Advances in the Diagnosis and Management of Cystic Fibrosis in the Genomic Era.
CF results from mutations within the CF transmembrane conductance regulator (CFTR)  gene (Online Mendelian Inheritance in Man *602421) (3). The CFTR gene is located on the long arm of chromosome 7. A 6.5-kb mRNA produced by the normal allele encodes CFTR, a 1490-amino acid integral membrane protein that functions as a regulated ion channel for chloride in the epithelial cells of multiple organs (4). Since the gene was cloned in 1989, >2000 CFTR variants have been described in the CF Mutation Database (CFMD) (5). Over the years, this database has served as the reference for a mechanism-based classification system (classes I--VI) (Table 1) for CF, allowing for improved therapy targets. However, more recently the Clinical and Functional Translation of CFTR project (CFTR2) has been created to assess disease severity of patients with CF. Researchers of CFTR2 gathered global data from CF patient registries, functional research, and clinical data to harmonize annotation terminology of CFTR variants (6).
Through aggressive multidisciplinary medical efforts, CF management has targeted chronic respiratory infection, airway clearance, and supplementation of pancreatic enzymes to correct nutritional deficits (7). Additional therapies target the molecular defects associated with production and function of the CFTR protein (8, 9). Further understanding of impaired mucus clearance as a consequence of abnormal ion transport in airway epithelium has led to the development of mucolytic agents and antipseudomonal antibiotics for patients with CF (10). Because of advances in treatment, the most common cause of death for patients with CF today is respiratory failure in individuals who are unable to receive a timely lung transplant.
As children with CF age, newer challenges are emerging in the adult CF population. Patients with CF require appropriate sex- and age-specific expertise, as well as multidisciplinary care. In countries lacking these necessary adult provisions, adults with CF will continue to be seen in CF centers traditionally equipped to handle pediatric patients (11, 12). Recent studies show there are different survival rates between developed countries. From 2009 to 2013, a comparison of the survival rates of patients with CF in the US and Canada revealed a median survival rate of 40.6 years in the US compared with 50.6 years in Canada. The 10-year variation in survival rates was recognized as an effect of differential access to lung transplantation, increased posttransplant survival, and overall differences in healthcare systems (11, 12).
Since CF was first recognized, there has been a substantial increase in new phenotypic and genotypic information about the disorder (13). With international collaborations and the widespread collection of CF data, there have been many novel laboratory developments and drastic improvements in the interpretation and classification of CF status. This growth of information has led to the review and revision of the 2008 CF Foundation diagnostic guidelines by an international consortium of CF experts (13, 14). Several goals of the 2017 CF Foundation guidelines were to harmonize the diagnostic criteria and terminology used in CF multidisciplinary care centers all over the world, as well as updating references intervals used for sweat chloride measurements. In this review, we address historical milestones for CF, together with the recent changes to the diagnostic criteria published in the 2017 CF Foundation consensus guidelines.
Clinical Manifestations of CF
The pathophysiology of CF presents as a multiorgan, multisystem disease process characterized by a classic triad of clinical symptoms: chronic obstruction and infection of the respiratory tract, exocrine pancreatic insufficiency, and increased sweat chloride concentrations (15). Since its discovery, different CFTR mutations have been determined to have varying effects on overall production, function, and stability of the CFTR protein (6, 16). The transmembrane protein is a complex regulated ion channel expressed on the apical surface of epithelial cells lining lumina within the airways, pancreas, intestine, hepatobiliary system, male genital tract, and the sweat glands (4). Physiologically, CF causes a reduction in the transport of chloride and bicarbonate. This impaired transport predisposes individuals to increased viscosity of mucus secretions in many tissues of the body, with the lungs being one of the primary organ systems involved, resulting in significant morbidity and mortality (16).
The altered CFTR protein in those with CF leads to viscous secretions that accumulate in the lungs. This generates a cascade of pulmonary complications that starts with persistent coughing and leads to inflammatory states of bronchiectasis and, ultimately, respiratory failure (Fig. 1). The mucus buildup also becomes a perfect milieu for persistent pulmonary infections. Patients with CF become infected early in life with Staphylococcus aureus (including methicillin-resistant Staphylococcus aureus) and Hemophilus influenzae, and later in life with Pseudomonas aeruginosa (17). Inhaled tobramycin and azithromycin are commonly used as antibiotic treatments (18, 19). Other physical techniques are also available to augment the normal mucociliary clearance mechanism of the lungs and to facilitate expectoration. These techniques may involve a variety of methods, such as mechanical percussion and vibration of the chest wall or wearable inflatable vests that perform high-frequency chest compressions (20, 21).
The development of neonatal intestinal obstruction secondary to meconium ileus is the earliest clinical manifestation of CF (22). Approximately 13% to 17% of newborns with CF form meconium ileus, although it is not unique to the disease. Meconium ileus is highly viscid, desiccated, and inspissated causing proximal dilation, bowel-wall thickening, and congestion in the midileum (22-24).
The pancreas is one of the initial organs that is seriously affected by CF. The CFTR protein is abundantly expressed in the pancreatic duct epithelia. Its role serves to permit anions and water into the ductal lumen. Malfunction of the CFTR protein in those with CF has catastrophic effects on the pancreas, leading to digestion and nutritional problems. At birth, approximately 66% of CF patients develop pancreatic insufficiency, whereas by 1 year of age 90% have signs of fat malabsorption (25). Patients are typically identified with pancreatic insufficiency owing to the clinical presentation of steatorrhea, failure to thrive, decreased fecal elastase, and decreased response to enzyme replacement therapy. Pancreatitis is caused by enzyme buildup of ductular and acinar pancreatic enzyme secretions that eventually damage the pancreas. This process is believed to be involved in the progression of glucose intolerance and CF-related diabetes (26).
Screening and Diagnosis of CF
Internationally, newborn screening (NBS) is recognized as a necessary public health initiative for the identification of genetic disorders in newborns who could benefit from early interventions. Historically, CF was diagnosed through the recognition of classic phenotypic signs and symptoms of the disease (27, 28). The origins of NBS for CF can be traced back to 1979 in New Zealand (29), and since 2010, all 50 states in the US have included CF screening in their NBS program (30). Individuals born in the US before this period most likely have never been screened. Consequently, individuals with milder cases of undiagnosed CF usually present with variable clinical manifestations that are not initially recognized with the overt CF disease. Through several standard approaches, newborns are screened for CF by serial, 2-tiered, or, more recently, 3-tiered/hybrid-based methods (31-33). Various positive predictive values, sensitivities, and specificities for these methods have been reported but reflect the specific population being screened (Table 2) (34-36).
There are several 2-tiered screening procedures being performed. One approach starts with the measurement of immunoreactive trypsinogen (IRT). Sample measurements above a defined cutoff are then followed up by genetic analysis to determine the presence of specific mutations in the CFTR gene (34). The second approach involves an initial IRT measurement with defined cutoffs, which is then followed with a subsequent IRT measurement on an additional specimen collected 2 weeks from the original test. These protocols are often referred to as the IRT/DNA and IRT/IRT protocols, respectively (34). A third protocol uses pancreatitis-associated protein following an increased IRT measurement and is commonly referred to as the IRT/ pancreatitis-associated protein method (37).
All current CF NBS protocols measure IRT as an initial screen. Pancreatic trypsinogen is produced as an inactive precursor of the enzyme trypsin and recognized by antibodies. Normally, IRT is found at low concentrations in the body. Newborns with CF have IRT increases as a consequence of thick mucus buildup in the pancreatic ducts, although this may also occur because of pancreatic injury, prenatal asphyxia, and other stresses (38). As infants reach approximately 8 weeks of age, the concentration of IRT generally decreases and testing would produce a negative result. As such, positive results are considered more indicative for a diagnosis of CF than a negative result for ruling out the disease.
Direct sandwich immunoassays have been designed to measure IRT (38). These assays incorporate either monoclonal or polyclonal antibodies and may also be multiplexed. The ability to test multiple patient samples rapidly and the relatively low cost made the IRT assay ideal for use in NBS laboratories. As a result of differing specificity between assays, there have been challenges in the overall standardization of IRT measurement. The 99th to 99.5th percentile is used as a concentration cutoff in the initial testing screen of the IRT/IRT protocol. Newborns with positive results within this interval must have another specimen collected ideally around 2 weeks for additional IRT analysis. If the results of the second IRT test are above the 95th to 97th percentile cutoff, the newborn is subsequently referred to a CF center for appropriate diagnosis. Although the protocol is less expensive than the IRT/DNA protocol, return visits for second specimen collection and pairing of medical data could be potentially fraught with logistical errors and delay diagnosis and treatment (39). In addition, the ethnicity of the newborn has been shown to cause variations in IRT concentrations (40).
The IRT/DNA protocol is commonly used in the US and other countries. The first step is to screen newborns by IRT at a defined cutoff around the 96th to 99th percentile. If the IRT measurement is above this cutoff, an additional DNA screening test is used to identify specific CFTR gene mutations. Based on each testing site's population, adjustments are usually made to the IRT cutoff as needed. With >2000 unique CF mutations identified, steps must be taken to ensure that individuals with rare cases are not being missed. To address this issue, NBS programs historically have widely adopted the 23-mutation pan-ethnic panel for CF recommended by the American College of Medical Genetics/American College of Obstetricians and Gynecologists (41-43). On occasion, additional mutations are added to the core screening panel that more accurately reflect the ethnic background of the population being tested. The 23-variant panel covers approximately 90% of all CFcausing variants identified but only 68.5% in the Hispanic population (43). Therefore, in ethnically diverse California, newborns with high IRT (>62 ng/mL, top 1.6%) are tested with a 28- to 40-selected CFTR variant panel to incorporate the range of diversity in the state's geographical area (33). Additionally, if only 1 CF mutation variant is found from the DNA panel, DNA sequencing is performed to determine any missed CFTR variants. This process is referred to as the 3-tiered approach for CF screening and has led to the identification of many unrecognized CFTR mutations, increased detection of CF, and decreased false-positive results (33). Other states like Colorado, Wyoming, and Texas have used the original IRT/IRT procedure, but they also have added an additional DNA screening step, commonly referred to as the IRT/IRT-DNA model (36). This additional screening step has led to higher detection rates with the IRT/IRT-DNA panel, although many of the same caveats with the IRT/IRT protocol still exist as previously described. The use of next-generation sequencing technology (IRT/next-generation sequencing) is also being assessed for improving NBS within the US (44). With recent diagnostic guidelines, the use of the CFTR2 project database is recommended as the first resource for assessing mutation pathogenicity following NBS (13, 45).
SWEAT CHLORIDE MEASUREMENT
Because of the heterogeneous nature of CF and the possibility of false-positive results, newborns identified with a positive NBS result are recommended to have their sweat chloride measured to confirm the diagnosis of CF (13). Likewise, in cases of newborns with a family history and clinical features consistent with CF, sweat chloride should be measured and interpreted (Fig. 2). For a positive NBS, recent guidelines recommend the newborn be >10 days of age and weigh >2 kg before sweat chloride measurement. Newborns identified with meconium ileus should also have sweat testing performed before a diagnosis of CF may be made (13).
Measurement of sweat chloride should be performed as outlined in approved procedural guidelines, such as the 2009 C34-A3 Sweat Testing: Sample Collection and Quantitative Chloride Analysis, Clinical and Laboratory Standards Institute (CLSI)-approved guideline document (46). Briefly, the sweat test requires the use of iontophoresis to deliver pilocarpine, which causes sweat glands to secrete sweat. The sweat is carefully collected and analyzed for chloride content, with results and an interpretation reported to the clinical team and family members as soon as possible. There are 2 commonly used methods for sweat collection. One uses microbore tubing; the other, gauze or filter paper. The specifics of each method are detailed in CLSI C34-A3 (46). The equipment used in the gauze or filter paper collection has been discontinued and will eventually become obsolete. Consequently, many laboratories have begun to validate or have already validated the microbore tubing method. Adequate sweat collection can be difficult, and the CF Foundation requires documentation of sweat weights that do not meet the minimum 75 mg for the gauze or filter paper collection process or a minimum of 15 [micro]L for the microbore method. These collections are commonly referred to as quality not sufficient specimens. For children >3 months of age, laboratories are to maintain an annual quality not sufficient rate of 5%, whereas there is a 10% requirement for neonates and infants <3 months of age (47).
There are several challenges in this techniquedependent collection process. The CLSI document helps identify the proper technique to minimize and, it is hoped, eliminate falsely increased or decreased chloride results (46). Some challenges include proper cleansing of stimulated site, proper attachment of electrodes, and proper techniques used to prevent evaporation of collected sweat, among many others. The quality not sufficient rate is the primary quality indicator used for determining whether the sweat collection process is working properly.
The sweat test is the gold standard in the diagnosis of CF because of its wide availability, standardization, and high level of sensitivity and specificity (48). Recent consensus guidelines reiterate that sweat testing is always needed to confirm CF diagnosis (13). However, this has not always been the case because there are reports of newborns being diagnosed with CF after only a positive NBS result (13). In addition, diagnostic sweat testing and follow-up genetic testing should always be performed, as many errors can arise from the NBS procedure (13, 49). Instances of problems with CF NBS have occurred with Guthrie card labeling, mutation panel changes, misinterpretations, and the detection of 2 CFTR mutations in cis (i.e., on the same chromosome) (13, 49). Therefore, before a definitive diagnosis of CF can ever be determined, it is always warranted to demonstrate CFTR dysfunction with a sweat test.
Sweat chloride test reference intervals have been updated into 3 categories (13). These revisions include the incorporation of all ages in the interpretation of intermediate sweat chloride results vs the previous age-based model owing to recently available data (6, 13, 48).
The 2017 CF Foundation Consensus Guidelines for all ages are as follows:
* [less than or equal to] 29 mmol/L: normal, CF unlikely (exceptions occur)
* 30 to 59 mmol/L: intermediate, possible CF
* [greater than or equal to] 60 mmol/L: abnormal, indicative for diagnosis of CF
Normal sweat chloride results are not typically repeated. However, if a patient has a strong/positive family history and continued symptoms of CF, additional sweat chloride determinations and/or CFTR genotyping may be performed (13). Rare cases of CF have also been reported in patients with sweat chloride <30 mmol/L (50). In individuals with an intermediate sweat chloride concentration, the sweat test should be repeated on a separate occasion (typically 2 weeks apart). If both sweat chloride values are determined in the intermediate range (30-59 mmol/L), the individual should be considered for CFTR functional or gene analysis. In ambiguous cases, the use of nasal potential difference (NPD) or intestinal current measurement (ICM) may also be performed on a screen-positive newborn (13, 51, 52). Screen-positive individuals with an abnormal sweat chloride concentration (e.g., >60 mmol/L) likely have CF. These patients are recommended to undergo CFTR genetic testing to confirm CF-causing mutations that could otherwise help to determine appropriate therapy selection (13, 45).
OTHER CF-ASSOCIATED TESTS
Additional functional tests may be used to diagnosis CF. During the past 30 years, NPD and ICM have been used as CFTR functional tests in addition to sweat chloride measurement. In the respiratory epithelium, CFTR function is measured in vivo by NPD and ex vivo in superficial rectal biopsies by ICM (52). Both tests have established international standard operating procedures and have been shown to discriminate healthy controls and CF patients. Unfortunately, these tests require specialized procedures and training, are costly, and are not widely available (13). The sweat chloride test, by comparison, is relatively easy, less costly, effective, and noninvasive. In addition, the process is standardized and transferrable among laboratories; thus, widely available.
The measurement of noninvasive fecal elastase has been shown to identify pancreatic insufficiency in CF children with high sensitivity and specificity but remains a third-line diagnostic test for CF because of poor performance in cases of mild and moderate pancreatic insufficiency (53). Other tests associated with CF, but considered unacceptable for diagnosis, include sweat osmolality and conductivity, sweat sodium or potassium, skin precipitation patches, and direct application of chloride electrodes to the skin (48). Sweat conductivity performed with approved equipment, however, is acceptable to screen for CF (48).
On the basis of data from NBS programs, the disease incidence for CF is estimated at 1 in 2000 to 4000 live births, with a prevalence of approximately 30000 individuals in the US population and 70 000 individuals worldwide (54). Current estimates of CFTR mutation carrier frequency in the US is estimated to be 1 in 38 and is expected to increase with improvements in newborn and carrier screening programs. However, the carrier frequency of CF may vary depending on ethnicity (Table 3) (42). Because of the high carrier frequency, the CF Foundation recommends all pregnant couples or couples considering pregnancy undergo CF genetic (carrier) testing. Couples who screen positive are recommended to receive genetic counseling (55). In addition, CF carriers can also be problematic for NBS programs because the detection process does not identify all carriers, particularly newborns who are unaffected heterozygous carriers (56). For this group of patients, additional genetic testing and counseling are necessary to lay the foundation for future potential clinical manifestations.
The investigation of CFTR variants is usually determined by direct DNA mutation analysis. Traditionally, CFTR mutations were analyzed through panel tests that could identify only a set number of known mutations (approximately 5-150 mutations). These panels could then be modified to fit the needs of the local population. The widespread adoption of next-generation sequencing has allowed for a comprehensive check of all nucleotides identified in the coding region of the gene, as well as noncoding regions where known mutations exist. Although sequencing is more sensitive, mutations in the noncoding region and large deletions or duplications in the gene may present diagnostic challenges depending on the laboratory method. In addition, multiple CFTR variants may occur on the same chromosome, e.g., the variant p.Arg117His (R117H) and accompanying polythymidine tract results in a varying genotype-phenotype relationship. With these cases, parent testing may be needed to determine cis/trans variants (57). In general, genetic tests are designed to have high specificity to CFTR variants that are known to cause CF, which are also reflective of the population being tested. Inevitably, because of decreasing cost, genetic sequencing for CF is being widely implemented for screening and diagnosis (44).
HARMONIZED DIAGNOSTIC TERMINOLOGY
Newborns with an inconclusive diagnosis of CF are now recognized with specific terminology as recommended by the CF Foundation guidelines (13). Although the two terms are nearly identical, the expression CFTR-related metabolic syndrome (CRMS) is used in the US for newborns, and the phrase CF screen positive, inconclusive diagnosis (CFSPID) is used in other countries (13, 58-60). Going forward, the combined terminology CRMS/ CFSPID is recommended to describe screen-positive newborns who have either (a) <2 CFTR gene mutations and 2 intermediate sweat chloride results on different occasions or (b) 2 CFTR gene mutations with no more than 1 known CF-causing variant and a normal sweat chloride result (13, 58-60). Individuals with CRMS/ CFSPID may develop signs of CF, such as infertility or pancreatic insufficiency; however, the disease course is much milder in infancy, and others may never develop symptoms. Individuals designated as CRMS/CFSPID should be referred to a CF specialist to monitor the possible development of clinical symptoms of CF and to establish an appropriate plan of care (58, 61). Extended CFTR gene analysis and functional analysis are highly recommended in the CRMS/CFSPID individual (50).
Individuals who have not undergone screening should also be approached with the same recommended diagnostic criteria that are used with the population who has been screened. Patients born before the implementation
of NBS who present with signs or symptoms of CF should have their sweat chloride measured, CFTR genetic analysis performed, and CFTR functional testing assessed to confirm CF. However, the terminology CRMS/ CFSPID should not be used to describe patients who do not meet the diagnostic criteria for CF but for whom the disease cannot be ruled out (13, 49). These patients typically are referred to as having a CFTR-related disorder and may present with clinical sequelae including pancreatitis, respiratory symptoms, chronic sinusitis, or male infertility (62-64).
To avoid confusion among parents, patients, and caregivers, the descriptive terminologies need to be harmonized. Therefore, clinicians and caregivers should avoid use of the terms classic/nonclassic CF, typical/atypical CF, and delayed CF whenever possible, as these terms are considered to have no harmonized definitions (13).
CFTR GENETIC DATABASES
Thousands of CFTR variants have been identified since the CFTR gene was cloned in 1989 (3, 5, 6). Shortly after, the first repository for CFTR variants was created, termed the CFMD (5). The information in the CFMD was constructed through voluntary contributions of clinicians, genetic facilities, and research laboratories and has served as an excellent fountain of CF nucleotide variant information. At first, individuals with outright clinical indications ofCF or complete CFTR dysfunction, as determined by sweat testing, underwent CFTR genotyping. As mutations were found in samples from individuals with CF, they were assumed to be CF-causing. Most of the time, this was an accurate assessment for well-recognized mutations, although several examples of variants were identified that were not independently associated with CF disease. As genetic analysis for CFTR spread to more patients with CF and the overall general population, there grew a need for more consistent annotation of CFTR variants (13).
Overall there are 6 mutation classes that are used to define the defective CFTR protein function (Table 1) (65). The most severe cases of CF were commonly found as a result of class I and II mutations. These patients commonly presented with progressive pulmonary problems and pancreatic insufficiency. Class I mutations are the result of defective CFTR protein production as a result of frameshifts or premature stop codons, whereas class II mutations disrupt protein processing and little or no CFTR protein function is present in the individual. Patients with class III, IV, or V mutations have a less severe CF phenotype because of residual CFTR function. Class III mutations result in the development of a defective channel regulation/gating, whereas class IV and V mutations present with either defective chloride conductance or reduced amounts of functional protein, respectively. Class VI was an additional class proposed that classified CFTR mutations that were contributed to increased protein turnover.
The CFMD has served as an exceptional resource on nucleotide variation in the CFTR gene. However, many believed with significant growth ofgenetic data that there needed to be a consistent and comprehensive approach to assess the functional implications of CFTR variants and the patient's subsequent phenotype (13). To address this task, an international research group was formed by the US CF Foundation to define CF disease severity and annotate the mutations seen in CF registries around the world. As part of the process, the CFTR2 research group has assembled data from CF registries and large clinical data sets from regions all over the world (6). Mutations seen most commonly were ranked by occurrence to prioritize the analysis and annotation. The CFTR2 defined 4 categories of mutations based on clinical criteria, functional analysis, and population/penetrance data. The 4 classes are (a) CF-causing, (b) non-CF-causing, (c) variants of varying clinical consequence, or (d) variants of unknown significance (Table 4). This novel classification has provided a means of differentiating disease burden with the association of specific molecular variants and their phenotypic expression (6, 13).
As of December 2017, 374 variants have been annotated by the CFTR2 project (6). Of those, 312 are considered CF-causing, 36 are variants of varying clinical consequence, 13 are non-CF-causing, and 13 are considered variants of unknown significance. The fourth category lists variants of unknown significance because the analysis for all CFTR variants has not yet been completed, and as more genetic data are being gathered, the project will be constantly updated (6, 13).
Traditional therapies, previously described, include pancreatic enzyme replacement therapy to alleviate the pancreatic insufficiency, prophylactic antibiotic use for the prevention of recurrent respiratory infections, bronchodilators and agents such as DNase to maintain adequate airflow, and chest physiotherapy for secretion clearance.
Advancements in molecular diagnostics and human genome mapping have led to the development of targeted therapies in CF medicine (66). In 2012, the small molecule ivacaftor (VX-770) became the first in an era of genotype-specific therapeutics shown to modulate CFTR function (67). In a subgroup of patients with at least 1 p.Gly551Asp (G551D) (class III mutation) mutation in their CFTR genes (5% of CF cases), ivacaftor showed significant health improvements. The success of this drug has enabled the development of additional targeted therapies used to improve CFTR function (66).
Three major approaches exist for targeted CF treatments known as potentiators, correctors, and production correctors (read-through agents) (68, 69). Potentiators, like ivacaftor, are used for class III or IV CFTR mutations and have been shown to increase CFTR function that is expressed at the surface of epithelial cells (67). Lumacaftor (VX-809), an example of a CFTR corrector, was designed to improve intracellular processing and delivery of mutant CFTR protein associated with class II CFTR mutations (70, 71). This therapy is used to increase the amount of CFTR protein that reaches the cell surface. The last novel compounds known as production correctors (ataluren) allow for more manufacturing of the CFTR protein in class I CFTR mutations (72). These compounds promote read-through of premature termination codons that would otherwise result in a truncated CFTR protein. Based on all these modulator therapies and their specific targets, it is estimated that up to 50% of all CF patients could be treated (73).
The variant p.Phe508del (F508del), caused by the deletion of phenylalanine, is found in approximately 85% of people with CF. Studies with monotherapies of either ivacaftor (potentiator) or lumacaftor (corrector) have shown little success in these patients (68). Because of the F508del, the CFTR protein is misfolded and degraded before significant amounts of protein make it to the cell surface (class II mutation). However, if the mutated protein is expressed (albeit in small amounts) at the membrane, it acts like a gating mutation (class III mutation), such as the G551D mutation. Because of the low risk-to-benefit ratio, ivacaftor combined with lumacaftor or novel tezacaftor (VX-661, corrector) are now both approved for patients [greater than or equal to] 12 years of age with CF and F508del homozygosity (8, 74). The combination therapy of ivacaftor and lumacaftor is also being prescribed for children 6 to 11 years of age with the same homozygous mutation (75).
Gene replacement and editing therapies are being developed as a future treatment of CF. The CRISPR (Classes of Regularly Interspaced Palindromic Repeats) and Cas9 (Crispr-ASsociated) nuclease is an editing system designed to target genes for correcting or modulating their expression (76). With respect to recent CF research, the CRISPR/Cas9 system was shown to repair the function of the CFTR protein in intestinal stem cell organoids of CF patients (77). Strategies using viral and nonviral vectors are currently under investigation (78). However, current gene therapy strategies have been hindered by limited expression of CFTR over time. Although treatment is likely years away, gene replacement and editing therapies are emerging as an important area of discussion
New information about the diagnosis and management of CF is constantly being updated. Newborn and carrier screening programs are evolving to identify children and adults with CF variants earlier in life before the onset of disease. Likewise, recent advancements in harmonized CF nomenclature, the CFTR2 project database, and targeted CF therapies are allowing for novel personalized-medicine approaches in the fight against CF. Sustained and future improvements in CF care will continue to rest on screening and diagnostic testing, as well as access to affordable healthcare.
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contribution to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
Authors' Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest.
(1.) Andersen DH. Cystic fibrosis of the pancreas and its relation to celiac disease. Am J Dis Child 1938;56:344-99.
(2.) Gibson LE, Cooke RE. A test for concentration of electrolytes in sweat in cystic fibrosis of the pancreas utilizing pilocarpine by iontophoresis. Pediatrics 1959;23:545-9.
(3.) Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, Grzelczak Z, et al. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 1989;245:1066-73.
(4.) Bear CE, Li CH, Kartner N, Bridges RJ, Jensen TJ, Ramjeesingh M, Riordan JR. Purification and functional reconstitution of the cystic fibrosis transmembrane conductance regulator(CFTR). Cell 1992;8:809-18.
(5.) Cystic Fibrosis Mutation Database, 2017. http://www. genet.sickkids.on.ca/Home.html (Accessed October 2017).
(6.) US CF Foundation, Johns Hopkins University, Hospital for Sick Children. CFTR2. Clinical and functional translation of CFTR. http://www.cftr2.org/ files/CFTR2_13August2015.pdf (Accessed October 2017).
(7.) Cohen-Cymberknoh M, Shoseyov D, Kerem E. Managing cystic fibrosis: strategies that increase life expectancy and improve quality of life. Am J Respir Crit Care Med 2011;183:1463-71.
(8.) Wainwright CE, Elborn JS, Ramsey BW, Marigowda G, Huang X, Cipolli M, et al. Lumacaftor-ivacaftor in patients with cystic fibrosis homozygous for Phe508del CFTR. N Engl J Med 2015;373:220-31.
(9.) Bell SC, De Boeck K, Amaral MD. New pharmacological approaches for cystic fibrosis: promises, progress, pitfalls. Pharmacol Ther 2015;145:19 -34.
(10.) Agent P, Parrott H. Inhaled therapy in cystic fibrosis: agents, devices and regimens. Breathe2015;11:110-8.
(11.) Burgel PR, Bellis G, Olesen HV, Viviani L, Zolin A, Blasi F, et al. Future trends in cystic fibrosis demography in 34 European countries. Eur Respir J 2015;46:133-41.
(12.) Stephenson AL, Sykes J, Stanojevic S, Quon BS, Marshall BC, Petren K, et al.Survival comparison of patients with cystic fibrosis in Canada and the United States: a population-based cohort study. Ann Intern Med 2017;166:537-46.
(13.) Farrell PM, White TB, Ren CL, Hempstead SE, Accurso F, Derichs N, et al. Diagnosis of cystic fibrosis: consensus guidelines from the Cystic Fibrosis Foundation. J Pediatr 2017;181:S4-15.
(14.) Farrell PM, Rosenstein BJ, White TB, Accurso FJ, Castellani C, Cutting GR, et al. Guidelines for diagnosis of cystic fibrosis in newborns through older adults: Cystic Fibrosis Foundation consensus report. J Pediatr 2008;153:S4-14.
(15.) Stoltz DA, Meyerholz DK, Welsh MJ. Origins of cystic fibrosis lung disease. N EnglJ Med 372;2015:1574-5.
(16.) Welsh MJ, Ramsey BW, Accurso F, Cutting GR. Cystic fibrosis. In: Scriver CR, Beaudet AL, Valle D, Sly WS, editors. The metabolic and molecular basis of inherited disease. 8th ed. New York(NY): McGraw-Hill; 2001. p. 5121-88.
(17.) Rosenfeld M, Ramsey BW, Gibson RL. Pseudomonas acquisition in young patients with cystic fibrosis: pathophysiology, diagnosis, and management. Curr Opin Pulmonol Med 2003;9:492-7.
(18.) Ramsey BW, Pepe MS, Quan JM, Otto KL, Montgomery AB, Williams-Warren J, et al. Cystic Fibrosis Inhaled Tobramycin Study Group. Intermittent administration of inhaled tobramycin in patients with cystic fibrosis. N Engl J Med 1999;340:23-30.
(19.) Saiman L, Marshall BC, Mayer-Hamblett N, Burns JL, Quittner AL, Cibene DA, et al. Macrolide Study Group. Azithromycin in patients with cysticfibrosis chronically infected with Pseudomonas aeruginosa: a randomized controlled trial. JAMA 2003;290:1749-56.
(20.) Rowe SM, Clancy JP. Advances in cystic fibrosis therapies. Curr Opin Pediatr 2006;18:604-13.
(21.) Osman LP, Roughton M, Hodson ME, Pryor JA. Short-term comparative study of high frequency chest wall oscillation and European airway clearance techniques in patients with cystic fibrosis. Thorax 2010;65:196-200.
(22.) Gorter RR, Karimi A, Sleeboom C, Kneepkens CM, Heij HA. Clinical and genetic characteristics of meconium ileus in newborns with and without cystic fibrosis. J Pediatr Gastroenterol Nutr 2010;50:569 -72.
(23.) Kerem E, Corey M, Kerem B, Durie P, Tsui L-C, Levison H. Clinical and genetic comparisons of patients with cystic fibrosis, with or without meconium ileus. J Pediatr 1989;114:767-73.
(24.) Kappler M, Feilcke M, Schroter C, Kraxner A, Griese M. Long-term pulmonary outcome after meconium ileus in cystic fibrosis. Pediatr Pulmonol 2009;44:1201- 6.
(25.) Nousia-Arvanitakis S. Cystic fibrosis and the pancreas: recent scientific advances. J Clin Gastroenterol 1999;29:138-42.
(26.) Brunzell C, Schwarzenberg SJ. Cystic fibrosis-related diabetes and abnormal glucose tolerance: overview and medical nutrition therapy. Diabetes Spectrum 2002;15:124-7.
(27.) Report of the committee for a study for evaluation of testing for cystic fibrosis. J Pediatr 1976;88(4 Pt 2):711-50.
(28.) Sosnay PR, White TB, Farrell PM, Ren CL, Derichs N, Howenstine MS, et al. Diagnosis of cystic fibrosis in nonscreened populations. J Pediatr 2017;181S:S52-7.
(29.) Crossley JR, Elliott RB, Smith PA. Dried blood spot screening for cystic fibrosis in the newborn. Lancet 1979;1:742-4.
(30.) Therrell BL, Hannon WH, Hoffman G, Oiodu J, Farrell PM. Immunoreactive trypsinogen (IRT) as a biomarker for cystic fibrosis: challenges in newborn dried blood spot screening. Mol Genet Metab 2012;106:1-6.
(31.) Comeau AM, Parad RB, Dorkin HL, Dovey M, Gerstle R, Haver K, et al. Population-based newborn screening for genetic disorders when multiple mutation DNA testing is incorporated: a cystic fibrosis newborn screening model demonstrating increased sensitivity but more carrier detections. Pediatrics 2004;113:1573-81.
(32.) Currier RJ, Sciortino S, Liu R, Bishop T, Alikhani Koupaei R, Feuchtbaum L. Genomic sequencing in cystic fibrosis newborn screening: what works best, two-tier predefined CFTR mutation panels or second-tier CFTR panel followed by third-tier sequencing? Genet Med 2017;19:1159-63.
(33.) Kharrazi M, Yang J, Bishop T, Lessing S, Young S, Graham S, et al. Newborn screening for cystic fibrosis in California. Pediatrics 2015;136:1062-72.
(34.) Rock MJ, Hoffman G, Laessig RH, Kopish GJ, Litsheim TJ, Farrell PM. Newborn screening for cystic fibrosis in Wisconsin: nine-year experience with routine trypsinogen/ DNA testing. J Pediatr 2005;147:S73-77.
(35.) Vernooij-van Langen AM, Loeber JG, Elvers B, Triepels RH, Gille JJ, Van der Ploeg CP, et al. Novel strategies in newborn screening for cystic fibrosis: a prospective controlled study. Thorax 2012;67:289-95.
(36.) Sontag MK, Lee R, Wright D, Freedenberg D, Sagel SD. Improving the sensitivity and positive predictive value in a cystic fibrosis newborn screening program using a repeat immunoreactive trypsinogen and genetic analysis. J Pediatr 2016;175:150-8.
(37.) Sommerburg O, Hammermann J, Lindner M, Stahl M, Muckenthaler M, Kohlmueller D, et al. Five years of experience with biochemical cystic fibrosis newborn screening based on IRT/PAP in Germany. Pediatr Pulmonol 2015;50:655- 64.
(38.) Lindau-Shepard BA, Pass KA. Newborn screening for cystic fibrosis by use of a multiplex immunoassay. Clin Chem 2010;56:445-50.
(39.) Wells J, Rosenberg M, Hoffman G, Anstead M, Farrell PM. A decision-tree approach to cost comparison of newborn screening strategies for cystic fibrosis. Pediatrics 2012;129:e339-47.
(40.) Fritz A, Farrell P. Estimating the annual number of false negative cystic fibrosis newborn screening tests. Pediatr Pulmonol 2012;47:207- 8.
(41.) Grody WW, Cutting GR, Klinger KW, Richards CS, Watson MS, Desnick RJ, Subcommittee on Cystic Fibrosis Screening, Accreditation of Genetic Services Committee, ACMG. American College of Medical Genetics. Laboratory standards and guidelines for population-based cystic fibrosis carrier screening. Genet Med 2001;3:149-54.
(42.) Watson MS, Cutting GR, Desnick RJ, Driscoll DA, Klinger K, Mennuti M, et al. Cystic fibrosis population carrier screening: 2004 revision of American College of Medical Genetics mutation panel. Policy statement. Genet Med 2004;6:387-91.
(43.) American College of Obstetricians and Gynecologists and American College of Medical Genetics. Preconception and prenatal carrier screening for cystic fibrosis, clinical and laboratory guidelines. Washington (DC): American College of Obstetricians and Gynecologists; 2001.
(44.) Baker MW, Atkins AE, Cordovado SK, Hendrix M, Earley MC, Farrell PM. Improving newborn screening for cystic fibrosis using next-generation sequencing technology: a technical feasibility study. Genet Med 2016;18:231-8.
(45.) Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 2015;17:405-24.
(46.) LeGrys VA, Applequist R, Briscoe DR, Farrell P, Hickstein R, Lo SF, et al. Sweat testing: sample collection and quantitative chloride analysis; approved guideline. 3rd Ed. CLSI document C34-A3. Wayne (PA): Clinical and Laboratory Standards Institute; 2009.
(47.) Aqil B, West A, Dowlin M, Tam E, Nordstrom C, Buffone G, Devaraj S. Implementation of a quality improvement program to improve sweat test performance in a pediatric hospital. Arch Pathol Lab Med 2014;138:920-2.
(48.) LeGrys VA, Yankaskas JR, Quittell LM, Marshall BC, Mogayzel PJ Jr. Diagnostic sweat testing: the Cystic Fibrosis Foundation guidelines. J Pediatr 2007;151:85-9.
(49.) Rock MJ, Levy H, Zaleski C, Farrell PM. Factors accounting for a missed diagnosis of cystic fibrosis after newborn screening. Pediatr Pulmonol 2011;46:1166 -74.
(50.) Sosnay PR, Salinas DB, White TB, Ren CL, Farrell PM, Raraigh KS, et al. Applying cystic fibrosis transmembrane conductance regulator genetics and CFTR2 data to facilitate diagnoses. J Pediatr 2017;181S:S27-32.
(51.) Farrell PM, White TB, Howenstine MS, Munck A, Parad RB, Rosenfeld M, et al. Diagnosis of cystic fibrosis in screened populations. J Pediatr 2017;181S:S33-44.
(52.) Bagheri-Hanson A, Nedwed S, Rueckes-Nilges, Naehrlich L. Intestinal current measurement versus nasal potential difference measurements for diagnosis of cystic fibrosis: a case- control study. BMC Pulm Med 2014;14:156.
(53.) Daftary A, Acton J, Heubi J, Amin R. Fecal elastase-1: utility in pancreatic function in cystic fibrosis. J Cyst Fibros 2006;5:71- 6.
(54.) Palomaki GE, FitzSimmons SC, Haddow JE. Clinical sensitivity of prenatal screening for cystic fibrosis via CFTR carrier testing in a United States panethnic population. Genet Med 2004;6:405-14.
(55.) Langfelder-Schwind E, Kloza E, Sugarman E, Pettersen B, Brown T, Jensen K, et al. Cystic fibrosis prenatal screening in genetic counseling practice: recommendations of the National Society of Genetic Counselors. J Genet Couns 2005;14:1-15.
(56.) Dungan JS. Carrier screening for cystic fibrosis. Obstet Gynecol Clin North Am 2010;37:47-59.
(57.) Kiesewetter S, Macek M Jr, Davis C, Curristin SM, Che CS, Graham C, et al. A mutation in CFTR produces different phenotypes depending on chromosomal background. Nat Genet 1993;5:274-8.
(58.) Cystic Fibrosis Foundation, Borowitz D, Parad RB, Sharp JK, Sabadosa DA, Robinson KA, et al. Cystic Fibrosis Foundation practice guidelines for the management of infants with cystic fibrosis transmembrane conductance regulator-related metabolic syndrome during the first two years of life and beyond. J Pediatr 2009;155 (6 Suppl):S106 -16.
(59.) Munck A, Mayell SJ, Winters V, Shawcross A, Derichs N, Parad R, et al. Cystic fibrosis screen positive, inconclusive diagnosis (CFSPID): a new designation and management recommendations for infants with an inconclusive diagnosis following newborn screening. J Cyst Fibros 2015;14:706 -13.
(60.) Ren CL, Borowitz DS, Gonska T, Howenstine MS, Levy H, Massie J, et al. Cystic fibrosis transmembrane conductance regulator-related metabolic syndrome and cystic fibrosis screen positive, inconclusive diagnosis. J Pediatr 2017;181S:S45-51.
(61.) Ooi CY, Castellani C, Keenan K, Avolio J, Volpi S, Boland M, et al. Inconclusive diagnosis of cystic fibrosis after newborn screening. Pediatrics 2015; 135:e1377-85.
(62.) Gilljam M, Ellis L, Corey M, Zielenski J, Durie P, Tullis DE. Clinical manifestations of cystic fibrosis among patients with diagnosis in adulthood. Chest 2004;126:1215-24.
(63.) Masaryk TJ, Achkar E. Pancreatitis as initial presentation of cystic fibrosis in young adults. A report of two cases. Dig Dis Sci 1983;28:874-8.
(64.) Marshak T, Rivlin Y, Bentur L, Ronen O, Uri N. Prevalence of rhinosinusitis among atypical cystic fibrosis patients. Eur Arch Otorhinolaryngol 2011;268:519-24.
(65.) Elborn JS. Cystic fibrosis. Lancet 2016;388:2519-31.
(66.) Quon BS, Rowe SM. New and emerging targeted therapies for cystic fibrosis. BMJ 2016;352:1859.
(67.) Ramsey BW, Davies J, McElvaney NG, Tullis E, Bell SC, Drevinek P, et al. A CFTR potentiator in patients with cystic fibrosis and the G551D mutation. N Engl J Med 2011;365:1663-72.
(68.) Brodlie M, Haq IJ, Roberts K, Elborn JS. Targeted therapies to improve CFTR function in cystic fibrosis. Genome Med 2015;7:101.
(69.) Ledford H. Drug bests cystic-fibrosis mutation. Nature 2012;482:145.
(70.) Van Goor F, Hadida S, Grootenhuis PD, Burton B, Stach JH, Straley KS, et al. Correction of the F508del-CFTR protein processing defect in vitro by the investigational drug VX-809. Proc Natl Acad Sci USA 2011;108:18843-8.
(71.) Clancy JP, Rowe SM, Accurso FJ, Aitken ML, Amin RS, Ashlock MA, et al. Results of a phase IIa study of VX-809, an investigational CFTR corrector compound, in subjects with cystic fibrosis homozygous for the F508del-CFTR mutation. Thorax 2012;67:12- 8.
(72.) Sermet-Gaudelus I, Boeck KD, Casimir GJ, Vermeulen F, Leal T, Mogenet A, et al. Ataluren (PTC124) induces cystic fibrosis transmembrane conductance regulator protein expression and activity in children with nonsense mutation cystic fibrosis. Am J Respir Crit Care Med 2010 15;182:1262-72.
(73.) Lopes-Pacheco M. cftr modulators: shedding light on precision medicine for cystic fibrosis. Front Pharmacol 2016;7:275.
(74.) Taylor-Cousar JL, Munck A, McKone EF, van der EntCK, Moeller A, Simard C, et al. Tezacaftor-ivacaftor in patients with cystic fibrosis homozygous for Phe508del. N Engl J Med 2017;377:2013-23.
(75.) Milla CE, Ratjen F, Marigowda G, Liu F, Waltz D, Rosenfeld M. Lumacaftor/ivacaftor in patients aged 6-11 years with cystic fibrosis and homozygous for F508del-CFTR. Am J Respir Crit Care Med 2017;195:912-20.
(76.) Colemeadow J, Joyce H, Turcanu V. Precise treatment of cystic fibrosis-current treatments and perspectives for using CRISPR. Expert Rev Precis Med Drug Dev 2016;1:169-80.
(77.) Schwank G, Koo B-K, Sasselli V, Dekkers JF, Heo I, Demircan T, et al. Functional repairof CFTR by CRISPR/ Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell 2013;13:653-8.
(78.) Burney TJ, Davies JC. Gene therapy for the treatment of cystic fibrosis. Appl Clin Genet 2012;5:29-36.
Joesph R. Wiencek  and Stanley F. Lo  *
 Division of Laboratory Medicine, Department of Pathology, University of Virginia School of Medicine and Health System, Charlottesville, VA;  Department of Pathology, Medical College of Wisconsin and Department of Pathology and Laboratory Medicine, Children's Hospital of Wisconsin, Milwaukee, WI.
* Address correspondence to this author at: Department of Pathology and Laboratory Medicine, Children's Hospital of Wisconsin, 9000 W. Wisconsin Ave., Milwaukee, WI 53226. Fax 414-266-2779; e-mail firstname.lastname@example.org.
The authors declare no competing financial interests. There is no conflict of interest.
Received November 1, 2017; accepted January 17, 2018.
Previously published online at DOI: 10.1373/clinchem.2017.274670
 Nonstandard abbreviations: CF, cystic fibrosis; CFMD, CF Mutation Database; CFTR2, Clinical and Functional Translation of CFTR project; NBS, newborn screening; IRT, immunoreactive trypsinogen; CLSI, Clinical and Laboratory Standards Institute; MVCC, mutations of varying clinical consequences; NPD, nasal potential difference; ICM, intestinal current measurement; CRMS, CFTR-related metabolic syndrome; CFSPID, CF screen positive, inconclusive diagnosis.
 Human gene: CFTR, cystic fibrosis transmembrane conductance regulator.
Caption: Fig. 1. Pathology cascade for cystic fibrosis.
The pathophysiological progression of CF with various types of Food and Drug Administration-approved therapeutic strategies. Modified from Agent et al. (10).
Caption: Fig. 2. Updated testing algorithm for CF diagnosis.
Evidence of disease and CFTR dysfunction is used to diagnosis CF. Sweat testing remains essential for CF diagnosis, followed by CFTR genetic analysis and CFTR physiologic tests. The 2017 CF consensus guidelines recommend all individuals diagnosed with CF should undergo testing for sweat chloride and CFTR genetic analysis. Reproduced from Farrell et al. (13).
Table 1. CFTR protein mutation classification with known therapy. Class Function I No functional CFTR protein II Little or no functional CFTR protein III Defective transport of chloride via regulation orgating IV Defective chloride conductance; some functional CFTR protein V Decreased amount of functional CFTR protein VI Increased functional CFTR protein turnover Class Mutation I G542, R1162X, R553X, W1282X II F508del, N1303 III G551D, G551S, G1349D IV R117H, R347P, R334W V A455E, 3120 + 1 G[right arrow]A, 2789 + 5G[right arrow]A, VI 120del23, N287Y Class Therapy I Production correctors (ataluren) II Corrector plus potentiator (lumacaftor plus ivacaftor, tezacaftor plus ivacaftor) III Potentiator (ivacaftor) IV Potentiator (ivacaftor) V No data available VI No data available Table 2. Sensitivity, specificity, and positive predictive values of various NBS methods for CF. Positive predictive Sensitivity, Specificity, Method value, % % % a 12.5 87.0 99.0 IRT/DNA (F508del) (a,b) 10.0 94.0 99.0 IRT/DNA (CFTR) (a,b) 9.0 99.0 99.0 IRT/PAP (a,c) 12.3 95.0 99.9 IRT/IRT-DNA (a,d) 19.7 96.2 NRe (a) Based on population-specific cutoffs. (b) Rocket al. (34). (c) Vernooij-van Langen et al. (35). (d) Sontag et al. (36). (e) NR depicts not reported in the study. PAP, pancreatitis-associated protein; F508del, F508del mutation. Table 3. Current estimates of CFTR mutation frequency in the US. Ethnicity Frequency Whites 1 in 28 Hispanic Americans 1 in 59 African Americans 1 in 84 Asian Americans 1 in 242 Table 4. Clinical definitions of mutations, from the CFTR2 project. CF-causing Mean sweat chloride [greater than or equal to] 60 mmol/L in patients with the mutation present in trans with a known CF-causing mutation Variants of varying clinical Variants may or not meet CF-causing consequence criteria Variant does not satisfy both clinical and functional criteria (but may satisfy one or the other, or may satisfy neither) Non-CF-causing Clinical evidence not considered Variants of unknown Analysis incomplete significance OR Unable to assign a disease liability characterization
|Printer friendly Cite/link Email Feedback|
|Author:||Wiencek, Joesph R.; Lo, Stanley F.|
|Date:||Jun 1, 2018|
|Next Article:||Noninvasive Detection of Cocaine and Heroin Use with Single Fingerprints: Determination of an Environmental Cutoff.|