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C ystic fibrosis (CF) is the most common lethal genetic disease in Caucasians, occurring in 1/2,500 births.1,2 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 develop-ments in newborn screening for CF in the context of our current standards for diagnosis, therapy, and improved outcomes.

The CFTR geneThe pathophysiology of CF results from mutations in the cys-tic-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, leav-ing 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 CFTR protein has multiple membrane-spanning regions, two nucleotide-binding domains (NBD), and

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Cystic fibrosis: newborn screening in AmericaBy Daniel T. Kleven, MD;

Christopher R. McCudden, PhD;

and Monte S. Willis, MD, PhD

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

LEARNING OBJECTIVES

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

describe the specific classes of mutations related to CF 1. phenotype variations.identify diagnostic criteria and specific tests for CF.2. describe the basis for serious complications related to 3. CF.describe newborn-screening tests used by most states.4.

C o N t I N U I N G e D U C A t I o N

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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 mutat ions that are present.3 Greater than 1,250 mutations have been identified to date (www.genet.sickkids. on.ca/cftr). These mu-tations have been di-vided 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 (DF508), which resides in the first nucleotide-binding domain (see Figure 3). Patients with two of these muta-tions 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 muta-tions 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, fac-tors 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 identi-

fying 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 mul-tiorgan nature of CF. Defects in CFTR function within these organs results in lung disease, pancreatic insufficiency, multifocal biliary cirrhosis, male infer-tility, and increased sweat ion loss.

Lungs. The most serious complica-

C y s t I C f I b r o s I s

Classification of CFTR mutations

Class I: Defective protein synthesis No CFTR

Class II: Defective processing CFTR degraded

Class III: Defective regulation Impaired response to ATP

Class IV: Transmembrane mutations Diminished ion flow

Class V: Intronic splice sites/CFTR pro mutations Decreased abundance

Table 1.

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tions 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 so-dium (and water), re-sulting in a dehydrated mucous. These changes impede the necessary ciliary clearance of mi-croorganisms and debris in the lungs, promoting obstruction of the air-

ways 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, Pseudomo-nas 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

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C o v e r s t o r y

disease, which progresses as a slower place.9 Lung involve-ment is responsible for greater than 90% of the mortality in CF patients.

Pancreas. The exocrine function of the pancreas is respon-sible for the secretion of enzymes essential for the breakdown of food. In the pancreas, defective CFTR protein causes reduced HCO

3- secretion (see Figure 2), leading to congestion of acini

and inappropriate activation of pancreatic proteases. This pro-cess 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 pancre-atic 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.11 In the hepatic biliary system, CFTR is expressed in cholangiocytes and gall-bladder epithe-lial 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.11 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.11 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 azoo-spermia (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 associ-ated morbidities in patients with CF (see Figure 1). These

Figure 1. Clinical effects of cystic fibrosis. Cystic fibrosis can affect numerous organs; the most severely affected of these are the lung and pancreas. In mild cases, affected males may be infertile. Abnormal sweat secretion is a hallmark of cystic fibrosis, and is the gold standard for detecting disease.

Continues on page 22

Figure 2. Cystic-fibrosis disease mechanisms. In the sweat glands, a de-fective CFTR channel results in excess sodium and chloride concentration in secretions. This abnormality is the biochemical basis for sweat testing. In the lung, impaired chloride transport causes increased sodium trans-port into the cell. Water follows sodium and the mucus layer to become dehydrated and more viscous, trapping pathogenic bacteria and leading to chronic respiratory illness. In the pancreas, it is proposed that CFTR impairment causes clogging of the acini and inappropriate activation of enzymes leading to pancreatic insufficiency and malabsorption. CFTR protein is red, other channels and transporters are grey. Adapted from References 11, 58, and 59.

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Sweat-chloride testing. The gold standard for CF diagnosis is sweat-chloride testing. Sweat-chloride test-ing will detect ~99% of pa-tients, is relatively inexpen-sive, and has a high degree of accuracy. Sweat testing is performed by stimulat-ing sweat production by pilocarpine iontophoresis; sweat is collected on pre-weighed gauze pads, and coulometric titration is used

to measure the chloride concentra-tion. A sweat-chloride value >60 mEq/L distinguishes most patients with cystic fibrosis; however, nor-mal 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 per-form 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 consider-ably more complex than detecting sweat-chloride concentra-tions 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.

manifestations affect the intestine and upper airway, and include sinusitis, nasal polyps, distal ileum obstruc-tion, 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 pathogno-monic 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 de-velop 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 inflam-mation and/or nasal polyps may prompt the screening for CF.

Clinical diagnosis of cystic fibrosisThe 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).19 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, allele-specific 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, iden-tifying 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.

Continues on page 24

Diagnostic criteria of CF

1. Presence of risk factors or clinical presentation:

a. one or more phenotypic features

b. or history of sibling with CF

c. or positive newborn screening

2. Laboratory evidence of CFTR dysfunction:

a. two abnormal sweat-chloride concentrations

b. or the presence of two disease-causing mutations in CFTR

c. or demonstration of abnormal nasal potential difference measurement

Table 2.

Medical conditions associated with elevated sweat-chloride levels other than cystic fibrosis64-69

1. Addison’s

2 Pulmonary edema/sepsis

3. Pseudo-aldosteronism

4. Hypoparathyroidism

5. Hypothyroidism

Table 3.

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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parents’ risks should be considered. Currently, the American College of Obstetricians and Gynecologists (ACOG) recom-mends 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. Screen-ing 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) CF or 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 seek-ing 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 screeningWhy screen newborns? 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

Screening for CFPre-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

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Figure 4. Severity of cystic fibrosis as related to mutation, CFTR activity, and sweat-test positivity. The most severe clinical phenotypes are associated with the absence of expression (<1% CFTR activity) and defective processing and regulation. In these cases, sweat-chloride tests are almost always abnormal. In less severe cases, there may only be mild disease and infertility associated with reduced activity (5% to 10% CFTR activity) or reduced expression. Sweat testing may not be abnormal in these mild clinical phenotypes. CBAVD-congenital bilateral absence of vas deferens. Based on References 61 and 62.

Figure 3. CFTR gene, protein, and mutations. The CFTR gene spans 250 kilobases and is encoded by 24 exons that transcribe a 1,480-amino-acid protein. The CFTR protein has 12 membrane-spanning regions bisected by a regulatory region (R) and a nucleotide-binding domain (NBD-1). This binding catalyzes hydrolysis of ATP in order to open the chloride chan-nel; it is counter-regulated by another nucleotide-binding region (NBD-2) at the C-terminus that closes the channel. The location and frequency of the five most common mutations are indicated; the frequency is based on the screening of 43,849 CF chromosomes (www.genet.sickkids.on.ca/cftr/resource/Table1.html). Adapted from References 59 and 60.

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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.33-39 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 im-proved 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 iden-tified 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 deci-sion 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 mul-tidisciplinary 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

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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 cur-rently 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 un-derlying pathology (www.cff.org/treatments/Pipeline/). These include therapies to replace defective genes (gene therapy); correction of abnormally folded proteins (i.e., DF508); induc-tion of alternative ion channels; suppression of inflammatory responses; and the development of antibiotics to continue to improve survival.

SummaryCystic 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 fam-ily 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 sig-nificantly (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.

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.

References

1. Wilcken B, Wiley V, Sherry G, Bayliss U. Neonatal screening for cystic fibrosis: a comparison of two strategies for case detection in 1.2 million babies. J Pediatr. 1995;127(6):965-970.

2. Massie RJ, Delatycki MB, Bankier A. Screening couples for cystic fibrosis carrier status: why are we waiting? Med J Aust. 2005;183(10):501-502.

3. Tsui LC, Durie P. Genotype and phenotype in cystic fibrosis. Hosp Pract (Min-neap). 1997;32(6):115-118, 123-119, 134, passim.

4. Groman JD, Karczeski B, Sheridan M, Robinson TE, Fallin MD, Cutting GR. Phe-notypic and genetic characterization of patients with features of “nonclassic” forms of cystic fibrosis. J Pediatr. 2005;146(5):675-680.

5. Groman JD, Meyer ME, Wilmott RW, Zeitlin PL, Cutting GR. Variant cystic fibrosis phenotypes in the absence of CFTR mutations. N Engl J Med. 2002;347(6):401-407.

6. Chmiel JF, Davis PB. State of the art: why do the lungs of patients with cystic fibrosis become infected and why can’t they clear the infection? Respir Res. 2003;4:8.

7. Parameswaran GI, Murphy TF. Infections in chronic lung diseases. Infect Dis Clin North Am. 2007;21(3):673-695, viii.

8. Mahenthiralingam E, Baldwin A, Dowson CG. Burkholderia cepacia complex bacteria: opportunistic pathogens with important natural biology. J Appl Mi-crobiol. 2008;104(6):1539-1551.

9. Yiallouros PK, Neocleous V, Zeniou M, et al. Cystic fibrosis mutational spectrum and genotypic/phenotypic features in Greek-Cypriots, with emphasis on dehydra-tion as presenting symptom. Clin Genet. 2007;71(3):290-292.

10. Baker SS, Borowitz D, Baker RD. Pancreatic exocrine function in patients with cystic fibrosis. Curr Gastroenterol Rep. 2005;7(3):227-233.

11. Colombo C. Liver disease in cystic fibrosis. Curr Opin Pulm Med. 2007;13(6):529-536.

12. Cohn JA, Strong TV, Picciotto MR, Nairn AC, Collins FS, Fitz JG. Localization of the cystic fibrosis transmembrane conductance regulator in human bile duct epithelial cells. Gastroenterology. 1993;105(6):1857-1864.

13. Cystic Fibrosis Foundation. Patient Registry 2003: Annual Report to the Center Directors. Bethesda, MD: Cystic Fibrosis Foundation; 2003.

14. Popli K, Stewart J. Infertility and its management in men with cystic fibro-sis: review of literature and clinical practices in the UK. Hum Fertil (Camb). 2007;10(4):217-221.

15. Khaitov S, Nissan A, Beglaibter N, Freund HR. Failure of medical treatment in an adult cystic fibrosis patient with meconium ileus equivalent. Tech Coloproctol. 2005;9(1):42-44.

16. Chaun H. Colonic disorders in adult cystic fibrosis. Can J Gastroenterol. 2001;15(9):586-590.

17. Ramsey B, Richardson MA. Impact of sinusitis in cystic fibrosis. J Allergy Clin Immunol. 1992;90(3, pt 2):547-552.

18. Yung MW, Gould J, Upton GJ. Nasal polyposis in children with cystic fibrosis: a long-term follow-up study. Ann Otol Rhinol Laryngol. 2002;111(12, pt 1):1081-1086.

19. Rosenstein BJ, Cutting GR. The diagnosis of cystic fibrosis: a consensus state-ment. J Pediatr. 1998;132(4):589-595.

20. Stern RC. The diagnosis of cystic fibrosis. N Engl J Med. 1997;336(7):487-491.21. Gibson RL, Burns JL, Ramsey BW. Pathophysiology and management of pulmo-

nary infections in cystic fibrosis. Am J Respir Crit Care Med. 2003;168(8):918-951.

22. Ratjen F, Doring G. Cystic fibrosis. Lancet. 2003;361(9358):681-689.23. Dreyfus DH, Bethel R, Gelfand EW. Cystic fibrosis 3849+10kb C > T mutation

associated with severe pulmonary disease and male fertility. Am J Respir Crit Care Med. 1996;153(2):858-860.

C o v e r s t o r y

Figure 5. Median predicted survival of CF patients. Data from cystic fibrosis foundation, 2008.63

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C y s t I C f I b r o s I s

24. LeGrys VA, Yankaskas JR, Quittell LM, Marshall BC, Mogayzel PJ Jr. Diag-nostic sweat testing: the Cystic Fibrosis Foundation guidelines. J Pediatr. 2007;151(1):85-89.

25. Middleton PG, Geddes DM, Alton EW. Protocols for in vivo measurement of the ion transport defects in cystic fibrosis nasal epithelium. Eur Respir J. 1994;7(11):2050-2056.

26. Schuler D, Sermet-Gaudelus I, Wilschanski M, et al. Basic protocol for tran-sepithelial nasal potential difference measurements. J Cyst Fibros. 2004;3(suppl 2):151-155.

27. ACOG Committee Opinion. Update on carrier screening for cystic fibrosis. Obstet Gynecol. 2005;106(6):1465-1468.

28. Watson MS, Cutting GR, Desnick RJ, et al. Cystic fibrosis population carrier screening: 2004 revision of American College of Medical Genetics mutation panel. Genet Med. 2004;6(5):387-391.

29. Grody WW, Cutting GR, Klinger KW, Richards CS, Watson MS, Desnick RJ. Laboratory standards and guidelines for population-based cystic fibrosis carrier screening. Genet Med. 2001;3(2):149-154.

30. Strom CM, Huang D, Chen C, et al. Extensive sequencing of the cystic fibrosis transmembrane regulator gene: assay validation and unexpected benefits of developing a comprehensive test. Genet Med. 2003;5(1):9-14.

31. Kammesheidt A, Kharrazi M, Graham S, et al. Comprehensive genetic analysis of the cystic fibrosis transmembrane conductance regulator from dried blood speci-mens — implications for newborn screening. Genet Med. 2006;8(9):557-562.

32. Grosse SD, Rosenfeld M, Devine OJ, Lai HJ, Farrell PM. Potential impact of newborn screening for cystic fibrosis on child survival: a systematic review and analysis. J Pediatr. 2006;149(3):362-366.

33. Robinson P. Cystic fibrosis. Thorax. 2001;56(3):237-241.34. Dankert-Roelse JE, Merelle ME. Review of outcomes of neonatal screening

for cystic fibrosis versus non-screening in Europe. J Pediatr. 2005;147(3)(suppl):S15-20.

35. Khoury MJ, McCabe LL, McCabe ER. Population screening in the age of genomic medicine. N Engl J Med. 2003;348(1):50-58.

36. Sims EJ, McCormick J, Mehta G, Mehta A. Newborn screening for cystic fibrosis is associated with reduced treatment intensity. J Pediatr. 2005;147(3):306-311.

37. Sims EJ, Mugford M, Clark A, et al. Economic implications of newborn screen-ing for cystic fibrosis: a cost of illness retrospective cohort study. Lancet. 2007;369(9568):1187-1195.

38. Siret D, Bretaudeau G, Branger B, et al. Comparing the clinical evolution of cystic fibrosis screened neonatally to that of cystic fibrosis diagnosed from clinical symptoms: a 10-year retrospective study in a French region (Brittany). Pediatr Pulmonol. 2003;35(5):342-349.

39. Waters DL, Wilcken B, Irwing L, et al. Clinical outcomes of newborn screening for cystic fibrosis. Arch Dis Child Fetal Neonatal Ed. 1999;80(1):F1-7.

40. Farrell PM, Kosorok MR, Rock MJ, et al. Early diagnosis of cystic fibrosis through neonatal screening prevents severe malnutrition and improves long-term growth. Pediatrics. 2001;107(1):1-13.

41. Koscik RL, Farrell PM, Kosorok MR, et al. Cognitive function of children with cystic fibrosis: deleterious effect of early malnutrition. Pediatrics. 2004;113(6):1549-1558.

42. Koscik RL, Lai HJ, Laxova A, et al. Preventing early, prolonged vitamin E defi-ciency: an opportunity for better cognitive outcomes via early diagnosis through neonatal screening. J Pediatr. 2005;147(3)(suppl):S51-56.

43. Milla CE. Association of nutritional status and pulmonary function in children with cystic fibrosis. Curr Opin Pulm Med. 2004;10(6):505-509.

44. Wagener JS, Sontag MK, Sagel SD, Accurso FJ. Update on newborn screening for cystic fibrosis. Curr Opin Pulm Med. 2004;10(6):500-504.

45. Farriaux JP, Vidailhet M, Briard ML, Belot V, Dhondt JL. Neonatal screening for cystic fibrosis: France rises to the challenge. J Inherit Metab Dis. 2003;26(8):729-744.

46. Price JF. Newborn screening for cystic fibrosis: do we need a second IRT? Arch Dis Child. 2006;91(3):209-210.

47. Sarles J, Berthezene P, Le Louarn C, et al. Combining immunoreactive trypsinogen and pancreatitis-associated protein assays, a method of newborn screening for cystic fibrosis that avoids DNA analysis. J Pediatr. 2005;147(3):302-305.

48. Grosse SD, Boyle CA, Botkin JR, et al. Newborn screening for cystic fibrosis: evaluation of benefits and risks and recommendations for state newborn screen-ing programs. MMWR Recomm Rep. 2004;53(RR-13):1-36.

49. Wilfond BS, Gollust SE. Policy issues for expanding newborn screening pro-grams: the cystic fibrosis newborn screening experience in the United States. J Pediatr. 2005;146(5):668-674.

50. Crossley JR, Elliott RB, Smith PA. Dried-blood spot screening for cystic fibrosis in the newborn. Lancet. 1979;1(8114):472-474.

51. Farrell PM, Li Z, Kosorok MR, et al. Bronchopulmonary disease in children with cystic fibrosis after early or delayed diagnosis. Am J Respir Crit Care Med. 2003;168(9):1100-1108.

52. Durie PR, Forstner GG, Gaskin KJ, et al. Age-related alterations of immunoreac-tive pancreatic cationic trypsinogen in sera from cystic fibrosis patients with and without pancreatic insufficiency. Pediatr Res. 1986;20(3):209-213.

53. Dalcin Pde T, Abreu ESFA. Cystic fibrosis in adults: diagnostic and therapeutic aspects. J Bras Pneumol. 2008;34(2):107-117.

54. Aurora P, Whitehead B, Wade A, et al. Lung transplantation and life extension in children with cystic fibrosis. Lancet. 1999;354(9190):1591-1593.

55. Liou TG, Adler FR, Huang D. Use of lung transplantation survival models to refine patient selection in cystic fibrosis. Am J Respir Crit Care Med. 2005;171(9):1053-1059.

56. Liou TG, Adler FR, Cox DR, Cahill BC. Lung transplantation and survival in children with cystic fibrosis. N Engl J Med. 2007;357(21):2143-2152.

57. Yankaskas JR, Marshall BC, Sufian B, Simon RH, Rodman D. Cystic fibrosis adult care: consensus conference report. Chest. 2004;125(1)(suppl):1S-39S.

58. Cohn JA. Reduced CFTR function and the pathobiology of idiopathic pancreatitis. J Clin Gastroenterol. 2005;39(4)(suppl 2):S70-77.

59. Mishra A, Greaves R, Massie J. The relevance of sweat testing for the diagnosis of cystic fibrosis in the genomic era. Clin Biochem Rev. 2005;26(4):135-153.

60. Scriver CR. The Metabolic & Molecular Bases of Inherited Disease. 8th ed. New York, NY: McGraw-Hill; 2001.

61. Kumar V, Abbas AK, Fausto N, Robbins SL, Cotran RS. Robbins and Cotran Patho-logic Basis of Disease. 7th ed. Philadelphia, PA: Elsevier/Saunders; 2004.

62. Wallis C. Diagnosing cystic fibrosis: blood, sweat, and tears. Arch Dis Child. 1997;76(2):85-88.

63. Cystic Fibrosis Foundation. Patient Registry Annual Data Report, 2006. Bethesda, MD: Cystic Fibrosis Foundation; 2008.

64. Chan HL, Gwee HM. Salty sweat and ichthyosis in Addison’s disease. Br Med J. 1977;1(6053):81-82.

65. Eisenhut M, Sidaras D, Barton P, Newland P, Southern KW. Elevated sweat sodium associated with pulmonary oedema in meningococcal sepsis. Eur J Clin Invest. 2004;34(8):576-579.

66. Foulston C, Gall G, Mitchell I, Cooper DM, Scott RB. Transient neutral fat steator-rhea, elevated sweat chloride concentration, and hypoparathyroidism in a child with celiac disease. J Pediatr Gastroenterol Nutr. 1985;4(1):143-145.

67. Hanukoglu A, Bistritzer T, Rakover Y, Mandelberg A. Pseudohypoaldosteronism with increased sweat and saliva electrolyte values and frequent lower respiratory tract infections mimicking cystic fibrosis. J Pediatr. 1994;125(5, pt 1):752-755.

68. Squires L, Dolan TF. Abnormal sweat chloride in auto-immune hypothyroidism. Clin Pediatr (Phila). 1989;28(11):535-536.

69. Brand PL, Gerritsen J, van Aalderen WM. A baby with eczema and an abnormal sweat test. Lancet. 1996;348(9032):932.

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Cystic-fibrosis products New Products New Technology New Services

Simplifies sweat test

Nanoduct is a complete, integrated system for inducing and analyzing sweat for cystic-fibrosis diagnosis that produces a result while attached to the patient. Nanoduct simplifies the CF sweat test and, for the first time, makes possible reliable CF diagnosis in the first days of life.

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External quality control

The INTROL Cystic Fibrosis Panel I Control is intended for use as a control product to monitor the analytical perfor-mance of the extraction, amplification, and detection of test systems used in the qualitative measurement of the cystic-fibrosis transmembrane conductance regulator (CFTR) gene.

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Mutation coverage

The xTAG Cystic Fibrosis Kit is for cystic-fibrosis genotyping. With vali-dated performance criteria, this highly accurate and reproducible (>99.9%) assay gives wide mutation coverage for carrier screening in adults and aid in the diagnosis in newborns.

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Carrier detection The eSensor CF Carrier Detection System offers highly accurate and re-producible cystic-fi-brosis-car-rier results delivered in an easy-to- in te r-p r e t r e -port. Each kit includes all reagents necessary for PCR amplification and mutation detec-tion in a single box. Reports include a summary “carrier” or “non-carrier.”

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Comprehensive test CF AMPLIFIED is a comprehensive CF test, detecting approximately 99% of mutations, in-cluding gross de-letions and dupli-cations, in patients of all ethnicities. The test begins with full gene se-quence analysis detecting 97% to 98% of mutations. Testing is done in steps to control costs and the average turnaround time is reduced from 15 to 35 days to 14 to 28 days

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Detect and identify

The InPlex CF Molecular Test has an easy multiplex format with few steps and few repeats, no necessary reflex testing, and very little hands-on time. After extraction, results are achieved in four hours or less. Low start-up costs, low hands-on time, fast time to results.

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Direct sequence analysisT h e f u l l C F G e n e Sequencing Test is direct s e q u e n c e analysis of the CFTR gene, identifying every nucleotide of all 27 exons, their associated splice site regions, and clinically relevant regions of introns that contain disease-causing mutations. Also offered is partial CF-gene sequencing for families with known mutations not detectable in a general screening assay.

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Expanded panel

The CF70 Genotype is a 70-mutation screening panel for cystic-fibrosis car-rier, pre-natal, and diagnostic testing. The broad mutation coverage of the CF70 Genotype exceeds the minimum recom-mendations of the American College of Medical Genetics and the American Col-lege of Obstetricians and Gynecologists for general population screening

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Interested in submitting your new product, service, or technology to MLO’s Industry Spotlight? Send only items that are currently available in the marketplace. Upgrades of exist-ing products and services are also acceptable. Submissions are accepted via e-mail at mlo-admin@nelsonpub.com or by regular mail. Limit copy to 50 words. (MLO reserves the right to edit.) Provide a color photograph (5x7, at least 300 dpi, JPG, EPS, or TIF). Ap-propriate products are kept on file for 120 days and are used when space is available. Deadline for submission is 60 to 90 days prior to the desired pub-lication issue dates. Note: Because of the number of submissions MLO re-ceives, its staff members cannot verify receipt of Industry Spotlight submis-sions by e-mail or by telephone.