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How do the CFTR alleles interact within an individual with Cystic Fibrosis when mutations of different classes are present?

How do the CFTR alleles interact within an individual with Cystic Fibrosis when mutations of different classes are present?


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So mutations in CF are classified by the severity of the impact on the production of the CFTR.

But an individual may have two different CFTR mutations.

I assume that the least severe mutation of the two is the most relevant to the phenotype. e.g It is more favourable to have a class I and class II mutation that to have 2 class I mutation.

However, I cannot find any references to support this or explain the molecular interaction between classes. Additionally, I fear that it may be more complex than this.

The purpose for knowing this is I want to control for CFTR class in regression analysis but I am removing individuals on ivacaftor or similar so I am talking about the effect purely at the baseline biological level rather than differential access to potentiator therapies.


7 Aug 2014: The PLOS ONE Staff (2014) Correction: CFTR Mutations Spectrum and the Efficiency of Molecular Diagnostics in Polish Cystic Fibrosis Patients. PLOS ONE 9(8): e105738. https://doi.org/10.1371/journal.pone.0105738 View correction

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane regulator gene (CFTR). In light of the strong allelic heterogeneity and regional specificity of the mutation spectrum, the strategy of molecular diagnostics and counseling in CF requires genetic tests to reflect the frequency profile characteristic for a given population. The goal of the study was to provide an updated comprehensive estimation of the distribution of CFTR mutations in Polish CF patients and to assess the effectiveness of INNOLiPA_CFTR tests in Polish population. The analyzed cohort consisted of 738 patients with the clinically confirmed CF diagnosis, prescreened for molecular defects using INNOLiPA_CFTR panels from Innogenetics. A combined efficiency of INNOLiPA CFTR_19 and CFTR_17_TnUpdate tests was 75.5% both mutations were detected in 68.2%, and one mutation in 14.8% of the affected individuals. The group composed of all the patients with only one or with no mutation detected (109 and 126 individuals, respectively) was analyzed further using a mutation screening approach, i.e. SSCP/HD (single strand conformational polymorphism/heteroduplex) analysis of PCR products followed by sequencing of the coding sequence. As a result, 53 more mutations were found in 97 patients. The overall efficiency of the CF allele detection was 82.5% (7.0% increase compared to INNOLiPA tests alone). The distribution of the most frequent mutations in Poland was assessed. Most of the mutations repetitively found in Polish patients had been previously described in other European populations. The most frequent mutated allele, F508del, represented 54.5% of Polish CF chromosomes. Another eight mutations had frequencies over 1%, 24 had frequencies between 1 and 0.1% c.2052-2053insA and c.3468+2_3468+3insT were the most frequent non-INNOLiPA mutations. Mutation distribution described herein is also relevant to the Polish diaspora. Our study also demonstrates that the reported efficiency of mutation detection strongly depends on the diagnostic experience of referring health centers.

Citation: Ziętkiewicz E, Rutkiewicz E, Pogorzelski A, Klimek B, Voelkel K, Witt M (2014) CFTR Mutations Spectrum and the Efficiency of Molecular Diagnostics in Polish Cystic Fibrosis Patients. PLoS ONE 9(2): e89094. https://doi.org/10.1371/journal.pone.0089094

Editor: Klaus Brusgaard, Odense University Hospital, Denmark

Received: September 14, 2013 Accepted: January 15, 2014 Published: February 26, 2014

Copyright: © 2014 Ziętkiewicz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by the grant from Polish Scientific Committee KBN-NN401-020-435 to EZ. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.


Targeted therapies in respiratory medicine — cystic fibrosis as a paradigm

Targeted therapies have evolved in medicine following advances in molecular technology and the successful mapping of the human genome. Such treatments are well recognized in oncology, where molecules required for tumor growth and spread are specifically targeted to stop the malignant process or prevent tumor progression [1, 2].

These therapies have been driven by the concepts of precision and stratified medicine, whereby molecular biomarkers can be used to select specific approaches for individuals or groups of individuals, respectively, enabling the production of highly effective and precise treatments [3]. Some of the advantages of targeted therapies include the ability to identify treatment responders, tailor treatment to an individual’s genetic profile, and avoid unwanted side effects [4]. This approach is in direct contrast to most drugs currently used in medical practice, which are used to treat large populations with the same broad disease label but with marked heterogeneity in response to treatment.

Recent advances in genome-wide association studies and an increased understanding of the genetic basis of complex diseases have enabled the concept of targeted therapies to be investigated in other areas, such as respiratory medicine. However, there are few examples of targeted therapies in this field outside of oncological problems, as most lung diseases are complex and polygenic. Therefore, developing strategies for specific molecular abnormalities in these conditions is challenging. An exception, however, is cystic fibrosis, in which the underlying genetic defect is well defined and lies within the CFTR gene [5]. The use of ivacaftor, a potentiator of CFTR function, has become a successful reality since 2012 as a targeted therapy for patients with cystic fibrosis caused by specific genotypes, and represents a powerful example of precision medicine [6]. Furthermore, the combination of a potentiator and a corrector (ivacaftor and lumacaftor) received US Food and Drug Administration (FDA) approval in 2015 for use in people with cystic fibrosis caused by the most common CFTR mutation, Phe508del [7, 8].

In this review we discuss the clinical and genetic basis of cystic fibrosis, the development of treatments targeted at specific classes of CFTR mutation to address the basic defects and improve CFTR function, and the advent of precision medicine in cystic fibrosis as a paradigm for other respiratory diseases.


Application of HindIII and EcoRI Restriction Endonucleases in Identifying and Diagnosing Cystic Fibrosis caused by the CFTR ∆F508 Mutation

Cystic fibrosis (CF) is caused by a mutation on the CFTR protein preventing transport of salts across epithelial cell surfaces leading to mucus hyperproduction and eventually death. The purpose of this experiment was to determine if a 3-year old patient has cystic fibrosis. The hypothesis stated that Jeff would have cystic fibrosis caused by the CFTR ∆F508 mutation. It was predicted that Jeff’s DNA and the positive/negative controls would be cut by EcoRI two times producing three bands with the sizes 2,150 bp, 2,150 bp, and 4,700 bp and that Jeff’s DNA and the positive control would be cut by HindIII once producing two bands with the sizes 7,200 bp and 1,800 bp. The experiment used EcoRI and HindIII restriction endonucleases in RFLP analysis and visualized the results with electrophoresis on an agarose gel, the molecular size of these DNA fragments was calculated from an equation produced from a standard curve graph. The DNA of a patient with the CFTR ∆F508 mutation was included as the positive control and the DNA of a patient without the CFTR ∆F508 mutation was included as the negative control. The results of the experiment showed that all of the DNA samples cut by EcoRI produced the similar-sized DNA fragments, and Jeff’s DNA and the positive control were cut nearly identically by HindIII, while the negative control was cut differently. These results led to the acceptance of the hypothesis, meaning that Jeff was diagnosed with cystic fibrosis caused by the CFTR ∆F508 mutation.

Introduction

In healthy individuals, the body responds to respiratory infections by increasing mucus levels in the lungs and respiratory tract. However, this mucus can also trap bacteria and foreign material so the cystic fibrosis transmembrane conductance regulator (CFTR) protein transports salts including chlorine ions, bicarbonate ions, and anions across epithelial lung cells to hydrate the lungs and clear away excess mucus (Gentzsch, 2018 Southern et al. 2018).

However, there are over 2000 mutations of the CFTR gene that can result in the deadly disease Cystic Fibrosis (Southern et al. 2018). These CFTR mutations are grouped into five different classes based on how they affect the CFTR protein: Class I mutations produce a dysfunctional CFTR protein by adding an early stop codon to the genetic sequence, Class II mutations produce an abnormal CFTR protein most of which is degraded by the cell before it reaches the lung lining, Class III mutations produce CFTR proteins that cannot transport ions across epithelial lung cells, Class IV mutations produce impaired CFTR proteins that have a difficult time transporting ions across epithelial lung cells, Class V mutations produce a functional CFTR protein but in lower numbers than usual so this reduces its efficiency (Southern et al. 2018).

The most common of the CFTR mutations, responsible for 90% of Cystic Fibrosis cases (Cooney, 2018) is a Class II defect called the ∆F508 mutation, this specific mutation occurs when phenylalanine is deleted at position 508 of the CFTR gene (Suaud et al. 2011). This is a very common and lethal autosomal recessive disease that affects 1 in 2000 North Americans or 70,000 individuals globally (Cutting, 2015 Southern et al. 2018). This disease is lethal due to a dysfunctional CFTR protein which means that mucus builds up in the lungs and pancreatic ducts which traps bacteria, weakens the immune system, damages organs, causes inflammation, and often leads to diabetes and/or malnutrition, usually, patients die from respiratory failure (Cutting, 2015 Southern et al. 2018). Fortunately, there are several treatments to mitigate the effects of this disease and extend the life of affected individuals, and since cystic fibrosis is only caused by mutations on the CFTR gene it is relatively easy to test and diagnose patients (Cutting, 2015).

These single-gene autosomal recessive disorders (Cutting, 2015) create a type of genetic variation of the CFTR gene within a population called polymorphism (Pare, 2012). One of the most common ways for diagnosing single-mutation polymorphisms is with a technique called restriction fragment length polymorphism (RFLP) analysis, which uses restriction endonucleases to cut DNA sequences into fragments at specific sites to aid researchers in identifying genetic variations (Loenen et al. 2014 Sapienza, 2012). Restriction endonucleases are enzymes that are naturally produced in several species of prokaryotes but have many applications in laboratory genetic experiments (Pingoud et al. 2014). Restriction endonucleases or REases are grouped into four categories (Type I, Type II, Type III, and Type IV) the most commonly used in genetic testing are Type II REases which function by cleaving the phosphate bands on or near the recognition sequence in the DNA, this process produces consistent DNA fragments (Pingoud et al. 2014 Sapienza, 2012).

Two of the most understood of the Type II REases include EcoRI and HindIII: EcoRI which was discovered from Escherichia coli and HindIII was discovered from Haemophilus influenzae rd (Pingoud et al. 2014). Specifically, EcoRI and HindIII locate their specific staggered nucleotide recognition sequences and cleave the phosphate bonds between the nucleotides: EcoRI recognizes GAATTC and cleaves the phosphate bond between the G and A, additionally, it will recognize and cut any sequences that differ by one base-pair, HindIII recognizes AAGCTT and cleaves the phosphate bond between the two A’s (Loenen et al. 2014 Sapienza, 2012). Additionally, since these two REases produces symmetrical staggered cuts, the DNA fragments can anneal to their complementary strand this has been exploited for genetic cloning applications (Loenen et al. 2014). When EcoRI is added to the blood or saliva DNA sample of a patient either with or without the CF∆F508 mutation, the EcoRI cleaves the CFTR gene twice to produce three fragments that are 2,150 bp, 2,150 bp, and 4,700 bp. However, HindIII produces different results when added to the blood or salvia DNA sample of patients with and without the CF∆F508 mutation. In patients without the CF∆F508 mutation, HindIII cleaves the CFTR gene twice to produce three fragments that are 1,500 bp, 5,700 bp, and 1,800 bp. On the other hand, in patients with the CF∆F508 mutation, HindIII does not identify the recognition sequence at 1,500 bp because of the phenylalanine deletion at position 508 messes up the rest of the sequence, so instead HindIII cleaves the CFTR gene only to produce two fragments that are 7,200 bp and 1,800 bp. The reason that the HindIII produces different results in patients with and without the mutation, while EcoRI produces identical results is that HindIII is more specific and can only identify exact recognition sequences whereas EcoRI is slightly less specific and can identify slightly varied sequences. The results of RFLP analysis can be analyzed with electrophoresis on an agarose gel, a fluorescent dye is added to the DNA samples so that it fluoresces under a UV transilluminator to reveal the distance that the fragments have traveled. The distance that the samples travel is proportional to their length, therefore, the distances that marker DNA fragments travel can be used to create an equation from a standard curve that will use the distances that the other DNA samples traveled to determine their molecular size.

This experiment is going to be testing an adopted 3-year old named Jeff for Cystic Fibrosis caused by the CFTR ∆F508 mutation. To determine if Jeff has cystic fibrosis his DNA will undergo RFLP analysis with the restriction endonucleases EcoRI and HindIII. A positive control (patient with the ∆F508 mutation) and negative control (patient without the ∆F508 mutation) will also undergo RFLP analysis with EcoRI and HindIII so that Jeff’s results can be compared to something. The results of the RFLP analysis will be visualized by electrophoresis on an agarose gel and then the equation produced from a standard curve will be used to calculate the molecular sizes of the DNA fragments after they are cut by the REases. Determining if Jeff has cystic fibrosis and what type of mutation his cystic fibrosis is caused by is important so he can receive the best treatment so that he can live a pain-free and extended life.

It is hypothesized that Jeff has cystic fibrosis because he displays several of the symptoms associated with cystic fibrosis including wheezing, crackling, persistent cough, greasy stools, and a runny nose. Furthermore, because cystic fibrosis caused by a CFTR ∆F508 mutation is an autosomal recessive disorder it is possible that both his parents were carriers of the mutation and that he inherited two mutated alleles, this explains why his parents would not have a medical history of cystic fibrosis.

If this hypothesis is correct and Jeff does have cystic fibrosis caused by the CFTR ∆F508 mutation, then it is predicted that when his DNA will be cut by EcoRI two times to produce three bands that have molecular weights of 2,150 bp, 2,150 bp, and 4,700 bp and that his DNA will be cut by HindIII once to produce two bands that have molecular weights of 7,200 bp and 1,800 bp. Also, the controls (patients with and without the ∆F508 mutation) will also be cut by EcoRI two times to produce three bands that have molecular weights of 2,150 bp, 2,150 bp, and 4,700 bp. Therefore, Jeff’s DNA and the patients with and without the ∆F508 mutation will have bands with identical molecular weights when cut by EcoRI. Additionally, the positive control (patient with the ∆F508 mutation) will also be cut by HindIII once to produce two bands that have molecular weights of 7,200 bp and 1,800 bp. Whereas, the negative control (patient without the ∆F508 mutation) will be cut twice to produce three bands that have molecular weights of 5,700 bp, 1,800 bp, and 1,500 bp. This means that the bands that appear when Jeff’s DNA sample is cut with HindIII should be identical in weight to the patient with the ∆F508 mutation whose DNA was also cut with HindIII, and both of these samples should be different than when HindIII is used with the patient who does not have the ∆F508 mutation.

Materials

To begin, six DNA samples were obtained: two samples from the patient being tested for cystic fibrosis (Jeff), two samples from an individual with the ∆F508 mutation, and two samples from an individual without the ∆F508 mutation. The samples from the individual with the ∆F508 mutation had previously been cut with EcoRI and HindIII respectively, nothing else was added to these samples until the loading dye was added. Reaction buffer was combined with the other samples, and then EcoRI was added to one DNA sample of the individual without the ∆F508 mutation and to one of Jeff’s DNA sample. Next, HindIII was added to one DNA sample of the individual without the ∆F508 mutation and to one of Jeff’s DNA sample. These four samples were incubated at 37 degrees Celsius for thirty minutes. A solution of 0.8% agarose gel was prepared with agarose, 1x TAE buffer, and 10,000x Sybr Safe DNA gel stain. This solution was poured into a gel tray locked into a casting rack, a comb was added and then the gel was placed in a refrigerator for thirty minutes to solidify. The comb was removed and then the gel tray with solid gel was lowed into the electrophoresis chamber and submerged in 1x TAE buffer. A loading dye containing Ficol was added to each of the six DNA samples. A marker was loaded into the first well/lane to display bands of the following molecular sizes: 12,000 bp, 7,000 bp, 3,000 bp, 2,500 bp, 2,000 bp, 1,800 bp, and 1,500 bp. Then the six DNA samples that had been cut with either EcoRI or HindIII were loaded into separate wells. The cover was placed on the electrophoresis chamber so that the charge would run from the negative to the positive end, the apparatus was allowed to run for 45 minutes at 120V until the due was half-way through the gel. The gel was removed from the apparatus and viewed on the UV transilluminator next to a ruler so that the distance that each dye traveled could be measured. A standard curve graph was created with the data from the measurements of the marker. The equation that this graph generated was used to approximate the molecular size of the bands from the six DNA samples. (DeCicco-Skinner, 2019).

Results

Figure 1: Displaying the gel on top of the UV transilluminator next to a metric ruler. Lane 1 contained the marker and produced 7 visible bands, lane 2 contained -E (DNA of the patient without the ∆F508 mutation, cut with EcoRI), lane 3 contained +E (DNA of the patient with the ∆F508 mutation, cut with EcoRI), lane 4 contained JE (Jeff’s DNA cut with EcoRI), lane 5 contained -H (DNA of the patient without the ∆F508 mutation, cut with HindIII), lane 6 contained +H (DNA of the patient with the ∆F508 mutation, cut with HindIII), lane 7 contained JH (Jeff’s DNA cut with HindIII). Lanes 2, 3, and 4 each produced two bands that were nearly identical across the three wells. Lane 5 produced four bands. Lanes 6 and 7 produced two bands that were nearly identical.

The distance the bands traveled on the gel was measured from the distance between the well and the bottom of the band. The marker from lane 1 contained bands of several molecular weights: the 12,000 bp band traveled 21.5 mm, the 7,000 bp band traveled 26.5 mm, the 3,000 bp band traveled 29.2 mm, the 2,500 bp band traveled 34.4 mm, the 2,000 bp band traveled 37.9 mm, the 1,800 bp band traveled 40.5 mm, and the 1,500 bp band traveled 44.1 mm (Figure 1). Lane 2 was loaded with the DNA of the patient without the ∆F508 mutation that was cut with EcoRI, lane 2 displayed two bands: one traveled 28.1 mm and the other traveled 35.2 mm (Figure 1). Lane 3 was loaded with the DNA of the patient with the ∆F508 mutation that was cut with EcoRI, lane 3 had two bands as well: one traveled 27.8 mm and the other 36.1 mm (Figure 1). Lane 4 was loaded with Jeff’s DNA and had been cut with EcoRI, lane 4 also had two bands in nearly the same location as the previous two wells: one band traveled 28.0 mm and the other traveled 35.9 mm (Figure 1). Lane 5 was loaded with the DNA of the patient without the ∆F508 mutation that was cut with HindIII, lane 5 had four bands traveling 27.8 mm, 35.8 mm, 42.3 mm, and 44.1 mm (Figure 1). Lane 6 was loaded with the DNA of the patient with the ∆F508 mutation that was cut with HindIII, lane 6 had two bands traveling 26.1 mm and 43.5 mm (Figure 1). Lane 7 was loaded with Jeff’s DNA and had been cut with HindIII, lane 7 had two bands that were very similar to lane 6, traveling 26.0 mm and 43.5 mm (Figure 1).

Figure 2: Displaying the Standard Curve for CFTR ∆F508 mutation restriction digestion. The log of the molecular weight of each marker band was plotted on the y-axis, and the distance in millimeters that each band migrated from the well that the marker was loaded into was plotted on the x-axis. From this data a linear trendline was added, the equation of the line of best fit was calculated and displayed (y=-0.0391x + 4.8133), and the coefficient of determination was calculated and displayed (R^2=0.8987).

The measurements that were collected from Figure 1 were used to create the standard curve graph in Figure 2. Adding a linear trendline produced an equation of the line of best-fit (y=-0.0391x + 4.8133) and yielded a 0.8987 coefficient of determination. The distance that each band traveled for the six samples was plugged into this equation and then the antilog was taken to calculate the approximate band size.

Table 1: CFTR ∆F508 mutation restriction digestion by EcoRI and HindIII results for Jeff, patient with the CFTR ∆F508 mutation, and patient without the CFTR ∆F508 mutation.
Table 1: Displaying the expected vs observed molecular weight for the marker and six DNA samples cut with EcoRI or HindIII. The molecular weight for each sample was calculated from the standard curve equation (y=-0.0391x + 4.8133). Lanes 2, 3, and 4 had bands of very similar sizes and the values were close to what was expected. Lane 5 had more bands than were expected. Lane 6 and 7 had bands that were very similar and close to what was expected.

From the equation in Figure 2 the observed band sizes were calculated for the six DNA samples and recorded in Table 1. The observed band sizes were also calculated for the marker so that the amount of error could be quantified. Table 1 shows that the calculated molecular weight is different than the expected molecular weight, this difference is most notable when the band has more base pairs. This means that the observed band size for the DNA samples should not exactly match the expected band size and that the value will be most accurate for bands with fewer base pairs. For the three DNA samples cut with EcoRI (the patients with (+E) and without (-E) the CFTR ΔF508 mutation, and Jeff (JE)) it was expected that the EcoRI would cut the DNA strand twice to produce three bands: two bands that were 2,150 bp and one band that was 4,700 bp. However, observation of these samples revealed only two bands. Patient -E had calculated bands of 2,500 bp and 5,183 bp patient +E had calculated bands of 2,522 bp and 5,325 bp patient JE had calculated bands of 2,568 bp and 5,229 bp. These values are very close and are essentially identical. The patient without the CFTR ΔF508 mutation (-H) whose DNA was cut with HindIII was expected to be cut twice to produce three bands: 1,500 bp, 5,700 bp, and 1,800 bp. However, four bands were observed indicating the DNA was cut in four places. This produced bands that were calculated to be 1,227 bp, 1,443 bp, 2,591 bp, and 5,325 bp. The patient with the CFTR ΔF508 mutation (+H) and Jeff (JH) whose DNA was cut with HindIII was expected to be cut once to produce two bands that were 7,200 bp and 1,800 bp. In actuality, these two samples were cut once, in the +H patient the band sizes were 6,206 bp and 1,296 bp, whereas in the JH patient the band sizes were 6,262 bp and 1,296 bp. These values are so similar that the band sizes could be considered identical.

Discussion

With over 2000 polymorphisms of cystic fibrosis mutations, there is certainly a lot that can go wrong for the CFTR gene. In the example of the CFTR ∆F508 mutation, a single amino acid (phenylalanine) is deleted at position 508 of the gene (Suaud et al. 2011). The deletion of phenylalanine at this position is so severe that the CFTR protein becomes dysfunctional, resulting in hyperproduction of mucus across epithelial surfaces (Kreda et al. 2012). The CFTR protein has been identified through several experiments as a cyclic adenosine monophosphate-dependent phosphorylation (cAMP) activated anion channel that transports salts (chloride ions and bicarbonate ions) and other anions across epithelial cells (Gentzsch, 2018). Healthy, fully functional CFTR proteins transport these salts across the plasma membrane of epithelial cells that line the lungs and other organs to clear away excess mucus by hydrating the surface (Gentzch, 2018 Suaud et al. 2011). In the case of the CFTR ∆F508 mutation, an abnormal CFTR protein which cannot fold properly is produced the protein cannot fold because the section of CFTR that interacts with ATP to bind nucleotides called the nucleotide-binding domain (NBDI) and the fourth cytosolic loop within another section of CFTR that anchor other protein into the plasma membrane (MSD 2) form hydrogen bonds with the arginine amino acid at the 1070 codon (Cutting, 2015). The cell detects the misfolded CFTR proteins and degrades most of them within the endoplasmic reticulum (Suaud et al. 2011). This means that very little or none of the CFTR protein reaches the surface of the epithelial cells therefore, chloride and bicarbonate ions cannot be transported across the plasma membrane (Suaud et al. 2011). Since these salt ions cannot be transported across the plasma membrane then the salts are in higher concentration on the basal side of the epithelial cells, this draws water away from the apical surface and leads to dehydration of the lung, gastrointestinal, and pancreatic surfaces (Gentzsch, 2018). When there is no water on the apical surfaces of these organs excess mucus cannot be cleared away and then dense sticky mucus creates obstructions and leads to respiratory illnesses and inflammation that eventually cause death in patients (Gentzsch, 2018).

This experiment tested 3-year old Jeff for Cystic Fibrosis caused by the CFTR ∆F508 mutation using RFLP analysis with EcoRI and HindIII restriction endonucleases. His results were compared to a positive control (patient with the ∆F508 mutation) and a negative control (patient without the ∆F508 mutation) who also were included in the RFLP analysis with EcoRI and HindIII so that Jeff’s results can be compared to something. The results of the RFLP analysis were visualized with electrophoresis on agarose gel (Figure 1) and then an equation was produced from a standard curve (Figure 2) which was used to calculate the molecular sizes (Table 1) of the DNA fragments that the restriction endonucleases produced. This early diagnosis is critical to ensuring that Jeff receives the care he needs so he can live a painless and long life. Furthermore, RFLP analysis is a much more reliable method to test for autosomal recessive disorders than direct-to-consumers genetic tests such as 23andMeTM because there is less room for misunderstandings. Companies like 23andMeTM measure genetic variation by comparing the customer's genetic information to their database of mutations, this database can detect 715,000 single nucleotide mutations (Lu et al. 2017). This means that the database would detect the three-nucleotide deletion that occurs when phenylalanine is deleted at position 508 of the CFTR gene. RFLP is different than this because it does not compare genetic sequences, rather it uses restriction endonucleases to cut DNA into fragments at recognition sequences, in this case, cystic fibrosis could be diagnosed if the DNA of a patient was cut at the same locations as a positive control. One advantage to genetic tests like those provided by 23andMeTM is that they can also tell if a patient is a carrier of a recessive mutation, this is something that RFLP analysis cannot do (Lu et al. 2017). However, one of the largest concerns with direct-to-consumer genetic testing is that most people do not know how to interpret the results and may take drastic behaviors as a result of not receiving genetic counseling (Pare, 2012), this is another reason why it is important to test for cystic fibrosis with RFLP instead of using 23andMeTM.

It was hypothesized that Jeff has cystic fibrosis caused by the CFTR ∆F508 mutation because he displays several of the symptoms associated with the disease. This would be possible if both of his parents were carriers of the CFTR ∆F508 mutation because carriers do not have symptoms since the disease only occurs when both alleles on this location of the CFTR gene are mutated. It was predicted that when Jeff’s DNA would be cut by EcoRI two times producing three bands with the sizes 2,150 bp, 2,150 bp, and 4,700 bp and that his DNA would be cut by HindIII once producing two bands with the sizes 7,200 bp and 1,800 bp. Also, the EcoRI samples (patients with and without the ∆F508 mutation) would be cut by EcoRI two times in an identical manner to Jeff’s DNA producing three bands with the sizes 2,150 bp, 2,150 bp, and 4,700 bp. Meaning that Jeff’s DNA and the DNA of the patients with and without the ∆F508 mutation should have bands identical in size when cut by EcoRI. Additionally, the positive control (patient with the ∆F508 mutation) would also be cut by HindIII once, similar to Jeff’s DNA producing two bands with sizes 7,200 bp and 1,800 bp. This is in contrast to the negative control (patient without the ∆F508 mutation) whose DNA was predicted to be cut twice, producing three bands with sizes 5,700 bp, 1,800 bp, and 1,500 bp. If the hypothesis is correct then the bands that appear when Jeff’s DNA sample is cut with HindIII would be identical in weight to the patient with the ∆F508 mutation whose DNA was also cut with HindIII, and both of these samples should be different than when HindIII was used with the patient who does not have the ∆F508 mutation.

The purpose of including the positive control was so that the HindIII restriction digestion results of an individual with ∆F508 mutation can be visualized and then compared to how Jeff’s DNA was cut, if they were cut the same way then it means he also has the same CFTR polymorphism. If his DNA is cut differently than the positive control, then it would mean he does not have the ∆F508 mutation. The purpose of including the negative control was so that the HindIII restriction digestion results of an individual without ∆F508 mutation can be visualized and then compared to how Jeff’s DNA was cut. If the negative control was cut differently than both the positive control and Jeff’s DNA then it would mean that he does not have the ∆F508 mutation. However, if Jeff’s results did not match either the positive or negative it would mean that the results of the experiment are invalid. Also, if the positive and negative controls are identical when cut by HindIII then the experimental results would be invalid. Furthermore, even though the results of the RFLP analysis will be the same for Jeff and the controls when cutting with EcoRI, the EcoRI was still included because if this did not produce identical results for all of the samples then the results of the experiment would have to be called into question.

The marker that was loaded into lane 1 successfully moved across the gel (Figure 1) and produced bands at 12,000 bp, 7,000 bp, 3,000 bp, 2,500 bp, 2,000 bp, 1,800 bp, and 1,500 bp as was expected (Table 1). The distance that each band migrated was measured and this data was plotted against the log of each molecular weight to produce a standard curve that yielded a 0.8987 coefficient of determination and line of best fit with the equation y=-0.0391x +4.8133 (Figure 2). The distance that each band traveled for the six samples was plugged into this equation and then the antilog was taken to calculate the approximate band size. This equation was used to calculate the size of the markers as well to determine how much mathematical error is present in this model. Table 1 shows that the calculated molecular weight is slightly different than the actual molecular weight, this effect is amplified when the DNA has more base pairs. Meaning that the results of the other DNA samples could also have some variation in the expected vs observed molecular weight of the bands and that even with such variation the results would still be valid. This equation was also used to calculate the molecular sizes of the bands shown on the gel in Figure 1. If the hypothesis is correct then Jeff’s DNA, the patient with the ∆F508 mutation, and the patient without the ∆F508 mutation would all be cut twice by EcoRI to produce bands with molecular weights of 2,150 bp, 2,150 bp, and 4,700 bp. Since two of these bands are the same molecular weight then only two bands would appear on the gel: 2,150 bp and 4,700 bp. Observation of these DNA samples showed that they all have two bands (Figure 1 – lanes 2,3,4) and calculations from the standard curve equation revealed that the molecular weight of these two bands in the three samples were very similar: in JE the sizes of the DNA fragments were calculated as 2,568 bp and 5,229 bp in -E the sizes of the bands were calculated as 2,500 bp and 5,183 bp in +E the sizes of the bands were calculated as 2,522 bp and 5,325 bp (Table 1). Since the EcoRI cut all of the samples in approximately the same place then it can be concluded that the RFLP analysis worked correctly and that the other results of the experiment should have also worked correctly. This also means that the CFTR gene was actually present in all of the DNA samples because if any of the samples lacked the CFTR gene the samples would not have produced identical DNA fragments. However, these results alone cannot provide a diagnosis for cystic fibrosis because the EcoRI enzyme cuts the positive and negative control the same way. If EcoRI and the positive control had been the only things used to diagnose Jeff, then the hypothesis would have been accepted because the CFTR gene is cut into the same DNA fragments for the positive control and Jeff. Paradoxically, if EcoRI and the negative control had been the only things used to diagnose Jeff then the hypothesis would have been rejected because the CFTR gene is cut into the same DNA fragments for the negative control and Jeff. Obviously, this is problematic because the hypothesis cannot both be accepted and rejected, therefore, to accurately diagnosis Jeff with cystic fibrosis a restriction endonuclease that cuts the positive and negative control differently must be used, this is why HindIII was used as well.

If the hypothesis was correct then Jeff’s DNA and the (positive control) patient with the ∆F508 mutation would be cut once by HindIII to produce bands with molecular weights of 7,200 bp and 1,800 bp. Observation of these DNA samples showed that they both were cut once and had two bands (Figure 1 – lanes 6 and 7) and calculations from the standard curve equation revealed that the molecular weight of these two bands in both samples was similar: in JH the sizes of the bands were calculated as 6,262 bp and 1,296 bp and in +H the sizes of the bands were calculated as 6,206 bp and 1,296 bp (Table 1). Since the HindIII cut both Jeff’s DNA and the DNA of the patient who has the ∆F508 mutation it is likely that Jeff also has the ∆F508 mutation which causes cystic fibrosis. To confirm that these results are valid both these samples must be compared to the negative control – the patient without the CFTR ∆F508 mutation. Observation revealed that the DNA of the patient (-H) without the ∆F508 mutation was cut three times by HindIII to produce four bands (Figure 1) and these bands were calculated to have the molecular weights of 1,227 bp, 1,443 bp, 2,591 bp, and 5,325 bp (Table 1). Obviously, these results do not match the prediction which stated that this patient’s DNA would be cut twice by HindIII to produce three bands with the molecular weights of 5,700 bp, 1,800 bp, and 1,500 bp. However, the top two bands appear to align with the bands from the samples cut with EcoRI. Meaning that this sample was likely contaminated with some EcoRI. This interferes with the analysis of the results however, some conclusions can still be drawn due to the placement of these bands in relation to the other HindIII samples. First, the results are different enough that it can be concluded that JH and +H were identical whereas -H was different. Second, the -H sample has a band that is below the lowest band in +H and JH (Figure 1– lanes 5, 6, 7). This would make sense if the hypothesis was correct because -H should have its smallest band be 1,500 bp and +H/JH should have their smallest bands at 1,800 BP (Table 1). Third, the only way that Jeff’s DNA sample could be cut by HindIII at 7,200 bp would be if he had the CFTR ∆F508 mutation (Figure 1 and Table 1). Therefore, the hypothesis is accepted, Jeff has cystic fibrosis caused by the CFTR ∆F508 mutation.

If the positive control had been omitted then the diagnosis would be very difficult to make and the hypothesis could not be confirmed, because the calculated molecular size of Jeff’s DNA fragments is different than the expected molecular sizes. Observing that the calculated molecular size of the also differed from the expected size for the positive control’s DNA fragments and that the molecular size of these fragments and Jeff’s were nearly identical when cut by HindIII was the ultimate factor that led to accepting the hypothesis. If the negative control had been omitted, then the hypothesis would have still been accepted because Jeff’s DNA would have still matched the positive control. However, in the alternative scenario where he did not have cystic fibrosis, missing a negative control would mean that it would be impossible to confirm that Jeff did not have cystic fibrosis because his DNA fragments would not have matched anything when cut by HindIII.

This experiment only used the segment of Jeff’s genome that included the CFTR gene. If we had used his entire genome of a patient with or without the mutation then EcoRI and HindIII would have located many more recognition sites (GAATTC and AAGCTT respectively) and cleaved phosphate groups at these sites, this would produce many more DNA fragments. In fact, so many DNA fragments that it would be difficult to tell what you were looking at, Instead of seeing a few bands on Figure 1 instead there might be hundreds of bands. This would make it impossible to identify whether or not Jeff had a mutation in CFTR. The lab protocol would need to be modified if entire genomes were used so that a specific region of DNA could be examined. One way to achieve this is through southern blotting: where the DNA fragments from electrophoresis are transferred to a membrane by upward capillary transfer and then immobilized, allowing for the bands matching the CFTR sequence to be identified with a probe (Brown, 2001).

In conclusion, these findings are significant because it reveals that HindIII is a very useful restriction endonuclease for diagnosing cystic fibrosis and Jeff’s cystic fibrosis diagnosis (caused by the CFTR ∆F508 mutation) means that he can receive personalized-treatment, lumacaftor, for example, is a drug that might benefit Jeff by preventing the degradation of the misfolded CFTR proteins caused by the CFTR ∆F508 mutation, this helps the proteins reach the apical epithelial cell surface so that function can be partially restored (Kreda et al. 2012). This experiment could be improved doubling the number of samples to ensure that the samples have not been contaminated by the incorrect restriction endonuclease and by using a different trendline that produces an equation that calculates molecular sizes with improved accuracy. Future studies can explore the application of other restriction endonucleases such as BamHI, EcoRII, Hand HaeIII to see how they cut the CFTR in the positive and negative controls.

References

Brown, T (2001) Southern Blotting. Curr Protoc Immunol 10(6a).

Cooney, A.L., P.B. McCray Jr, and P.L. Sinn (2018) Cystic Fibrosis Gene Therapy: Looking Back, Looking Forward. Genes (Basel) 9(11).

Cutting, G. R. (2015) Cystic fibrosis genetics: from molecular understanding to clinical application. Nat Rev Genet 16(1): 45-56.


3 FROM DIAGNOSIS VIA SYMPTOMS TO CF NEWBORN SCREENING

If CF goes undiagnosed and untreated in early life, severe bronchiectasis can already be present at diagnosis. Despite symptoms like chronic coughs, poor weight gain or complications due to malnutrition, the median age at diagnosis ranges from a few months to several years. 4 Making the diagnosis after a few weeks of life is already too late for optimal outcomes. 22 In CF, prevention of lung disease progression and complications is key, but it still took many decades to prove that CF was a disease that fitted the criteria for newborn screening. We now know that the benefits of CF newborn screening outweigh the harm and that CF newborn screening leads to better survival. 23

Cystic fibrosis newborn screening started with measuring serum immunoreactive trypsinogen in a dry blood spot. But this strategy had a low positive predictive value, of around 10%, and patients had to be recalled for a second test. Adding detection of the most frequent CFTR mutations to the immunoreactive trypsinogen measurement has improved the positive predictive value of CF newborn screening, and this is now the preferred strategy in most countries. 24 A positive screening test must be followed by proving the diagnosis via a positive sweat test (sweat chloride >60 mmol/L) or the presence of two mutations that cause CF. 2

At present, nationwide CF newborn screening has been implemented in nearly all European countries, in the United States, Canada, Australia and even in Russia, Turkey and Brazil. Some cases are missed by CF newborn screening. Algorithms should strive for a sensitivity above 95% and a positive predictive value above 30%. 25 The major side effect of CF newborn screening is detecting infants in whom the diagnosis CF cannot be made or excluded with certainty. These children, designated as having a CF screen positive inconclusive diagnosis, are asymptomatic but follow-up is needed to detect the minority who will develop CF symptoms, which is about 10%. 26

In regions with a long-standing history of CF newborn screening linked to carrier detection, the incidence of CF is decreasing due to cascade screening in relatives and preconception counselling. 27


Fixing cystic fibrosis by correcting CFTR domain assembly

For cystic fibrosis (CF) patients most therapies focus on alleviating the disease symptoms. Yet the cellular basis of the disease has been well studied mutations in the CF gene can impair folding, secretion, cell surface stability, and/or function of the CFTR chloride channel. Correction of these basic defects has been a challenge, but indicates that a deeper understanding of the molecular and cellular mechanism of mutations is a prerequisite for developing more efficient therapies.

CF is an autosomal recessive genetic disease with incidence of 𢏁 in 2,500 Caucasians, affecting �,000 people in North America and Europe (Riordan, 2008). The clinical features include pancreatic insufficiency, male infertility, meconium ileus in the newborn, and chronic lung infection with excessive inflammation, leading to progressive deterioration of lung function (Zielenski, 2000). The loss of lung function is the main cause of death in CF patients. Most current therapies treat the symptoms of these aspects of the disease and have increased the median life expectancy for individuals with CF to � years (Ashlock and Olson, 2011).

In 1989, the CF gene that encodes the CF transmembrane conductance regulator (CFTR), a member of the ABC transporter superfamily, was isolated (Rommens et al., 1989). More than 1,900 mutations have been identified in the CF gene (http://www.genet.sickkids.on.ca/cftr). CFTR, a polytopic membrane protein, is composed of five domains: two nucleotide-binding domains (NBDs), two membrane-spanning domains (MSDs) and a regulatory (R) region (Riordan, 2008). Biochemical, cell biological, and functional studies have shown that CFTR is an ATP- and phosphorylation-regulated chloride channel (Riordan et al., 1989). CFTR is confined to the apical plasma membrane of secretory epithelia in the airways, intestine, pancreas, testis, and exocrine glands and besides chloride, transports bicarbonate and regulates other ion transporters (Gadsby et al., 2006).

What is wrong with mutant CFTRs?

CF mutations have been grouped into six categories based on their cellular/molecular pathogenesis (Zielenski, 2000). Class I mutations include nonsense mutations (G542X and R553X), generating premature termination codons and frame-shift mutations that lead to truncated and/or and nonfunctional protein ( Fig. 1 ). Class V mutations cause mRNA mis-splicing or interfere with the promoter activity. Both classes impair CFTR protein production and plasma membrane expression, causing a severe CF phenotype.

Cellular mechanism and therapeutics of prevalent classes of CF-causing mutations. (A) Class I mutations (e.g., G542X) impair production of CFTR full-length protein by induction of premature termination codons (PTC). Aminoglycosides and an investigational drug, Ataluren, can rescue this phenotype by inducing read-through of the PTC and allow translation of full-length CFTR protein. (B) The most common � mutation (class II) impairs the channel conformational maturation and misfolded CFTR is recognized by the endoplasmic reticulum (ER) quality control system and is targeted for degradation via the ubiquitin–proteasome system. Correctors (e.g., VX-809) can partially rescue the misprocessing, probably by improving folding at the ER and delaying turnover at the plasma membrane (PM) with a presently poorly understood mechanism. Although rescued �-CFTR retains partial Cl − channel function, it is conformationally unstable and eliminated by the PM QC system via ubiquitination-dependent lysosomal delivery (Okiyoneda et al., 2010). (C) Class III mutations (e.g., G551D) do not affect CFTR biosynthesis and PM expression, but impair the channel gating. CFTR potentiators, including the FDA-approved Ivacaftor, correct this phenotype.

Class II mutations, despite normal transcript levels, have little or no detectable CFTR at the plasma membrane as a consequence of misfolding of the newly translated polypeptide. This category includes the most common mutation, deletion of phenylalanine 508 (�) in the NBD1, identifiable in one or both alleles in �% of CF patients (Riordan, 2008). �-CFTR is largely retained in the ER and degraded by the ubiquitin–proteasome system ( Fig. 1 Cheng et al., 1990 Ward et al., 1995).

Class III (e.g., G551D, 𢏄%) and class IV (e.g., R117H) mutations impair the CFTR channel opening-closing (or gating) cycle and conductance, respectively, without recognizable conformational or trafficking defects. Class III mutations are primarily associated with NBD1-2, whereas class IV mutations are localized to the channel pore (Riordan, 2008). Class VI mutations reduce CFTR expression by facilitating the channel removal from the plasma membrane. Notably, some mutations have a mixed phenotype. For example the � mutation causes folding, and gating, as well as plasma membrane stability impairments (Dalemans et al., 1991 Denning et al., 1992 Lukacs et al., 1993).

Correction of the basic defects

Because CF is a monogenic disease, it is postulated that the clinical phenotype would be alleviated by correcting the basic defects caused by various mutations impeding or preventing CFTR function, expression, or both (Cai et al., 2011). Efforts to correct the basic defects of CFTR biogenesis and function have been primarily focused on the most prevalent mutations: �, G551D, and premature termination codons ( Fig. 1 ).

Read-through of premature termination codons.

Aminoglycosides such as gentamycin interact with eukaryotic rRNA within the ribosomal subunits and reduce the fidelity of translation by interrupting the normal proofreading function (Burke and Mogg, 1985). Consequently, aminoglycosides allow insertion of a near-cognate amino acid at a premature termination codon and the translation of the entire coding region. Aminoglycosides have been used to suppress premature termination codons, resulting in the synthesis of full-length CFTR in CF patients with class I mutations (Wilschanski et al., 2003). Ataluren (PTC124), an orally bioavailable drug with diminished toxicity, was developed by a cell-based high-throughput screening assay (Welch et al., 2007). Although Ataluren selectively suppresses the premature termination codon in a mouse model (Welch et al., 2007), it showed variable efficiency among patients with different genotypes (Rowe et al., 2007). Orally administered Ataluren has been reported to rescue the activity of CFTR with premature termination codons in phase II trials (Kerem et al., 2008 Sermet-Gaudelus et al., 2010). A phase III clinical study is currently underway to evaluate long-term efficacy and safety (http://clinicaltrials.gov/ct2/show/ <"type":"clinical-trial","attrs":<"text":"NCT00803205","term_id":"NCT00803205">> NCT00803205).

Are �-CFTR folding, processing, and functional defects correctable?

In principle, the CFTR folding defect could be counteracted by pharmacological chaperones (PCs), similar to other misfolding diseases, where a variety of ligands or substrates can stabilize the target protein functional conformation (Bernier et al., 2004). Although this approach would be highly specific, and maintain the endogenous regulation and expression pattern of CFTR, high affinity CFTR ligands are not available. Altering the cellular folding environment could also be exploited to overcome the mutant misfolding/misprocessing and has shown some success in preclinical settings (Balch et al., 2011). Chemical chaperones similar to reduced temperature can also counteract the �-CFTR misfolding and elicit modest accumulation of partially functional but unstable channels at the plasma membrane (Denning et al., 1992 Sato et al., 1996 Sharma et al., 2001).

The revelation that �-CFTR misfolding can be rescued prompted the development of a cell-based high-throughput screening assay using the yellow fluorescent protein�sed halide indicator, which monitors the cAMP-activated plasma membrane chloride permeability, including CFTR activity, by sensing changes in the cytoplasmic halide concentration (Galietta et al., 2001). This assay became instrumental in the identification of not only 𠇌orrectors” that improve �-CFTR plasma membrane expression, but also “potentiators” to activate plasma membrane–resident CFTR channels. Interestingly, the assay also identified inhibitors of chloride channels, which may be potential therapeutics in secretory diarrheas (Verkman and Galietta, 2009).

Screening of diverse chemical libraries has produced several classes of small-molecule �-CFTR correctors, including corr-4a and its analogues (Pedemonte et al., 2005a). Additional correctors were also obtained by high-throughput screening and computational methods (Kalid et al., 2010 Robert et al., 2010 Sampson et al., 2011). However, the efficacy of these correctors in restoring chloride conductance was limited and reached only 㰐% of normal human primary epithelia, which is significantly lower than the predicted requirement for therapeutic efficiency (Pedemonte et al., 2005a, 2010 Van Goor et al., 2006). A similar approach by Vertex Pharmaceuticals, Inc. with Cystic Fibrosis Foundation Therapeutics’s (CFFT) support has yielded new classes of correctors, including VX-809, the most promising compound being evaluated in phase III clinical trials (Van Goor et al., 2006, 2011). VX-809 restores �% CFTR channel activity in primary respiratory epithelia expressing �-CFTR, but appears to have marginal clinical benefits (Van Goor et al., 2011 Clancy et al., 2012).

Reactivation of defective plasma membrane CFTR channels.

Cell-based functional high-throughput screening assays have also isolated several potentiators that improve the channel function of class II and III mutants (Pedemonte et al., 2005b Van Goor et al., 2006). The most promising potentiator, VX-770 (Ivacaftor), isolated by Vertex Pharmaceuticals, Inc., restores G551D-CFTR activity to �% of wild-type level (Van Goor et al., 2009). Clinical studies confirmed short-term safety and clinical benefits, including 55% reduced pulmonary exacerbation frequency and 10% increased lung function (Ramsey et al., 2011). Ivacaftor is the first FDA-approved drug for treatment of G551D-CFTR patients (𢏄% of CF population), representing a landmark translational achievement, exploiting the basic biology of CFTR and years of research and development in both academia and industry. Remarkably, Ivacaftor also restores the gating defect of several other class II mutations therefore, it may benefit �% of CF patients (Yu et al., 2012).

Combination therapy of potentiators and correctors could be useful for improving �-CFTR function given the persisting gating defect of rescued �-CFTR at the plasma membrane. Indeed, interim results of a phase II clinical trial suggest that � CF patients treated with a combination of VX-809 and Ivacaftor seem to display better lung function than those treated with either drug alone (http://clinicaltrials.gov/ct2/show/ <"type":"clinical-trial","attrs":<"text":"NCT01225211","term_id":"NCT01225211">> NCT01225211).

Challenges ahead: Efficacious therapy of �-CFTR

Despite recent advances of CFTR research, further improvement in functional expression of �-CFTR, the most common mutation in CF patients, is necessary because correction of the CF phenotype likely requires restoring �% of wild-type CFTR plasma membrane activity. Elucidating CFTR folding/misfolding and the available corrector mechanisms should help to achieve this goal. Here, we focus on efforts to understand and correct the folding defects of the �-CFTR.

CFTR domain folding and misfolding.

Compelling evidence supports the coupled domain-folding model of CFTR. Accordingly, individual domains can fold cotranslationally to metastable states but attaining the CFTR native fold requires post-translational domain assembly and inter-domain interactions that are critical to proper folding ( Fig. 2 A Du et al., 2005 He et al., 2008, 2010 Du and Lukacs, 2009). The slow post-translational conformational maturation is assisted by chaperones (Rosser et al., 2008) and reflected by the delayed formation of NBD–MSD interfaces in the mature wild-type CFTR (He et al., 2008 Serohijos et al., 2008). The energetic instability of individual domains and the slow domain assembly with the fast ER-associated degradation kinetics of folding intermediates all contribute to the inefficient folding (�%) of wild-type CFTR ( Fig. 2 B ) and are further sensitized by point mutations in CF (Rabeh et al., 2012).

Working models of CFTR folding, misfolding, and mechanism of �-CFTR correction by pharmacological chaperones. (A) Hypothetical folding and misfolding models of the multidomain CFTR channel. Each CFTR domain, such as MSD1, NBD1, MSD2, and NBD2 (M1, N1, M2, and N2), folds to variable extents cotranslationally to form metastable states. Formation of domain𠄽omain interfaces energetically facilitates further coupled-domain folding and assembly, a prerequisite for CFTR native tertiary structure. Progressive enthalpic stabilization of individual domains during co- and posttranslational folding is indicated by pseudocolors. � mutation (Δ) impairs both NBD1 energetics and domain𠄽omain interactions (especially via the NBD1–MSD2 interface) due to conformational and topological defects, rendering all four major domains structurally impaired in the �-CFTR. Adapted from Rabeh et al. (2012) with permission from Elsevier. (B) Genetic rescue of �-CFTR folding defect. Progressive stabilization of �-NBD1 by a panel of suppressor mutations (e.g., 3S) achieves only modest improvement in the marginal folding efficiency of �-CFTR (𢏀.4%). Representative data points and correlations between NBD1 stability and CFTR folding were obtained from Rabeh et al. (2012). Comparable changes in the conformational stability of the WT NBD1 (e.g., 3S) caused nearly twofold increase in WT CFTR folding efficiency. Stabilization of the NBD1–MSD2 interface by second site suppressor mutations (e.g., R1070W) largely restored the WT-like coupling efficiency between NBD1 stability and �-CFTR folding (Rabeh et al., 2012). This indicates that correction of two distinct structural defects is essential to achieve robust restoration of �-CFTR folding and function. (C) Predicted features of �-CFTR pharmacological rescues by structural defect-specific correctors. We speculate that a subset of correctors, yet to be identified, as pharmacological chaperones may either stabilize the NBD1 (1, blue dashed line) and/or the NBD1–MSD2 interface (2, red dashed lines) via direct binding to �-CFTR. Individual compound would result in modest increase in the mutant folding efficiency, but complementary pairs targeting both primary structural defects would synergistically improve the �-CFTR folding, PM expression, and function similar to suppressor mutations. For reference the WT- and �-CFTR folding efficiency are indicated (black lines).

How does the � mutation affect the channel? Homology modeling and cysteine cross-linking experiments have revealed the unique three-dimensional architecture of CFTR and the possible role of F508 (He et al., 2008 Mornon et al., 2008 Serohijos et al., 2008). In native CFTR, the F508 residue and surrounding area in the NBD1 forms an interface with the coupling helix of cytoplasmic loops 4 (CL4) and 1 (CL1) in MSD2 and MSD1, respectively, which creates a hydrophobic patch. NBD2 associates with CL2 and CL3 of MSD1 and MSD2, respectively. These interfaces relay conformational changes of the NBDs to the MSDs during channel gating, and are essential for CFTR folding (Wang et al., 2007 He et al., 2008, 2010 Mornon et al., 2008 Serohijos et al., 2008 Loo et al., 2010 Thibodeau et al., 2010 Grove et al., 2011). Destabilization of the interface by missense mutations in the CLs or by mutagenesis of the F508 side chain disrupts folding (Du et al., 2005 Mornon et al., 2008 He et al., 2010 Loo et al., 2010 Thibodeau et al., 2010). These observations are in support of the emerging model of co- and post-translational conformational maturation of CFTR that involves energetic and/or kinetic domain stabilization during coupled-domain folding (Du and Lukacs, 2009), similar to that of certain soluble multi-domain proteins and the BtuCD transporter ( Fig. 2 A Han et al., 2007 DiBartolo and Booth, 2011).

Targeting more than one folding defect in �-CFTR.

Recent studies revealed that the �-NBD1 is thermodynamically and kinetically destabilized at physiological temperature and suggested that the NBD1 stabilization would effectively counteract �-CFTR misprocessing (Protasevich et al., 2010 Wang et al., 2010). Surprisingly, this was not the case. Even substantial conformational stabilization of �-NBD1 by second site mutations led to modest rescue (㰠%) of �-CFTR processing, plasma membrane expression, and function, and failed to reinstate coupled domain folding ( Fig. 2 B Mendoza et al., 2012 Rabeh et al., 2012). Likewise, reversing the NBD1–MSD2 interface instability by second site mutations (e.g., R1070W) only marginally rescued the �-CFTR phenotype ( Fig. 2 B ). Remarkably, simultaneous genetic stabilization of NBD1 energetics and the NBD1–MSD2 interface led to robust, synergistic rescue (65�%) of �-CFTR folding and function ( Fig. 2 B Rabeh et al., 2012). A similar conclusion was reached by the analysis of evolved sequences coupled to the F508 residue (Mendoza et al., 2012). These unexpected findings suggest that correction of two primary structural defects is necessary and sufficient to restore CFTR function in most CF patients.

Translational implications of the �-CFTR misfolding mechanism

The discovery of two primary folding defects in �-CFTR highlighted three pharmacological implications ( Fig. 2 C ): (1) the plural folding defects provide a reasonable explanation for the modest efficacy of single correctors that may target only one of them with a presently unknown mechanism(s) (Sampson et al., 2011 Van Goor et al., 2011) (2) second site mutations counteracting one of the primary folding defects could promote mechanistic classification of existing corrector molecules, as well as the identification of new ones by second generation of structural defect–targeted high-throughput screening assays (3) correction of both NBD1 energetic and interface instability is likely required to robustly normalize �-CFTR processing, expression, and function ( Fig. 2 C ). Whether this could be achieved by one or two small molecules alone, or in combination with proteostasis network regulators that indirectly modulate �-CFTR folding, trafficking, and function (Balch et al., 2011) awaits further experimentation.

Other major challenges to translation

In this paper we have described the cellular consequences of CFTR mutations and recent efforts to understand the folding defects underlying the �-CFTR mutation in order to improve channel folding, stability, and function. However, many critical issues remain. Our understanding of how the loss of channel function results in CF, particularly the lung symptoms, is incomplete (Ashlock and Olson, 2011) and compounded by the fact that mouse models fail to recapitulate the CF lung disease, though the development of transgenic pig and ferret might address this issue (Rogers et al., 2008 Sun et al., 2010). Mechanistic studies of the channel are hampered by the low copy number and instability of mutants and the difficulties to monitor their structural alteration, protein–protein interaction, and trafficking at high spatiotemporal resolution in the appropriate cellular environment (Riordan, 2008 Balch et al., 2011). In addition to the drug discovery programs described in this paper, there are ongoing efforts to replace the mutant by gene therapy and/or activating alternative chloride secretion. However, multiple cellular mechanisms impede the nuclear delivery of CFTR transgene packaged either into cationic lipid complexes or viruses, though overcoming these processes may allow genotype-independent therapy (Griesenbach and Alton, 2009). Identification of the epithelial isoform of the Ca 2+ -activated chloride channel (TMEM16A) has opened the possibility to pharmacological activation of an alternative chloride secretory pathway (Ferrera et al., 2010).

The ultimate success of translational research most often relies on our detailed understanding of the basic biological problem at hand. We hope that this short perspective will help inspire further biological research, a prerequisite for translational successes in curing basic defects in CF and other genetic diseases such as diabetes insipidus and familial hypercholesterolemia.


Barriers and Future Directions for Precision Medicine to Reach All Individuals With CF

CFTR modulators have become transformative therapeutic approaches for many CF patients, as mentioned above. Despite several breakthroughs, further research is needed to continue optimizing therapies and to identify novel modulators for patients carrying rare, ultra-rare, or even unique CFTR mutations, who still face an unmet need for efficient, corrective therapies. Furthermore, some barriers still pose substantial challenges in the equitable availability of these pharmacotherapies, including the excessive costs and regulatory national issues. The collaborative environment composed by academic researchers, healthcare professionals, pharmaceutical companies, and patient representatives has been crucial in developing better treatments for people with CF.

Continuing the Optimization of Therapeutic Regimens to Increase the Adherence and Reduce the Burden

The multifaceted nature of CF requires complex and time-consuming therapeutic regimens that should be periodically adapted according to disease progression. Furthermore, CF patients are subjected to substantial clinical, psychosocial, and economic burdens, which pose challenges to achieve optimal, lifelong treatment adherence. The adherence varies largely depending on treatment type, route of administration, duration, and number of distinct medications, as well as patient age and socioeconomic status (Sawicki et al., 2013 Angelis et al., 2015 Quittner et al., 2016 Narayanan et al., 2017). Moreover, CF patients usually spend twice and 20 times more time in daily treatment activities than diabetic and asthmatic patients, respectively (Ziaian et al., 2006), which may considerably affect adherence. Poor adherence has also been associated with higher healthcare costs, more frequent hospitalizations, and worse quality of life and clinical manifestations (Sawicki et al., 2013 Quittner et al., 2016 Narayanan et al., 2017). Establishing a closer relationship among patient, families/caregivers and the multidisciplinary healthcare team may be a first step to overcome key barriers to treatment adherence.

To date, only few publications have evaluated the adherence to ivacaftor treatment and adherence to modulator combinations remains yet to be demonstrated. From a clinical perspective, a life-transforming oral medication with a simple dosing schedule would supposedly be taken as prescribed. Adherence to ivacaftor has nevertheless varied from suboptimal (Siracusa et al., 2015) to optimal (Suthoff et al., 2016). As these studies had a small sample size and applied distinct methods, it is still difficult to extrapolate the results to a broader CF population, and further studies are needed to better address this issue. Certain therapeutic benefits may also be more modest in a real-world setting compared to clinical trials, as patients should take these oral medications following specific recommendations, including dietary to ensure better drug absorption and availability in the body. Furthermore, recent studies have demonstrated that abrupt interruption of CFTR modulator therapy may cause severe clinical consequences. Ivacaftor withdrawal resulted in accelerated deterioration of lung function consistent with a pulmonary exacerbation episode in a case series (Trimble and Donaldson, 2018). Patients who discontinued treatment with lumacaftor/ivacaftor, mainly due to early adverse effects, also demonstrated a higher risk of worsening clinical manifestations compared to patients who continued treatment or those who restarted it after temporary discontinuation in a real-world study (Burgel et al., 2020). As patients have different lifestyles and socioeconomic conditions, the development of educational and motivational interventions at an individual level may help in ensuring optimal adherence to achieve the greatest clinical outcomes.

CFTR modulators have been added to therapeutic regimens of eligible patients, rather than replacing some symptomatic therapies. This appears to be the optimal approach for most CF patients, although it also increases the burden of medications in use. Once the safety and efficacy of novel therapies are demonstrated in adults, extension clinical trials are pursued to evaluate the effects on younger patients, as adverse effects may vary across distinct age groups (Davies et al., 2016 Rosenfeld et al., 2018 McNamara et al., 2019 Rosenfeld et al., 2019). Starting these transformative therapies in milder disease severity and earlier in life may offer more chances of significantly improving long-term outcomes or even preventing certain injury of affected organs, which may also result in a lower burden of medications in a long-term perspective. Some reports have also demonstrated that younger patients are more adherent to therapies than adolescents and adults, possibly due to higher parental supervision (Quittner et al., 2014 Shakkottai et al., 2014). Providing educational and supporting approaches to young children and their parents may result in optimal, lifelong treatment adherence. As patients should be transferred from pediatric to adult care at a certain age (generally between 18 and 21 years old), a planned transition is greatly helpful to maximize independence, minimize chances of interruption in the therapies and continue improving their quality of life (Goralski et al., 2017).

Continuing the Development of Transformative Therapeutics to Reach All Individuals With CF

Most development programs of CFTR modulators has been initially focused on the correction of F508del mutation, since fully overcoming the defects in this mutation would result in an effective therapy for approximately 82% of the CF patient population worldwide. There are still nevertheless 10%�% of patients without any CFTR-directed therapeutics. This percentage is even higher in countries where the prevalence of F508del is much lower, such as Brazil, Israel, Italy and Turkey (Figure 3).

Identifying the putative binding sites of CFTR-directed modulators using the novel insights of CFTR structure may facilitate the rational design of novel compounds with enhanced pharmacological properties. The pipeline of CFTR modulators continues to expand and some recent drug development programs have also been pursuing the identification of modulators to less common CF-causing mutations. Identification of specific therapies for rare and ultra-rare mutations poses nevertheless several challenges due to the great variability of CF-causing mutations and the very small number of patients. In addition to CFTR-directed modulators, CFTR dysfunction might be compensated by targeting alternative ion channels, such as ENaC (Moore and Tarran, 2018), the calcium-activated chloride channel transmembrane protein membrane 16A (TMEM16A) (Sondo et al., 2014), and the solute carrier 26A9 (SLC26A9) (Balázs and Mall, 2018). Strategies that modulate these alternative ion channels might be efficient therapies for all patients, regardless of their CF genotypes. These strategies might also be used alone or in combination with CFTR modulators to enhance clinical outcomes. Nevertheless, as CF patients are already subjected to a substantial burden of medications, drug-drug interaction profiles should be further exploited to avoid adverse effects or inhibitory effects of one therapy on another. In this line, itraconazole, an antifungal commonly used for the treatment of allergic bronchopulmonary aspergillosis, was demonstrated to significantly increase systemic exposures of tezacaftor and ivacaftor (Garg et al., 2019). Caution and appropriate monitoring are recommended when these therapies are used at the same period.

Traditional trials with a placebo-controlled design have been providing evidence for the safety and efficacy of CFTR modulators (Habib et al., 2019) (Table 1) however, alternatives will be needed in the near future, as more modulator options become available and the number of patients without any modulator therapy will certainly reduce. Furthermore, clinical trials in sicker or younger patients, and those carrying rarer CFTR mutations are more challenging due to small sample size, specific inclusion/exclusion criteria, or even for some hesitation on the part of the investigators. Strategies to adapt and optimize trial design and deliver for speed and efficacy have been discussed, including the use of patient-derived specimens, power calculations to compensate for group sampling, and N-of-1 and �sket” trials (Matthes et al., 2018 Amaral et al., 2019 Davies et al., 2019b).

The use of patient-derived specimens to comparatively evaluate drug efficacies may be a feasible starting point to identify the best candidate drug(s) in vitro and predict the magnitude of therapeutic responses for following clinical testing (Strauss and Blinova, 2017 Amaral et al., 2019). In fact, a significant but variable clinical responsiveness was observed in clinical trials with CFTR modulators in patients carrying at least one G551D mutation (Ramsey et al., 2011 Rowe et al., 2014) or in F508del-homozygous patients (Boyle et al., 2014 Wainwright et al., 2015 Donaldson et al., 2018a), which suggests that patient responsiveness to a certain therapy is influenced not only by the CF genotype but also by the genetic background and/or epigenetic factors. In this line, some reports have demonstrated that single nucleotide polymorphisms in SLC26A9 gene contribute to heterogeneity in inter-individual responsiveness to CFTR modulator therapies (Strug et al., 2016 Corvol et al., 2018). Such findings denote the relevance of assessing the drug effectiveness at an individual level in patient-derived specimens.

A report pairing in vitro measurement of CFTR function in cell lines and clinical features demonstrated a strong correlation between CFTR function and sweat chloride concentration, and to a lesser extent but still significant with lung function and pancreatic status (McCague et al., 2019). Correlations between responses in patient-derived specimens and clinical parameters/biomarkers have been investigated to establish reliable prediction of drug effectiveness. A consistent correlation was found among forskolin-induced swelling of intestinal organoids, sweat chloride concentration and intestinal current measurements of infants with CF (de Winter-de Groot et al., 2018). Despite the clinical heterogeneity in adults with CF and homozygous for F508del mutation, forskolin-induced swelling of intestinal organoids positively correlated with FEV1 and BMI (de Winter-de Groot et al., 2019). Responses from intestinal organoids were also demonstrated to correlate with intestinal current measurements, reduction in sweat chloride concentration and improvement in lung function of patients after CFTR modulator therapies (Dekkers et al., 2016a Berkers et al., 2019). In N-of-1 trial series, an increase in CFTR-dependent chloride transport in nasal epithelial cell cultures was only found in the three patients who also demonstrated a reduction in sweat chloride concentration after ivacaftor treatment (McGarry et al., 2017). Furthermore, responses in F508del-homozygous patient-derived nasal epithelial cells were correlated to improvements in ppFEV1 and intestinal current measurements, but not with nasal potential difference after co-treatment with lumacaftor/ivacaftor (Pranke et al., 2019). Nevertheless, no significant correlations were found among responses in intestinal current measurement, nasal potential difference and sweat chloride concentration, despite high concordance for all CFTR-dependent biomarkers in another study evaluating the co-treatment with lumacaftor/ivacaftor (Graeber et al., 2018). Further studies are certainly needed to better correlate and validate drug effectiveness in patient-derived specimens with clinical features, and identification of novel biomarkers may also enrich strategies in efficacy trials.

In an era of drugs targeting the underlying defects in CF-causing mutations, the development of symptomatic therapies might appear less attractive. Nevertheless, these therapies must continue to be developed as most (if not all) existing CF population will need them at some point, and CFTR modulators are very unlikely to reverse lung tissue remodeling already established (Davies et al., 2019b). A recent study demonstrated that six months of ivacaftor treatment was unable to significantly change airway microbiome and several inflammation measurements in patients carrying at least one G551D mutation. Such findings indicate that antibiotics and anti-inflammatory drugs will still be required to control disease symptoms and prevent complications (Harris et al., 2019). As the disease progresses, patients may also develop comorbidities and thus require even more complex therapeutic regimens, adding further burdens. Ivacaftor treatment was demonstrated to improve exocrine pancreatic function as well as insulin secretion profile, which may alleviate or even reverse CF-related diabetes (Hayes et al., 2014 Davies et al., 2016 Tsabari et al., 2016 Kelly et al., 2019). Ivacaftor treatment was also demonstrated to improve bone health (Sermet-Gaudelus et al., 2016) and vascular tone abnormalities (Adam et al., 2016). A recent review nicely summarizes the current understanding of CFTR modulators on extra-pulmonary complications in CF (Sergeev et al., 2019). Nevertheless, most studies are case reports or have a small sample size, and further studies are warranted to investigate the impact of CFTR modulator therapies on CF comorbidities.

Treatment with more than one CFTR modulator appears to be the optimal approach for many CF-causing mutations. As heterozygous carriers are asymptomatic, fully overcoming CFTR dysfunction in one allele might be enough to halt disease progression, if treatment is started early in life and before severe lung injury occurs. Based on in vitro evidence (Zhang et al., 2009), rescue of 25-50% of WT-CFTR function in both alleles might also be sufficient to restore normal rates of mucociliary clearance. It remains nevertheless unclear how many CFTR modulators would be needed to reach such threshold in patients. In addition to CFTR modulators, progress has been made in developing cell-based (Barical et al., 2019 Hayes et al., 2019) and gene-based therapies (Donnelley and Parsons, 2018 Duncan et al., 2018 Lopes-Pacheco et al., 2018 Osman et al., 2018) for CF lung disease.

Identifying Feasible Solutions for a CF Healthcare Cost Sustainable

A major limitation of these novel pharmaceutical treatments for CF patients, such as the CFTR modulators, is the excessive costs when they reach the market (over US$250,000 per patient per year), which renders difficulties in their availability for many patients worldwide (O'Sullivan et al., 2013 Ferkol and Quinton, 2015 Orestein et al., 2015), especially for those living in low- and middle-income countries (Cohen-Cymberknoh et al., 2016). In developed countries, certain health authorities have also been slow in approving reimbursement (Bush and Simmonds, 2012 Whiting et al., 2014 Sharma D. et al., 2018) and the cost-effectiveness of these pharmacotherapies has yet been questioned (Gulland, 2016 Balk et al., 2018). Even though the quality-adjusted life-year (QALY) analysis might not adequately address all concerns for rare diseases, such as CF (Schlander et al., 2014 Pearson et al., 2018), these therapies pose a substantial burden on national healthcare systems, as they are expensive and lifelong. It remains nevertheless unclear if such prices will persist over time, as several novel molecules are on the horizon and probably will reach the market over the next years, if they prove to be safe and to have efficacy in clinical studies. Further discussion should certainly be undertaken with patient representatives, healthcare providers, policymakers, government authorities and pharmaceutical companies to identify feasible and sustainable solutions that would enable equitable access to eligible patients for these “on-target” therapies. Hopefully, market competition will also reduce modulator prices with the approval of novel ones.

Over the past three decades, the human disease target landscape considerably expanded, as approximately 40% of approved pharmaceuticals received an orphan designation (Attwood et al., 2018). Drug discovery and development for a new molecule may be nevertheless far slower than expected, as it is a costly process with high attrition rates that also depends on several regulatory requirements. Drug repurposing (also known as drug repositioning or reprofiling) has become an increasingly attractive strategy that may save valuable time and funding investments in drug development for common and rare diseases. As approved drugs have already undergone extensive toxicological evaluations in both experimental and early-stage clinical studies, the time frame to obtain a new disease indication may be reduced, if safety and efficacy is demonstrated for the repurposed use in late-stage clinical studies (Pushpakom et al., 2019). Furthermore, drug repurposing may unravel effective therapies for patients with common and rare CF-causing mutations in an expedited way and at a feasible cost for national healthcare systems. In experimental models, certain underlying defects in CFTR mutations have been rectified by administering clinically approved drugs, such as gentamicin (Howard et al., 1996), amlexanox (Gonzalez-Hilarion et al., 2012), escin (Mutyam et al., 2016), ibuprofen (Carlile et al., 2015), and genistein (Illek and Fischer, 1998). These findings indicate that other existing and approved drugs for unrelated disease indications might have the potential to correct or circumvent CFTR dysfunction and should be exploited in the pre-clinical setting. Both cysteamine and thymosin α-1 were also claimed to restore functional expression of F508del-CFTR (Tosco et al., 2016 Romani et al., 2017). Nevertheless, several independent CF research groups failed to demonstrate rescue of F508del-CFTR PM expression and function by either cysteamine or thymosin α-1 (Tomati et al., 2018b Armirotti et al., 2019 Awatade et al., 2019). Although the immunomodulatory effect of these molecules in CF remains to be further exploited, they did not demonstrate F508del-CFTR correction. As the identification of highly efficient treatments often draws the attention of both scientific and lay audiences, a note of caution should be considered before such findings are diffused in the press to avoid creating premature expectations, especially in CF patients and their relatives.


HISTORY OF CF

The disease now known as CF has long been identified by health workers as indicated by the old adage from Northern European folklore “Woe to that child which when kissed on the forehead tastes salty. He is bewitched and soon must die” (35). The first major contribution to determining the cause of CF occurred in 1938 when Dorothy Andersen (after performing autopsies on infants and children with the disease and reviewing their case histories) provided a comprehensive description of their symptoms and the changes produced by the disease in various organs. Andersen noted that there was almost always destruction of the pancreas accompanied by infection of and damage to airways in the lungs. Andersen named the disease “cystic fibrosis of the pancreas.” Subsequently in 1946, researchers deduced that CF was inherited and results from an autosomal recessive mutation. In 1948, there was a devastating heat wave in New York City and hospitals saw a disproportionate number of children with CF who had become dehydrated from losing excessive salt in their sweat (26). This observation by P. A. di Sant’Agnese at Columbia Hospital led to his presentation of his findings to the American Pediatrics Society in 1953 and the development of the cornerstone diagnosis for CF, the sweat chloride test.


Fixing cystic fibrosis by correcting CFTR domain assembly

For cystic fibrosis (CF) patients most therapies focus on alleviating the disease symptoms. Yet the cellular basis of the disease has been well studied mutations in the CF gene can impair folding, secretion, cell surface stability, and/or function of the CFTR chloride channel. Correction of these basic defects has been a challenge, but indicates that a deeper understanding of the molecular and cellular mechanism of mutations is a prerequisite for developing more efficient therapies.

CF is an autosomal recessive genetic disease with incidence of ∼1 in 2,500 Caucasians, affecting ∼70,000 people in North America and Europe (Riordan, 2008). The clinical features include pancreatic insufficiency, male infertility, meconium ileus in the newborn, and chronic lung infection with excessive inflammation, leading to progressive deterioration of lung function (Zielenski, 2000). The loss of lung function is the main cause of death in CF patients. Most current therapies treat the symptoms of these aspects of the disease and have increased the median life expectancy for individuals with CF to ∼39 years (Ashlock and Olson, 2011).

In 1989, the CF gene that encodes the CF transmembrane conductance regulator (CFTR), a member of the ABC transporter superfamily, was isolated (Rommens et al., 1989). More than 1,900 mutations have been identified in the CF gene (http://www.genet.sickkids.on.ca/cftr). CFTR, a polytopic membrane protein, is composed of five domains: two nucleotide-binding domains (NBDs), two membrane-spanning domains (MSDs) and a regulatory (R) region (Riordan, 2008). Biochemical, cell biological, and functional studies have shown that CFTR is an ATP- and phosphorylation-regulated chloride channel (Riordan et al., 1989). CFTR is confined to the apical plasma membrane of secretory epithelia in the airways, intestine, pancreas, testis, and exocrine glands and besides chloride, transports bicarbonate and regulates other ion transporters (Gadsby et al., 2006).

What is wrong with mutant CFTRs?

CF mutations have been grouped into six categories based on their cellular/molecular pathogenesis (Zielenski, 2000). Class I mutations include nonsense mutations (G542X and R553X), generating premature termination codons and frame-shift mutations that lead to truncated and/or and nonfunctional protein (Fig. 1). Class V mutations cause mRNA mis-splicing or interfere with the promoter activity. Both classes impair CFTR protein production and plasma membrane expression, causing a severe CF phenotype.

Class II mutations, despite normal transcript levels, have little or no detectable CFTR at the plasma membrane as a consequence of misfolding of the newly translated polypeptide. This category includes the most common mutation, deletion of phenylalanine 508 (ΔF508) in the NBD1, identifiable in one or both alleles in ∼90% of CF patients (Riordan, 2008). ΔF508-CFTR is largely retained in the ER and degraded by the ubiquitin–proteasome system (Fig. 1 Cheng et al., 1990 Ward et al., 1995).

Class III (e.g., G551D, ∼4%) and class IV (e.g., R117H) mutations impair the CFTR channel opening-closing (or gating) cycle and conductance, respectively, without recognizable conformational or trafficking defects. Class III mutations are primarily associated with NBD1-2, whereas class IV mutations are localized to the channel pore (Riordan, 2008). Class VI mutations reduce CFTR expression by facilitating the channel removal from the plasma membrane. Notably, some mutations have a mixed phenotype. For example the ΔF508 mutation causes folding, and gating, as well as plasma membrane stability impairments (Dalemans et al., 1991 Denning et al., 1992 Lukacs et al., 1993).

Correction of the basic defects

Because CF is a monogenic disease, it is postulated that the clinical phenotype would be alleviated by correcting the basic defects caused by various mutations impeding or preventing CFTR function, expression, or both (Cai et al., 2011). Efforts to correct the basic defects of CFTR biogenesis and function have been primarily focused on the most prevalent mutations: ΔF508, G551D, and premature termination codons (Fig. 1).

Read-through of premature termination codons.

Aminoglycosides such as gentamycin interact with eukaryotic rRNA within the ribosomal subunits and reduce the fidelity of translation by interrupting the normal proofreading function (Burke and Mogg, 1985). Consequently, aminoglycosides allow insertion of a near-cognate amino acid at a premature termination codon and the translation of the entire coding region. Aminoglycosides have been used to suppress premature termination codons, resulting in the synthesis of full-length CFTR in CF patients with class I mutations (Wilschanski et al., 2003). Ataluren (PTC124), an orally bioavailable drug with diminished toxicity, was developed by a cell-based high-throughput screening assay (Welch et al., 2007). Although Ataluren selectively suppresses the premature termination codon in a mouse model (Welch et al., 2007), it showed variable efficiency among patients with different genotypes (Rowe et al., 2007). Orally administered Ataluren has been reported to rescue the activity of CFTR with premature termination codons in phase II trials (Kerem et al., 2008 Sermet-Gaudelus et al., 2010). A phase III clinical study is currently underway to evaluate long-term efficacy and safety (http://clinicaltrials.gov/ct2/show/NCT00803205).

Are ΔF508-CFTR folding, processing, and functional defects correctable?

In principle, the CFTR folding defect could be counteracted by pharmacological chaperones (PCs), similar to other misfolding diseases, where a variety of ligands or substrates can stabilize the target protein functional conformation (Bernier et al., 2004). Although this approach would be highly specific, and maintain the endogenous regulation and expression pattern of CFTR, high affinity CFTR ligands are not available. Altering the cellular folding environment could also be exploited to overcome the mutant misfolding/misprocessing and has shown some success in preclinical settings (Balch et al., 2011). Chemical chaperones similar to reduced temperature can also counteract the ΔF508-CFTR misfolding and elicit modest accumulation of partially functional but unstable channels at the plasma membrane (Denning et al., 1992 Sato et al., 1996 Sharma et al., 2001).

The revelation that ΔF508-CFTR misfolding can be rescued prompted the development of a cell-based high-throughput screening assay using the yellow fluorescent protein–based halide indicator, which monitors the cAMP-activated plasma membrane chloride permeability, including CFTR activity, by sensing changes in the cytoplasmic halide concentration (Galietta et al., 2001). This assay became instrumental in the identification of not only “correctors” that improve ΔF508-CFTR plasma membrane expression, but also “potentiators” to activate plasma membrane–resident CFTR channels. Interestingly, the assay also identified inhibitors of chloride channels, which may be potential therapeutics in secretory diarrheas (Verkman and Galietta, 2009).

Screening of diverse chemical libraries has produced several classes of small-molecule ΔF508-CFTR correctors, including corr-4a and its analogues (Pedemonte et al., 2005a). Additional correctors were also obtained by high-throughput screening and computational methods (Kalid et al., 2010 Robert et al., 2010 Sampson et al., 2011). However, the efficacy of these correctors in restoring chloride conductance was limited and reached only <10% of normal human primary epithelia, which is significantly lower than the predicted requirement for therapeutic efficiency (Pedemonte et al., 2005a, 2010 Van Goor et al., 2006). A similar approach by Vertex Pharmaceuticals, Inc. with Cystic Fibrosis Foundation Therapeutics’s (CFFT) support has yielded new classes of correctors, including VX-809, the most promising compound being evaluated in phase III clinical trials (Van Goor et al., 2006, 2011). VX-809 restores ∼15% CFTR channel activity in primary respiratory epithelia expressing ΔF508-CFTR, but appears to have marginal clinical benefits (Van Goor et al., 2011 Clancy et al., 2012).

Reactivation of defective plasma membrane CFTR channels.

Cell-based functional high-throughput screening assays have also isolated several potentiators that improve the channel function of class II and III mutants (Pedemonte et al., 2005b Van Goor et al., 2006). The most promising potentiator, VX-770 (Ivacaftor), isolated by Vertex Pharmaceuticals, Inc., restores G551D-CFTR activity to ∼50% of wild-type level (Van Goor et al., 2009). Clinical studies confirmed short-term safety and clinical benefits, including 55% reduced pulmonary exacerbation frequency and 10% increased lung function (Ramsey et al., 2011). Ivacaftor is the first FDA-approved drug for treatment of G551D-CFTR patients (∼4% of CF population), representing a landmark translational achievement, exploiting the basic biology of CFTR and years of research and development in both academia and industry. Remarkably, Ivacaftor also restores the gating defect of several other class II mutations therefore, it may benefit ∼10% of CF patients (Yu et al., 2012).

Combination therapy of potentiators and correctors could be useful for improving ΔF508-CFTR function given the persisting gating defect of rescued ΔF508-CFTR at the plasma membrane. Indeed, interim results of a phase II clinical trial suggest that ΔF508 CF patients treated with a combination of VX-809 and Ivacaftor seem to display better lung function than those treated with either drug alone (http://clinicaltrials.gov/ct2/show/NCT01225211).

Challenges ahead: Efficacious therapy of ΔF508-CFTR

Despite recent advances of CFTR research, further improvement in functional expression of ΔF508-CFTR, the most common mutation in CF patients, is necessary because correction of the CF phenotype likely requires restoring ∼35% of wild-type CFTR plasma membrane activity. Elucidating CFTR folding/misfolding and the available corrector mechanisms should help to achieve this goal. Here, we focus on efforts to understand and correct the folding defects of the ΔF508-CFTR.

CFTR domain folding and misfolding.

Compelling evidence supports the coupled domain-folding model of CFTR. Accordingly, individual domains can fold cotranslationally to metastable states but attaining the CFTR native fold requires post-translational domain assembly and inter-domain interactions that are critical to proper folding (Fig. 2 A Du et al., 2005 He et al., 2008, 2010 Du and Lukacs, 2009). The slow post-translational conformational maturation is assisted by chaperones (Rosser et al., 2008) and reflected by the delayed formation of NBD–MSD interfaces in the mature wild-type CFTR (He et al., 2008 Serohijos et al., 2008). The energetic instability of individual domains and the slow domain assembly with the fast ER-associated degradation kinetics of folding intermediates all contribute to the inefficient folding (∼30%) of wild-type CFTR (Fig. 2 B) and are further sensitized by point mutations in CF (Rabeh et al., 2012).

How does the ΔF508 mutation affect the channel? Homology modeling and cysteine cross-linking experiments have revealed the unique three-dimensional architecture of CFTR and the possible role of F508 (He et al., 2008 Mornon et al., 2008 Serohijos et al., 2008). In native CFTR, the F508 residue and surrounding area in the NBD1 forms an interface with the coupling helix of cytoplasmic loops 4 (CL4) and 1 (CL1) in MSD2 and MSD1, respectively, which creates a hydrophobic patch. NBD2 associates with CL2 and CL3 of MSD1 and MSD2, respectively. These interfaces relay conformational changes of the NBDs to the MSDs during channel gating, and are essential for CFTR folding (Wang et al., 2007 He et al., 2008, 2010 Mornon et al., 2008 Serohijos et al., 2008 Loo et al., 2010 Thibodeau et al., 2010 Grove et al., 2011). Destabilization of the interface by missense mutations in the CLs or by mutagenesis of the F508 side chain disrupts folding (Du et al., 2005 Mornon et al., 2008 He et al., 2010 Loo et al., 2010 Thibodeau et al., 2010). These observations are in support of the emerging model of co- and post-translational conformational maturation of CFTR that involves energetic and/or kinetic domain stabilization during coupled-domain folding (Du and Lukacs, 2009), similar to that of certain soluble multi-domain proteins and the BtuCD transporter (Fig. 2 A Han et al., 2007 DiBartolo and Booth, 2011).

Targeting more than one folding defect in ΔF508-CFTR.

Recent studies revealed that the ΔF508-NBD1 is thermodynamically and kinetically destabilized at physiological temperature and suggested that the NBD1 stabilization would effectively counteract ΔF508-CFTR misprocessing (Protasevich et al., 2010 Wang et al., 2010). Surprisingly, this was not the case. Even substantial conformational stabilization of ΔF508-NBD1 by second site mutations led to modest rescue (<20%) of ΔF508-CFTR processing, plasma membrane expression, and function, and failed to reinstate coupled domain folding (Fig. 2 B Mendoza et al., 2012 Rabeh et al., 2012). Likewise, reversing the NBD1–MSD2 interface instability by second site mutations (e.g., R1070W) only marginally rescued the ΔF508-CFTR phenotype (Fig. 2 B). Remarkably, simultaneous genetic stabilization of NBD1 energetics and the NBD1–MSD2 interface led to robust, synergistic rescue (65–80%) of ΔF508-CFTR folding and function (Fig. 2 B Rabeh et al., 2012). A similar conclusion was reached by the analysis of evolved sequences coupled to the F508 residue (Mendoza et al., 2012). These unexpected findings suggest that correction of two primary structural defects is necessary and sufficient to restore CFTR function in most CF patients.

Translational implications of the ΔF508-CFTR misfolding mechanism

The discovery of two primary folding defects in ΔF508-CFTR highlighted three pharmacological implications (Fig. 2 C): (1) the plural folding defects provide a reasonable explanation for the modest efficacy of single correctors that may target only one of them with a presently unknown mechanism(s) (Sampson et al., 2011 Van Goor et al., 2011) (2) second site mutations counteracting one of the primary folding defects could promote mechanistic classification of existing corrector molecules, as well as the identification of new ones by second generation of structural defect–targeted high-throughput screening assays (3) correction of both NBD1 energetic and interface instability is likely required to robustly normalize ΔF508-CFTR processing, expression, and function (Fig. 2 C). Whether this could be achieved by one or two small molecules alone, or in combination with proteostasis network regulators that indirectly modulate ΔF508-CFTR folding, trafficking, and function (Balch et al., 2011) awaits further experimentation.

Other major challenges to translation

In this paper we have described the cellular consequences of CFTR mutations and recent efforts to understand the folding defects underlying the ΔF508-CFTR mutation in order to improve channel folding, stability, and function. However, many critical issues remain. Our understanding of how the loss of channel function results in CF, particularly the lung symptoms, is incomplete (Ashlock and Olson, 2011) and compounded by the fact that mouse models fail to recapitulate the CF lung disease, though the development of transgenic pig and ferret might address this issue (Rogers et al., 2008 Sun et al., 2010). Mechanistic studies of the channel are hampered by the low copy number and instability of mutants and the difficulties to monitor their structural alteration, protein–protein interaction, and trafficking at high spatiotemporal resolution in the appropriate cellular environment (Riordan, 2008 Balch et al., 2011). In addition to the drug discovery programs described in this paper, there are ongoing efforts to replace the mutant by gene therapy and/or activating alternative chloride secretion. However, multiple cellular mechanisms impede the nuclear delivery of CFTR transgene packaged either into cationic lipid complexes or viruses, though overcoming these processes may allow genotype-independent therapy (Griesenbach and Alton, 2009). Identification of the epithelial isoform of the Ca 2+ -activated chloride channel (TMEM16A) has opened the possibility to pharmacological activation of an alternative chloride secretory pathway (Ferrera et al., 2010).

The ultimate success of translational research most often relies on our detailed understanding of the basic biological problem at hand. We hope that this short perspective will help inspire further biological research, a prerequisite for translational successes in curing basic defects in CF and other genetic diseases such as diabetes insipidus and familial hypercholesterolemia.


Affiliations

Clinical Research Services, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada

Dalla Lana School of Public Health, University of Toronto, Toronto, Ontario, Canada

Annie Dupuis PhD, Lisa J. Strug PhD, ScM & Tanja Gonska MD

Program in Physiology and Experimental Medicine, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada

Discipline of Paediatrics, School of Women's and Children's Health, Faculty of Medicine, University of New South Wales, Sydney, Australia

Department of Gastroenterology, Sydney Children's Hospital Randwick, New South Wales, Australia

GeneYouIn Inc., Toronto, Ontario, Canada

Department of Epidemiology, Colorado School of Public Health, University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado, USA

Department of Pediatrics, Justus-Liebig-University Giessen, Giessen, Germany

Cystic Fibrosis Center, Azienda Ospedaliera Universitaria Integrata, Verona, Italy

The Hospital for Sick Children and Division of Biostatistics, Program in Genetics & Genome Biology, Research Institute

Lisa J. Strug PhD, ScM & Johanna M. Rommens PhD

Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada

Department of Pediatrics, University of Toronto, Toronto, Ontario, Canada


Watch the video: Κυστική ίνωση. Cystic fibrosis (May 2022).


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