCO₃⁻. Without sufficient fluid and HCO₃⁻, digestive proenzymes are retained within small pancreatic ducts and are prematurely activated, ultimately leading to tissue destruction, fibrosis, and fatty replacement. The resulting malabsorption contributes to the failure to meet the increased energy demands because of the hypermetabolic state associated with endobronchial infection. Lung infections may lead to anorexia and vomiting, promoting malnutrition. These factors may exacerbate lung infection, leading to a vicious cycle of malnutrition and infection.

Clinical Features

Typical signs and symptoms for CF are listed in Box 44-1. Symptoms of CF may vary, with monosymptomatic cases often diagnosed late. It is therefore important to be aware of the spectrum of symptoms that may arise and to initiate adequate diagnostic steps.

Although the classic presentation of CF is the combination of chronic productive cough, steatorrhea, and failure to thrive, 10% to 15% of patients do not have pancreatic insufficiency clinically. Because the lungs of patients with CF are normal at birth, pulmonary symptoms may not be obvious. Between 10% and 15% of newborn infants with CF may fail to pass meconium, leading to meconium ileus, which is linked to pancreatic insufficiency but not directly associated with more severe clinical disease.

Less common presentations of CF, such as prolonged jaundice in the newborn and rectal prolapse in infants and young children, should trigger diagnostic tests. Occasionally, an infant may have severe malnutrition with anemia, hypoalbuminemia, and edema in the first 4 months of life.

Infertility can be a presenting symptom in adult patients with CF with limited pulmonary symptoms; 98% of males with CF are infertile, with azoospermia secondary to atretic or absent vas deferens. Spermatogenesis and sexual potency are normal. Female reproductive function is normal, although a lower rate of fertility has been postulated because of dehydrated cervical mucus.

Initially, pulmonary symptoms of cough will occur only at times of exacerbations, but eventually there is progression to a chronic daily cough productive of sputum. The sputum is initially white, but as infection continues, the mucus becomes thicker and purulent. Minor hemoptysis often occurs at exacerbation. Some patients have an “asthmatic” component to their disease, with wheezing, chest tightness, paroxysmal dry cough, and a degree of reversibility in airflow obstruction with bronchodilators. Over time, as pulmonary function declines, there is increasing dyspnea. Hypoxemia is not usually seen until forced expiratory volume in 1 second (FEV₁) is less than 35% of predicted value, and hypercarbia usually occurs when the FEV₁ is less than 25% or 30% predicted. Cor pulmonale occurs late in the illness.

Pansinusitis is found on sinus radiographs in most patients, although not all will have symptoms of recurrent sinusitis, headache, and postnasal drip. Nasal polyps are seen in approximately 20% of patients and tend to recur even after surgical removal.

Diagnostic Approach

The diagnosis of cystic fibrosis is established by clinical manifestations (see Box 44-1), a history of CF in a sibling, or a positive newborn screening result, in conjunction with laboratory evidence of CFTR dysfunction. CFTR dysfunction is documented by elevated sweat chloride or characteristic abnormalities in nasal potential difference or by CF-causing mutations in the CFTR gene.

A diagnostic algorithm is presented in Figure 44-5. Abnormal ion transport is reflected in high sweat NaCl levels, and measurement of chloride concentration in sweat after iontophoresis of pilocarpine is used for diagnosis. Sweat testing must be done using standardized methods, by qualified staff in an experienced laboratory. A sweat chloride concentration greater than 60 mmol/L on repeated analysis is diagnostic for CF; 30 to 60 mmol/L is considered a “borderline” result but may be seen in patients with CF.

Figure 44-5 Diagnostic algorithm for cystic fibrosis (CF). PD, potential difference.

Diagnosis can be confirmed by genotyping of the most common CFTR mutations, which vary by ethnic origin of the population tested. More than 1800 mutations in CFTR have been reported to the CFTR database. Most commercial screening panels test for less than 50 mutations and will identify 85% to 90% of CF alleles. Although CFTR mutation testing has had no clinical implications in the past, this may change with the introduction of mutation-specific therapy (see later discussion).

The diagnosis of CF requires the presence of two CF-causing mutations. To be considered CF-causing, the mutation must (1) cause a change in the amino acid sequence that severely affects CFTR synthesis or function, (2) introduce a premature termination signal (insertion, deletion, or nonsense mutations), (3) alter the “invariant” nucleotides of intron splice sites (first or last two nucleotides), or (4) cause a novel amino acid sequence that does not occur in the normal CFTR genes from at least 100 carriers of CF mutations from the patient’s ethnic group. Of the more than 1800 CFTR mutations reported, only approximately 25 are considered disease-causing to date.

If CFTR genotyping or sweat test is not diagnostic, a second test of CFTR function such as nasal potential difference (NPD) measurement can be performed. The transport of Na⁺ and Cl⁻ ions across the nasal mucosa creates a transepithelial electrical potential difference. Changes in NPD in response to stimulation or inhibition of ion channels by nasal perfusion can be measured, and a typical normal or CF response exists. The NPD test is technically difficult, requiring a skilled operator, and therefore is not available in all CF centers. Standard operating procedures and reference values have recently been determined. Other techniques to examine CFTR function include analysis of rectal mucosal biopsies in an Ussing chamber.

Clinical tests not directly assessing the CFTR defect can also aid in the diagnostic process. Most patients with CF are pancreatic insufficient, and a decreased concentration of chymotrypsin or pancreas-specific elastase in feces or 72-hour stool collection with fecal fat analysis can confirm this. Most patients with CF have total opacification of paranasal sinuses, and sinus radiography may be helpful. Bacterial pathogens typical for CF (e.g., mucoid Pseudomonas, S. aureus) can be detected in sputum or throat swabs and suggest a CF diagnosis. Obstructive azoospermia is found in 98% of men with CF and is a result of congenital bilateral absence of the vas deferens (CBAVD). The finding of azoospermia, or lack of vas deferens on careful urologic examination or transrectal ultrasound, suggests CF.

In the past, about 50% of patients with CF in North America were diagnosed by age 6 months and 90% by 8 years. Neonatal screening has been proposed as early diagnosis, and therefore earlier initiation of treatment may improve outcome. A randomized screening program in Wisconsin state found that weight gain and early growth were better in patients diagnosed by neonatal screening. Because good nutrition is linked to a better prognosis, these data would favor the introduction of population-wide neonatal screening. Neonatal screening programs have now been introduced in many countries, most often based on a two-step approach, with immunoreactive trypsinogen (IRT) in dried blood spots and confirmation by DNA analysis in positive cases. Blood trypsinogen is elevated in pancreatic-insufficient patients in the first weeks of life, but the rate of false-positive results of a single IRT test is high, which can be reduced substantially by inclusions of a second step, including genetic testing for the most common CFTR mutations.