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Advances in the Diagnosis and Management of Cystic Fibrosis in the Genomic Era.

Cystic fibrosis (CF) [3] is a life-shortening multisystem disease first described in 1938 (1). The disease presents itself with a classic clinical triad of pancreatic insufficiency, chronic pulmonary disease, and salt loss in sweat; however, other organs may also be involved. It would not be until the 1950s that increased sweat electrolytes (sodium and chloride) would be associated with CF. Around this era, the "sweat test" developed by Gibson and Cooke would become the first diagnostic test for CF (2). Since then, the sweat test has remained the gold standard for CF diagnosis even with recent advances in molecular diagnostics.

CF results from mutations within the CF transmembrane conductance regulator (CFTR) [4] 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

NEWBORN SCREENING

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).

GENETIC ANALYSIS

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).

Treatments

TARGETED THERAPIES

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 EDITING

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

(76).

Summary

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.

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Joesph R. Wiencek [1] and Stanley F. Lo [2] *

[1] Division of Laboratory Medicine, Department of Pathology, University of Virginia School of Medicine and Health System, Charlottesville, VA; [2] 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 slo@mcw.edu.

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

[3] 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.

[4] 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
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