July 31, 2008

Cystic Fibrosis: Newborn Screening in America

By Kleven, Daniel T McCudden, Christopher R; Willis, Monte S

Cystic fibrosis (CF) is the most common lethal genetic disease in Caucasians, occurring in 1/2,500 births.12 In its most common form, CF manifests as progressive lung dysfunction, pancreatic insufficiency, and intestinal disease (see Figure 1). The gene which harbors mutations responsible for disease was identified in 1989, after which the protein it encodes was determined to function as a chioride channel that indirectly controls sodium transport. Since then, genetic testing has expanded our appreciation of the spectrum of disease that CF represents. Our continued desire to improve outcomes in the quality and quantity of life of CF patients has led to the recent implementation of newborn screening for CF in the United States. In this review, we will discuss exciting new developments in newborn screening for CF in the context of our current standards for diagnosis, therapy, and improved outcomes. The CFTR gene

The pathophysiology of CF results from mutations in the cystic- fibrosis transmembrane regulator (CFTR) gene. The CFTR gene encodes a protein that regulates chloride transport. As a consequence of chloride transport, the CFTR protein regulates multiple ion channels and cellular processes, most notably the epithelial sodium (Na+) channel (also known as ENaC). In general, when mutations in CFTR result in a non-functional protein, ENaC activity increases, and sodium transport across the membrane is augmented. In the lungs and intestine, this results in the accelerated uptake of water from the lumen, leaving dehydrated mucous layers (see Figure 2), Conversely, in the sweat gland, defective chloride transport impairs sodium uptake in the sweat duct, resulting in elevated NaCl levels in sweat (see Figure 2). Accordingly, sweat chloride has allowed effective non-invasive diagnosis for decades.

The CFTR gene spans 250,000 bases encoding 1,480 amino acids (see Figure 3). The CKlK protein has multiple membranespanning regions, two nucleotide-binding domains (NBD), and an "R domain" which contains sites to which phosphate groups can be attached. The severity of disease in cystic fibrosis varies greatly, generally based on the specific types of CFTR mutations that are present.3 Greater than 1,250 mutations have been identified to date ( www. gene t.sickkids. on.ca/cftr). These mutations have been divided into five different classes, based on the fundamental defects that they cause in the CFTR protein (see Figure 4). Class I and II mutations (see Table 1 ) result in no CFTR protein at the cell surface and are present in patients with more severe disease.3 In contrast, Class III, IV, and V mutations (see Table 1 ) have diminished activity and can result in milder disease (see Figure 4).

The most common CF mutation is a deletion of a phenylalanine at position 508 (DeltaF508), which resides in the first nucleotide- binding domain (see Figure 3). Patients with two of these mutations suffer from classic CF symptoms: bronchiectasis, pancreatic insufficiency, male infertility, and hepatic cirrhosis (see Figure 3). Since CF is an autosomal recessive disorder, disease phenotypes are only observed in individuals with two inherited mutations in the CFTR gene. It is the effect of these two CFTR mutations on the function of the CFTR protein that, ultimately, determines the clinical phenotype seen in patients. CF, however, is a complex disorder and other factors such as modifying genes, environment, and treatment affect disease progression and severity.

Despite the large number of CFTR mutations that have been identified, a small number of patients have clinical evidence of CF, including a positive sweat-chloride test, but no identifiable CFTR gene defect. For example, in one study of non-classical (mild) CF, 40% of patients did not have any detectable mutations, despite exhaustive analysis.4,5 It is not exactly clear what the underlying defect is in these cases; however, factors other than CFTR mutations appear to lead to phenotypes indistinguishable from CF in some patients. It is clear, however, that even with exhaustive searches CF mutations may miss identifying the underlying cause (discussed in diagnostic genotyping of CF section).

Cystic-fibrosis pathophysiology

Cystic fibrosis affects epithelial cells in organs where the CFTR protein is found, including lungs, pancreas, intestine, vas deferens, liver, and sweat glands. It is the distribution of the CFTR in these organs that explains much of the multiorgan nature of CF. Defects in CFTR function within these organs results in lung disease, pancreatic insufficiency, multifocal biliary cirrhosis, male infertility, and increased sweat ion loss.

Lungs. The most serious complicalions of CF occur in the lungs, due to abnormal epithelial-cell transport of Cl-, resulting in altered surface fluid6 (see Figure 2). The airway surface fluid is decreased due to an increased uptake of sodium (and water), resulting in a dehydrated mucous. These changes impede the necessary ciliary clearance of microorganisms and debris in the lungs, promoting obstruction of the airways and infections. These recurrent infections lead to airway impairment and can cause permanent damage to the lungs.6 CF patients become infected with specific bacteria, such as Staphylococcus aureus or Haemophilus influenza, early in life. As disease progresses, Pseudomonas aeruginosa and Burkholderia spp. may infiltrate the lung.7,8 Despite current therapies, lung disease in CF patients still worsens over time; milder forms of CF are associated with a later onset of lung disease, which progresses as a slower place.9 Lung involvement is responsible for greater than 90% of the mortality in CF patients.

Pancreas. The exocrine function of the pancreas is responsible for the secretion of enzymes essential for the breakdown of food. In the pancreas, defective CFTR protein causes reduced HCO1- secretion (see Figure 2), leading to congestion of acini and inappropriate activation of pancreatic proteases. This process effectively impairs secretion of the pancreatic enzymes necessary for digestion. Approximately 85% of patients with CF have exocrine pancreas insufficiency, which manifests as poor nutrition and increased fat in stool. This results in weight loss, abdominal pain, and flatulence.10 Replacement of pancreatic enzymes and careful diet planning can overcome many of these problems.

Liver disease. While pulmonary and pancreatic disease occurs in 90% of CF patients, liver manifestations occur in no more than one- third of patients." In the hepatic biliary system, CFTR is expressed in cholangiocytes and gall-bladder epithelial cells but not hepatocytes.12 The main role of CFTR within these cells is to regulate the fluid and electrolyte content of bile; its absence or dysfunction results in impaired secretory function, secondary to increased bile viscosity and bile-duct occlusion." This stasis results in damage to the hepatocytes and increases pro-inflammatory cytokines, growth factors, and lipid peroxidation. These derangements prompt liver stellate cells to synthesize collagen, leading to fibrosis." Liver disease is the most common non- pulmonary cause of death resulting in approximately 2.5% of all CF mortality.13

Infertility. Most males with CF are infertile as a result of azoospermia (complete lack of sperm) secondary to the congenital bilateral absence of the vas deferens (CBAVD).14 In patients with mild disease, infertility may be the first indication that they may have CF. Due to advances in reproductive medicine, spermatozoa can be retrieved in order to overcome the infertility.14 While 1% to 2% of CBAVD occurs in infertile males without CF, as many as 80% of men with CBAVD have CFTR gene mutations.15

Other organ systems. There are a number of associated morbidities in patients with CF (see Figure 1). These manifestations affect the intestine and upper airway, and include sinusitis, nasal polyps, distal ileum obstruction, and meconium ileus. Up to one-fifth of newborns with CF present with meconium ileus, the retention of the meconium after birth. The identification of meconium ileus is nearly pathognomonic of CF, since it occurs so infrequently in patients without CF. Small-bowel obstruction can also occur in older children and adults in patients with severe disease, sometimes requiring surgical intervention to alleviate the obstruction.16 Most CF patients develop sinus disease (90% to 100%), while 10% to 32% develop abnormal lesions of the nasal mucosa (nasal polyps).17,18 In undiagnosed patients with mild CF, recurring sinus inflammation and/ or nasal polyps may prompt the screening for CF.

Clinical diagnosis of cystic fibrosis

The diagnosis of cystic fibrosis is made on the basis of two criteria: 1 ) one or more phenotypic features and 2) laboratory evidence of CFTR dysfunction (see Table 2).'9 In the absence of typical phenotypic features, positive newborn screen or history of a sibling can fulfill the first criteria. The second criteria can be met by either two positive sweat-chloride tests or the presence of two disease-causing alleles. These place the focus of laboratory testing on the genotypic analysis of mutations and sweat-chloride testing, the latter being the gold standard for diagnosis.

Diagnostic genotype analysis of CF. Direct analysis of CFTR gene mutations is performed by a variety of techniques, including allele- specific oligonucleotide hybridization, allelespecific amplification, ligase amplification, direct sequencing, and restriction enzyme analysis. Most laboratories in the United States screen for 20 to 30 of the most common mutations, identifying 80% to 90% of CF patients.20'22 Over 1,000 mutations then account for the remaining 10% to 20% of patients, making comprehensive testing impractical for everyone. Expanded testing can be performed to cover mutations more common in particular ethnic groups, but the size of the gene makes extensive genetic screening a time-consuming and expensive endeavor. Fortunately, sweat-chloride testing can identify up to 99% of patients with phenotypic CF. Sweat-chloride testing. The gold standard for CF diagnosis is sweat-chloride testing. Sweat- chloride testing will detect -99% of patients, is relatively inexpensive, and has a high degree of accuracy. Sweat testing is performed by stimulating sweat production by pilocarpine iontophoresis; sweat is collected on preweighed gauze pads, and coulometric titration is used to measure the chloride concentration. A sweat-chloride value >60 mEq/L distinguishes most patients with cystic fibrosis; however, normal sweat-chloride concentrations are observed in approximately 1% of patients, generally with specific uncommon genotypes.23 Sweat-chloride testing should be performed by laboratories that perform this test on an ongoing basis. The Cystic Fibrosis Foundation has published guidelines for diagnostic sweat testing, which are mandated for accreditation in CF centers.24 It is important to recognize that there are clinical conditions that falsely elevate sweat-chloride levels (see Table 3), in order not to misdiagnose cystic fibrosis. This includes transient elevations that occur during the first 24 hours after birth (in up to 25% of normal newborns). Since this transient elevation rapidly declines the second day after birth, sweat testing should not be performed on children less than 48 hours old. Sweat-test collection can be performed by The Gibson-Cooke sweat-test apparatus (C&S Electronics, Columbus, NE) or commercial Wescor systems (Macroduct/Nanoduct, Wescor Inc., Logan, UT).

Nasal potential difference (NPD). Impaired ion transport in respiratory epithelia can be determined by measuring the potential difference in nasal mucosa. NPD testing is considerably more complex than detecting sweat-chloride concentrations and is performed only in specialized centers.25 CF patients have reduced chloride transport and increased absorption of sodium, which results in a more negative potential difference at baseline.26 When the sodium channel-blocker amiloride is perfused with isoproterenol to stimulate CFTR function, no change in NPD occurs in patients with mutated or deficient CFTR channels. NPD may complement sweat testing, although it is technically difficult and is used mainly on adults. It cannot be used when nasal inflammation is present, including allergic rhinitis or infection, which can alter ion transport.26

Ancillary tests. Additional tests can be performed to assess organ involvement of CF patients. CT scanning or X-rays can be used to evaluate the paranasal sinuses; pancreatic-exocrine function can be assessed using fecal-fat analysis; sputum or broncho-alveolar lavage can be tested for the presence of bacteria: and semen analysis can be performed to determine if vas-deferens impairment is present.

Screening for CF

Pre-natal genetic screening. The goal of screening for CF mutations either before pregnancy or prenatally is to identify couples who are at risk for having a child with CF.27 Prenatal diagnosis is performed in order for prospective parents to have the best medical data for making medical and personal decisions. CF screening should be available to all couples planning a pregnancy who are at increased risk, particularly if of Caucasian, European, or Ashkenazi Jewish ancestry27; ideally, testing should be completed pre-pregnancy. Since the fetus must inherit an affected CF gene from each parent, both parents' risks should be considered. Currently, the American College of Obstetricians and Gynecologists (ACOG) recommends offering CF screening to 1) individuals with a family history of CF; 2) reproductive partners of individuals with CF; and 3) couples in which one or both are Caucasian and/ or European or Ashkenazi Jewish descent who are planning a pregnancy or seeking prenatal care.27 Either couple-based or sequential testing may be done, whereby the woman is tested first, and her partner is tested only if she is a carrier. Screening for a specific panel of the 25 most common CF mutations should be performed, and reflex tests performed as indicated.27 Consultation is recommended if either partner has 1) CPor a positive screening test for CF; 2) a family history of CF; 3) an affected fetus (female); or 4) infertility due to vas-deferens absence or atresia (male).27

Maternal screening. Currently, ACOG recommends that DNA testing for CFTR mutations be offered to all couples seeking prenatal or preconception care.28,29 This recommendation is extended beyond couples that belong to high-risk ethnic groups or individuals with a family history of CF. This recommendation is in conjunction with an American College of Medical Genetics (ACMG) recommendation for a standard panel of mutations to be detected by any specific screening modality. The ACMG set a standard that all screening panels include mutations that have a frequency of at least 0.1 % in the CF-patient population. Testing for these more frequent mutations detects 80% to 90% of CF carriers. The testing of more frequent mutations is necessary due to the large complex CFTR gene and the number of diverse mutations present. Testing is typically performed on a whole- blood sample where DNA is extracted from nucleated cells. The DNA sample is then subjected to multiplex PCR to amplify fragments of the CFTR gene; individual mutations are then identified by various molecular techniques indicated above. Additional reflex testing is required for some mutations due to interfering polymorphisms and so on. If one parent is discovered to be a carrier of CF, a more extensive screening of the other parent is initiated. This is typically accomplished using an extended-panel-mutation screen or whole-gene screening. Most of the whole-gene-screening assays available utilize high-performance liquid chromatography to targeting specific regions of the gene followed by sequencing. These methodologies promise to have increased detection rates but may be hampered by large deletions or interpretation of poorly characterized nucleotide changes.30,31

Newborn screening

Why screen newbornsl In brief, CF patients live longer and healthier lives if the disease is diagnosed earlier, as this enables therapies to be initiated sooner. An analysis of randomized trials evaluating newborn screening in Europe and Australia identified a 5% to 10% reduction in deaths by 10 years of age in CF patients.32 It has been reported in observational trials that CF patients identified by newborn screening have better lung function and growth with less intensive treatment compared to CF patients diagnosed clinically.31-39 CF patients identified earlier who have nutritional intervention also have improved brain function.38,40-43 From a healthcare perspective, screening programs may be less costly because the patients have improved outcomes. A study of the United Kingdom CF database compared annual costs of therapy in CF patients identified by newborn screening to patients identified after two months of age by clinical symptoms.37 The cost of therapy in patients identified by newborn screening was significantly lower, indicating unintended benefits from CF screening programs.

National programs to identify CF patients using circulating immunoreactive trypsinogen (IRT) exist in England, Scotland, France, Wales, Northern Ireland, New Zealand, and Australia.44-47 In Canada, as of April 2008, Alberta and Ontario were the only provinces to perform universal newborn screening. In the United States, the Centers for Disease Control and Prevention identified that CF- screening programs were justified but made the decision to implement these programs dependent on the resources and priorities of individual states.48,49 Currently, 37 states have adopted CF newborn screening, and the list is growing (www.cff.org/AboutCF/Testing/ NewbornScreening/#What_states_do_newborn_screening_for_CF).

Newborn screening of CF by immunoreactive trypsinogen (IRT) and mutational analysis. Newborns with cystic fibrosis have increased levels of circulating IRT, an enzyme produced in the exocrine pancreas. Using an immunoassay, dried-blood samples routinely taken for newborn screening allows the detection of at least 95% CF newborns.44,50,51 Since IRT levels drop precipitously during infancy, a negative result becomes less useful after eight weeks of age.44,51 It is reported, however, that both false-positive and false-negative test results can occur frequently.44,52 Therefore, this test is not diagnostic and requires confirmation by established diagnostic methods such as sweat-chloride testing or CFTR mutational analysis.

Therapy. Due to complexity and diversity of CF, a multidisciplinary approach to comprehensive therapy has been established to improve prognosis. While our understanding of the pathophysiology of disease improves, treatment still focuses on resolving symptoms and organ dysfunction in both children and the growing number of adult patients.22 Since pulmonary impairment is the principal cause of morbidity and death, treatment of the lung component of disease can help slow the progression. A standard pulmonary regimen includes antibiotics, bronchial hygiene, mucolytic agents, bronchodilators, anti-inflammatory agents, nutritional support, and oxygen.53 Lung transplant is related to greater survival and quality of life in patients with advanced lung involvement.54 Recent studies have suggested, however, that older patients benefit more than younger patients with transplantation.55,56 Exocrine-pancreas insufficiency is treated by supplementing pancreatic enzymes with meals and snacks. Since patients with pancreatic insufficiency have poor absorption of lipid soluble vitamins (A,D,E,K), supplementation is routinely recommended.57 Patients with liver disease have been treated with a secondary bile acid (ursodeoxycholic acid) to promote bile flow and reduce cholesterol update; although there is currently limited data regarding patient survival and drug safety, secondary bile-acid therapy is widely used. In the 6% to 8% of patients that evolve to liver failure, transplant has been the major therapeutic strategy.57 New treatment strategies. Most current treatments focus on resolving symptoms and organ dysfunction, however, a wide spectrum of approaches are being explored to improve the underlying pathology (www.cff.org/treatments/Pipeline/). These include therapies to replace defective genes (gene therapy); correction of abnormally folded proteins (i.e., DeltaF508); induction of alternative ion channels; suppression of inflammatory responses; and the development of antibiotics to continue to improve survival.


Cystic fibrosis is the most common lethal genetic disease in Caucasians, manifesting as progressive lung dysfunction, pancreatic insufficiency, and intestinal disease. CF was traditionally diagnosed clinically, either because of a family history or occurrence of meconium ileus, or as a result of intestinal malabsorption and chronic pulmonary disease. In 1979, it was discovered that immunoreactive trypsinogen was increased in neonatal dried-blood specimens on Guthrie cards,50 making it possible to screen neonates. During the past decades, survival rates of patients with CF have improved significantly (see Figure 5). To continue this progress, universal newborn screening has been implemented in many states as an addition to the arsenal of therapies and strategies to improve survival. National newborn-screening programs to identify CF patients after birth have been adopted for a number of years in Europe, Australia, and Canada. As expected, many benefits have been seen due to the early identification of CF patients, including improved survival, better lung function and growth with less intensive therapy, and reduced cost of therapy. To date, 37 states in the United States have adopted similar programs, in the hopes of improving CF outcomes. This welcome trend should help improve the lives of CF patients living in America.

To earn CEUs, see current test at www.mlo-online.com under the CE Tests tab.


Upon completion of this article, the reader will be able to:

1. describe the specific classes of mutations related to CF phenotype variations.

2. identify diagnostic criteria and specific tests for CF.

3. describe the basis for serious complications related to CF.

4. describe newborn-screening tests used by most states.

Despite the large number of CFTR mutations that have been identified, a small number of patients have clinical evidence of CF, including a positive sweat-chloride test but no identifiable CFTR gene defect.


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By Daniel T. Kleven, MD; Christopher R. McCudden, PhD; and Monte S. Willis, MD, PhD

Daniel T. Kleven, MD; Christopher McCudden, PhD; and Monte S. Willis, MD, PhD, are affiliated respectively with the University of North Carolina Hospitals, McLendon Clinical Laboratories, and Department of Pathology and Laboratory Medicine, and the University of North Carolina at Chapel Hill.

Copyright Nelson Publishing Jul 2008

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