CYSTIC FIBROSIS:
A DRIVE FOR ACQUIRING KNOWLEDGE
By
KATHERINE VIRGINIA BULLOCK
____________________
A Thesis Submitted to the Honors College
In Partial Fulfillment of the Bachelors degree
With Honors in
Physiology
THE UNIVERSITY OF ARIZONA
MAY 2016
Approved by:
_________________________
Dr. Douglas Keen
Department of Physiology
Abstract Cystic fibrosis (CF) detrimentally affects the pulmonary and digestive system of patients and only a
few years ago had a life expectancy of a mere 40 years. However, due to strides made in CF research
the life expectancy projection is now 58 years. Cystic fibrosis transmembrane conductance regulator
(CFTR), an anion channel located on the apical membrane of epithelia, is directly responsible for the
translocation of chloride and indirectly responsible for the paracellular translocation of water. A
mutation in CFTR causes systemic hyperosmotic epithelial secretions. My thesis focuses on existing
knowledge about CF with emphasis on the basic defect of the disease and the current standard of
care. Recent and noteworthy papers were collected and synthesized. Current treatments were
examined for specificity and success in reducing symptoms. CF research is making great
advancements in individualized treatment of this disease which has vast variation from patient to
patient due to the vast number of mutations that can affect CFTR. CF research is an excellent
example of how targeting specific mechanisms of a disease can decrease morbidity in CF patients.
Having comprehensive knowledge may help in the understanding necessary to continue focused
basic research and alleviate the health problems associated with CF.
2 Table of Contents ABSTRACT 1 TABLE OF CONTENTS 3 INTRODUCTION 4 CELLULAR & MOLECULAR BASIS OF CYSTIC FIBROSIS 5 GENETICS CLASSES OF MUTATIONS BIOGENESIS & MUTATIONS THE CFTR PROTEIN 5 6 7 11 DIAGNOSTICS 14 PRENATAL SCREENING NEWBORN SCREENING (NBS) TYPICAL SCREENING THROUGH IRT AND DNA TESTING FECAL ELASTASE NASAL POTENTIAL DIFFERENCE 14 15 16 17 17 TREATMENTS 18 BASIC DEFECT TREATMENTS CFTR POTENTIATORS CFTR CORRECTORS GENE REPLACEMENT TRANSLATIONAL READTHROUGH RESPIRATORY TREATMENTS AIRWAY CLEARANCE THERAPY PREVENTION & TREATMENT OF RESPIRATORY DISEASE NUTRITION 18 18 19 19 20 20 21 22 22 CONCLUSION 24 ACKNOWLEDGEMENTS 24 REFERENCES 25 3 Introduction “There is not a true life expectancy for this disease because there are so many varying
factors that contribute, but on average, people with CF are living into their 30's and 40's.
That being said, there are those who live much longer than that... my goal is to be one of
those.”
This quote was written by Kayla English, a blogger with cystic fibrosis (CF), and describes the
drive for not only survival but happiness that is apparent in many cystic fibrosis patients (9).
Cystic fibrosis is a multisystem genetic disease characterized by lung disease and pancreatic
insufficiency. It is the most common genetic disease among Caucasians with a live birth rate of 1
in 3,200 in the United States. This genetic mutation results in a nonfunctioning or absent protein
responsible for chloride movement in epithelial cells. The absence of this process results in thick
mucus secretions that causes pancreatic insufficiency and an increased susceptibility to lung
infections. This sounds discouraging and overwhelming; however, great advances have been
made in understanding and treating CF. The goal of this thesis is to summarize what knowledge
has been acquired about CF and the progress that’s been made towards individualized treatments.
While writing this thesis, I read many scientific articles that dispassionately describe
what cystic fibrosis is and how we treat it. However, it is the stories of dedication and
perseverance that have driven me forward. They are the reason I chose this disease in the first
place and why I’ve compiled this summary of the disease and the current diagnosis and treatment
options.
4 Cellular & Molecular Basis of Cystic Fibrosis Genetics Cystic fibrosis (CF) occurs in patients with an autosomal recessive gene, meaning that he or she
inherited two recessive genes, one from each parent (5). Because CF used to result in mortality at
such a young age and some symptoms may include infertility, it is extremely rare for patients to
reproduce (18). Therefore, cystic fibrosis is most often passed from two parents without CF, but
who are carriers for the recessive gene linked to CF. In Caucasians, the population with the
highest prevalence of CF, the carrier rate is 1 in 25 people (4). The CF mutations may either be
homozygous or compounded heterozygous, meaning that there must be a mutation present in
each copy of the gene, but the mutation does not necessarily have to be the same. There are
thousands of mutations linked to cystic fibrosis and therefore this phenomenon is not uncommon
in cystic fibrosis patients (31). This means that cystic fibrosis patients present with a relatively
large variety of phenotypes (5).
The DNA present in each of our cells contains all of the genetic information that allows
for the creation of proteins that are responsible for all bodily functions. DNA is organized into
chromosomes, which are made of genes responsible for individual proteins. Using a technique
known as molecular cloning, Bat-sheva et. al. determined there is a single CF locus that exists on
human chromosome 7 (16). The CF gene is about 230,000 base pairs long with 27 exons (41).
Genetic information is transcribed into a messenger RNA molecule (mRNA), which can then go
through splicing modifications that act to remove unnecessary information. The mRNA functions
as the directions for the formation of the proteins. Each protein has a specific function
contributing to complex bodily functions. It is known that protein malformations can be caused
by issues at one (or more) of many stages in protein formation listed above; however, according
to Riordan et. al., the crucial error that leads to the symptoms in CF patients is isolated to the
5 genetic information (25). This means that the error occurs from the deletion of one or more base
pairs and the subsequent protein malformation is consistent in all types of epithelial cells of the
patient. This channel is responsible for the translocation of negatively charged chloride across
the plasma membrane in epithelial cells. It has been shown that there are thousands of mutations
on the CF gene that can lead to CF symptoms (21). Genomic DNA samples of CF patients and
their parents have been used to determine the frequency of the various types of mutations (8).
Classes of Mutations Cystic Fibrosis mutations are placed into classes based on the effects of the mutation on
the biogenesis of the cystic fibrosis transmembrane conductance regulator (CFTR) channel (32).
Class I CFTR mutations are characterized by a defective production of the CFTR protein, which
leads to no function, nor any expression of the CFTR protein at the membrane. Class II CFTR
mutations result in impaired processing of the protein. These mutations result in nonfunctioning
proteins that are not expressed at the plasma membrane; the ΔF508 mutation, to be discussed
later, is an example of this type of mutation. Next, class III mutations result in defective
regulation of CFTR. This also means the patient has no functioning CFTR channel, but these
channels are expressed in the membrane. Class IV mutations are characterized by the
conductance of the channel being defective. This mutation is the first, thus far, that results in
functioning CFTR; however, their function is significantly reduced. Lastly, class V mutations
produce CFTR that is translocated to the membrane but smaller amounts of the protein are
produced (32). Classes I-III are considered severe mutations and classes IV and V are considered
mild based on the phenotypes and symptoms that occur in the presence of the corresponding
mutations.
6 Figure 1. CFTR classes of mutations. ER=endoplasmic reticulum, PM= plasma membrane (32).
Biogenesis & Mutations The presence of certain mutations affects the biogenesis of CFTR. However, the normal
biogenesis of CFTR starts with the translation of the CFTR protein in the rough endoplasmic
reticulum (ER). Simultaneously a glycan (sugar molecule) is attached to a nitrogen atom of the
protein in a process called N-linked glycosylation. Also during translation the protein is folded
and refolded in a process that maximizes the bioenergetics of the system. ER-associated
degradation (ERAD) is constantly interacting with the protein, checking for deleterious errors.
This quality control process is highly effective and accounts for about 80% of the degradation of
abnormal CFTR proteins (32). If a protein is abnormal, like in the case of a ΔF508 mutation,
chaperone complexes lead to the ubiquination and decomposition of the abnormal CFTR (6).
Next, CFTR is transported to the Golgi complex via intracellular translocation pathways. Here,
CFTR is prepared for translocation to the plasma membrane. Further glycosylation occurs in the
7 Golgi complex. The transportation of CFTR to the plasma membrane is modulated by PDZdomain-containing proteins (32). The healthy biogenesis of CFTR will lead to functional
chloride channels, but if there is a mutation present some step of the biogenesis may be
disrupted.
Based on the putative frequency of the mutations and the results of the various types of
mutations, I have chosen to focus on four common mutations that also have four unique
mechanisms that all lead to CF symptoms and cover classes I-IV. The four mechanisms describe
at what point in the process of the protein formation and activation the error affects the function
of the protein. The genetic mutation will prevent the CFTR channel from forming properly,
prevent the formed channel from being delivered to the membrane, prevent the channel from
functioning within the plasma membrane or prevent full function of the CFTR channel in the
plasma membrane.
The first type, a class I mutation, is the R553X nonsense mutation. It has a high
prevalence in Hispanic populations and accounts for about 1% of CF patients (41). This
nonsense mutation indicates that there is a single base pair substitution at the 553rd amino acid,
where normally an arginine resides (32). In the case of this mutation, a premature stop codon is
coded and the protein is truncated before the complete formation of the protein. The premature
stop codon terminates the translation of the protein from mRNA, therefore forming an
incomplete protein that cannot fold properly or function as a normal protein. Mechanisms within
the cell recognize this protein as insufficient and destroy it before it can be transported to the cell
membrane. Due to this, there is a defective production of CFTR and no CFTR channels at the
apical membrane of the epithelial cells.
8 Another type of mutation is a class II mutation called the ΔF508 mutation and is the most
prevalent mutation, occurring in about 66% in CF patients (41). This mutation leads to the
deletion of three nucleotides that make up one codon. The deletion of this codon results in a
missing phenylalanine in position 508 in the first nucleotide-binding domain on the gene. The
missing phenylalanine prevents the protein from folding properly because the subsequent
charged residue has taken the place of the phenylalanine, which has a nonpolar residue.
Therefore, the charged residue does not interact with the other hydrophobic residues. These lack
of interactions result in an inability of the protein to fold properly, which traps the protein in the
endoplasmic reticulum and prevents any further processing. Similar to the protein that results
from the R553X mutation, the protein does not get delivered to the membrane and therefore
those cells are completely missing the CFTR channel protein, preventing the conductance of
chloride. In the case of both the R553X and ΔF508 mutations, the misfolded proteins are
recognized first by the endoplasmic reticulum quality control (ERQC) mechanisms. These
proteins do not pass the criteria of the ERQC and therefore are degredated by the endoplasmic
reticulum associated degradation (ERAD) pathway. This pathway first recognizes the protein as
misfiled in the endoplasmic reticulum, using cytoplasmic and luminal chaperones. Next, through
a process called retrotranslocation, translocation machinery or E3 ligases transport the erroneous
protein into the cytoplasm. Next, E3 ubiquitin ligases polyubiquitylates the protein, thus labeling
it for degradation (7). De-ubiquitylating enzymes recognize the label and guide the protein
through the catalytic region of a proteasome. This decomposes the CFTR protein into
polypeptide fragments, ready for reuse (40).
The G551D mutation accounts for the third most common CF mutation in the world and
affects approximately 4 to 5% of CF patients (23). This is a class III mutation and therefore
9 results in a translocated, but non-functioning protein (41). The G551D is a missense mutation; a
missense mutation is caused by an error in one nucleotide and results in the translation of a
serine instead of a glycine at the 551st amino acid position on the protein (32). This is in the first
nucleotide-binding site of the protein. This amino acid replacement leads to the insertion of
polar side chains into the ATP-binding site (43). This abnormal protein will significantly
decrease the channel functionality of the CFTR protein such that it is essentially nonfunctioning.
In this case, the CTFR protein is present in the plasma membrane but chloride translocation is
significantly diminished or stopped completely.
The last mutation type discussed here is the missense R117H mutation. This occurs
when an arginine is replaced by a histidine at the 117th residue (34). This in considered a class IV
mutation due to its decreased conductance of anions through CFTR. This mutation is completely
transcribed and makes it to the plasma membrane of the epithelial cells, like the G551D
mutation. However, this mutation is different because it is somewhat functional. There is
conductance of chloride across through CFTR, but it is abnormal. This abnormality is due to an
effect from the mutation on the second and sixth membrane-spanning domain. Therefore, the
regulation of the activity of this channel is normal, but it’s been found that there is a decrease in
the movement of chloride across the channel or a decrease in conductance (34).
Though there are more than a thousand mutations that result in cystic fibrosis, the
examination of the types of mutations has inspired individualization of treatment and research
for cures (27). By examining the genetic mutations, one can predict the changes in the structure
of the CFTR and therefore how its functionality is affected. Though these mutation lead to
similar symptoms, the identification of precise locations in the pathway will lead to treatments
that terminate or repair the occurring errors.
10 The CFTR Protein CFTR is located in epithelial cells including the lungs, skin, pancreas, liver and the
digestive tract. Specifically, CFTR resides in the apical side of the membrane of the epithelial
cells (32) and is responsible for the movement of chloride and other anions out of epithelial cells
and into the lumen of various organs. The energy stored in ATP allows for a conformational
change in CFTR that regulates the passage of the ions down their electrochemical gradient. The
decrease in chloride transport is coupled with an accelerated sodium absorption by the epithelial
cells. Water also follows these ions across the cell layer paracellularly. This movement of
sodium and therefore water away from the mucosal membrane is what leads to the thick mucous
characteristic in CF patients (2). Therefore, this channel also regulates the transport of salt, ions
and the flow of fluids across the epithelium. CFTR is termed an ABC transporter, because it is an
ATP-dependent cassette transporter (13) and has 5 crucial domains: one regulatory domain, two
nucleotide-binding domains (NBD) and two membrane-spanning domains (MSD) (35).
Figure 2. Proposed model of CFTR. MSD: membrane-spanning domain; NBD: nucleotidebinding domain; R: regulatory domain; PKA: cAMP-dependent protein kinase (35).
11 The mechanism of opening the normal CFTR channel begins with the phosphorylation of
the regulatory domain by protein kinase A (PKA) at various serine residues. The regulatory
domain controls the activity of the channel and therefore can be phosphorylated by molecules
other than PKA, which leads to stimulatory or inhibitory regulation of the channel (13). Overall,
the control of the activity of the channel is regulated by kinase and phosphatase activity within
the cell. After phosphorylation of the regulatory domain, ATP binds with the first nucleotidebinding domain. This ATP molecule then forms a dimer with the first and second nucleotidebinding domains at which point the ATP molecule is then hydrolyzed. This allows for the
activation of the chloride channel. Lastly, protein phosphatases dephosphorylate the regulatory
domain and CFTR returns to a quiescent state (13). In a healthy individual, the building,
translocation and proper gating of CFTR will result in very important mucus secretion
throughout the body. However, in patients with CFTR gene mutations theses process are altered
in a way that hinders the healthy mucus secretions.
The first two mutations discussed, the R553X and ΔF508 mutations do not produce
functional proteins due to a lack of production and impaired processing, respectively. However,
the G551D and R117H mutations produce proteins that make it to the membrane, but have a
lower functionality. In the case of the G551D mutation, glycine, a small nonpolar amino acid, is
replaced with a serine, a larger polar amino acid, at the 551st amino acid on the CFTR protein.
This is the location of the first nucleotide-binding site and is called a gating mutation because it
affects the regulation of the protein (32). The location of this mutation is particularly important
because it occurs at the interface between the first and second nucleotide binding sites. This
severely inhibits the ATP-dependent gating and decreases the probability that the channel will
open by 100-fold when compared to a healthy CFTR (32). This is due to a decrease in the
12 frequency of the opening of the channels (35). In this case, there is nearly no movement of
chloride through this channel.
In the case of the R117H mutation, arginine has replaced histidine at the 117th amino acid
(32) on the outside of the second membrane-spanning segment (35). Sheppard and Welsh
determined that the R117H mutated protein was still activated by cAMP, but that the
conductance was 30% less than the wild-type CFTR (35). It is believed that the pH outside of the
cell affects the conductance of these channels as well. This is because in an experiment with
Sheppard et. al. (1993) the external pH was greater than 8.8, the conductance of the channel
significantly dropped, but this was reversed when the pH was brought back to a physiological
standard (34). This suggests that the location of the amino acid replacement is likely on the
external portion of the protein. Because there is some conductance of chloride across the channel
the phenotypic differences between the R117H CFTR and the wild-type CFTR are milder than in
any of the other mutations discussed. This is true for most class IV and V mutations.
13 Diagnostics The diagnosis of cystic fibrosis is a systemic two tier analysis of test results. The testing may
begin before or after birth. Family history and early symptoms may also initiate the screening
process for cystic fibrosis patients and their families. Prenatally the parents may chose to
genetically test the fetus and this may be due to family history along with a variety of other
reasons. On the other hand, newborn screening (NBS) is required for all newborns in the United
States. Figure 1 outlines the screening process.
Prenatal Screening Newborn Screening (NBS) Genetic Testing IRT Normal Results Abnormal Results Normal Results No diagnosis yet, continue to NBS Notify the parents and discuss options No CF diagnosis Abnormal Results Repeat IRT (+SC test) Genetic Testing (+SC test) Normal Results Abnormal Results Normal Results Abnormal Results No CF Diagnosis CF Diagnosis No CF Diagnosis CF Diagnosis Figure 3. Screening for Cystic Fibrosis. The general protocol for CF screening in the United
States. There are variations such as different screening techniques or a change in order
depending on the particular patient.
Prenatal Screening Prenatal screening is the first opportunity for the diagnosis of cystic fibrosis. Genetic screening is
available and recommended for all pregnant women in the United States (3). The genetic test
identifies 23 different mutations of the CFTR gene. The genetic testing can involve identifying a
fetus and/or parents with one or two mutations of the CFTR gene. If parents discover that one or
14 both are carriers of CFTR gene mutations then they are provided educational material about the
possibility of having a child with cystic fibrosis. Because there are thousands of mutations and
mutation combinations that can cause a wide range in the severity of symptoms, at this stage
there is still a large amount of uncertainty. Some patients with a CFTR mutation are not widely
affected, but in other cases a patient may have significant morbidity (10).
While this knowledge allows doctors and family members to act promptly in order to
slow any symptoms of cystic fibrosis, there are downsides to prenatal genetic screening. For
those carrier couples, this discovery could lead to changes in reproductive plans. This could lead
to the termination of a pregnancy, if prenatal testing is completed, which has vast ethical
implications. Parental responses may lead to depression or other adverse effects. While these
possibilities should be carefully considered, one survey of parents of children with CF and adult
CF patients found that 80% supported preconception carrier screening for CF gene mutations
(14).
Newborn Screening (NBS) In the United States, there is a standard newborn screening for congenital genetic
abnormalities (3). This early detection has been shown to improve the outcomes for patients and
families due to earlier onset of treatment and preventative measures (42). Furthermore, patients
detected via newborn screening techniques had improved scores on the Schwachman-Kulczycki
(SK) score. The SK score is the first and most widely used test used to determine the severity of
cystic fibrosis symptoms. It is a useful tool for determining the progression or changes of the
disease in cystic fibrosis patients (36).
The disadvantages to NBS are similar to those of prenatal screening. This includes
depression and anxiety in parents possibly due to a misunderstanding of a genetic carrier
meaning. This depression and anxiety can lead to issues with parent-child bonding. Another
15 disadvantage is that these patients may be unnecessarily given medication in the cases of
diagnostic errors (30). Overall, however, the newborn screening have been found to lead to an
improvement in patient outcomes (3).
Typical Screening through IRT and DNA testing The newborn screening process involves two tiers. In the first tier, the functionality of
immunoreactive trypsionogen (IRT) is measured. IRT is a pancreatic exocrine biomarker that is
typically elevated in infants with partial or nonfunctional CFTR. Immunoreactive trypsinogen
has two isoforms, IRT-1 and IRT-2, cationic and anionic, respectively (37). Current techniques
that test for both of these isoforms requires blood spots followed by assaying the samples for the
presence of IRT (24). The threshold for IRT levels indicating the necessity for further testing
varies per state, country and tier two protocol, but is generally within the 94-98th percentiles or
greater than or equal to 65 µg/L of IRT (3). These values are debated because of the
inconsistency in results due to seasonal and reagent variations (17). Historically, IRT testing has
high rates of false positives, up to 85.9% in some studies (26). This level requires further testing,
the second tier of diagnostics. Some cases of CF do not present with elevated IRT due to
meconium ileus, a bowl obstruction due to thicker and sticker ileum (3). This is indicative of CF
and therefore prompts further testing.
The next tier of testing is either a repeat the IRT test or complete a DNA test. The NBS
genetic tests for CF are very similar to the prenatal genetic screening described above.
Additionally, in either protocol, a sweat chloride (SC) test is also completed in order to confirm
the results. SC tests are well established as an assessment for CFTR abnormality and the
diagnosis of CF. Since CFTR is involved in sodium reabsorption, when it is not fully
functioning, sodium is not reabsorbed and the skin sweat has excess sodium. Many mothers will
first notice something when they kiss their baby and he/she tastes salty. This test stimulates
16 sweating and measures the sodium concentration. It is measured against standards to determine if
sodium levels are elevated. These observations can be somewhat subjective, as the “standards”
vary somewhat dependent on the state or country. The SC content also varies by age and
therefore there are different standards for those under 6 months. While this test is a good tool for
diagnosis of CF, there are multiple conditions under which a patient might get a false positive SC
test, which include: anorexia nervosa, autonomic dysfuction, Mauriac’s syndrome, nephrogenic
diabetes insipidus and psychosocial failure to thrive (3).
Fecal Elastase While these are the most commonly used and well known diagnostic tools, there are a
few others that can help to confirm a CF prognosis. The first is the measurement of fecal elastase
(FE). A positive result from the FE test would suggest pancreatic dysfunction. A common result
of non-functioning CFTR proteins. It is important to note with this test that the fecal elastase
levels vary during the first year of life, and a follow up test should be completed after 1 year of
age to decrease the chances of a false positive that leads to an incorrect diagnosis.
Nasal Potential Difference Another test, is the nasal potential difference (NPD) test. This test is often used in clinical
research to measure restored CFTR function, but can be used for diagnostics as well. This
process involves placing electrodes across nasal mucosa which measure the potential difference
as various solutions at set concentrations are perfused. Since, CFTR is responsible for chloride
(an anion) transport, the electrodes are able to pick up on differences with high sensitivity (28).
The benefits of this technique is the sensitivity to changes in potential difference of the nasal
mucosa, however, there are yet to be standardized procedures. This means that the results may be
interpreted differently or may not be comparable due to difference is protocol.
17 Treatments There are two main categories for cystic fibrosis treatment; treatments that target the
initial defect that is causing the malfunctioning protein and treatments that target symptom
management. In this paper, we delve into the basic defects, the treatment of respiratory
symptoms and the nutrition requirements. Both, the basic defect and symptom treatments, are
very important for the progress in CF treatment research, as one helps decrease the symptoms a
patient has and the other helps manage what symptoms they do have. Recently, there has been an
increase in research producing results suggesting individualized treatments for the initial defect.
This is crucial because unlike some genetic diseases, there are many combinations of mutations
that lead to CF through unique mechanisms. The development of these targeted treatments is
showing promise for not only CF treatments, but also other genetic diseases.
Basic Defect Treatments There are four major therapies that attempt to correct or make up for the CFTR
mutations: CFTR potentiators, CFTR correctors, gene therapy and translational readthrough.
These function through different mechanisms to increase the correct cellular processing and
delivery of the CFTR protein. The goal is to have treatments for all classes of CF mutations, but
due to the large variation between classes, most treatments are especially beneficial for a single
or couple classes.
CFTR Potentiators One key treatment that works to remedy the basic defect of CFTR is CFTR potentiators.
This treatment acts to activate pre-existing CFTR channels in the cell membrane. This is
particularly useful for Class III mutations, such as the G551D mutation discussed above, but may
be used for Classes I, II and IV (29). The mechanism of CFTR potentiators is to increase the
likelihood of cAMP dependent channels opening by decoupling with ATP hydrolysis (38). This
18 therapy potentiates CFTR function by increasing the open time of healthy and mutant CFTR
proteins already active in the cell membrane and is hypothesized to be because of the hydrolysis
of more than one ATP molecule per one opening/closing event (15). There are still unknowns
about the mechanism of specific CFTR potentiators such as Vx-770 and ivacaftor, but it is one
FDA approved therapy that targets the issue at a cellular and molecular level as opposed to
treating symptoms (29).
CFTR Correctors The goal of a CFTR corrector is to restore the processing of CFTR. While CFTR
potentiators work to increase the function of already existing CFTR, correctors work to increase
the number of CFTR that make it to the membrane. This is a particularly popular area of research
because the mutation ΔF508del, one of the most common CF mutations, is caused by an error in
CFTR processing, more specifically CFTR folding. Some forms of the correctors work to down
regulate cellular chaperones, whose functions are to breakdown misfolded proteins (39). Another
attempted mechanism is increasing the half life of CFTR so that it has more time to travel to the
cell membrane. Most of these therapies are attempting to target cellular pathways that prevent
CFTR from making it to the cell membrane due its misfolded nature. Some other CFTR
correctors focus on supporting the folding of CFTR. For example, trafficking assays are shown
to increase the translocation of misfolded CFTR to the membrane (29).
Gene Replacement Next, there is active research on gene replacement through viral and nonviral gene
therapies. The focus of the gene therapy is in the lungs due to the high mortality and morbidity
associated with lung pathologies that arise in CF patients. This is a difficult place to focus the
studies because of the lack of success in gene transfer into the lungs caused by the high number
of immunity barriers, both intracellular and extracellular (11). These studies would allow for
19 targeted therapies that are unique to the patient due to the complex heteroxygous combinations of
CFTR mutations. Similar to gene therapy, there is research examining the expression of CFTR
through mRNA transduction. This would allow for the proper processing and translocation of
CFTR despite mutated genes. There has been some research suggesting this may be a viable
treatment, as it has been shown to restore cAMP-induced CFTR currents similar to those in
healthy tissue (1).
Translational Readthrough Another therapy, translational readthrough, targets premature stop codons that occur in
nonsense mutations. This therapy has been explored for CF and other genetic diseases caused by
nonsense mutations (29). Results have shown that therapies such as aminoglycoside antibiotics
allow for the expression of full length CFTR (12). This mechanism occurs because the
aminoglycoside antibiotics interact with a proof-reading portion of the ribosome and decreases
its ability to properly recognize premature stop codons (29). There is also evidence that this
mechanism does not cause elongation of mRNA, which was a major concern for this type of
treatment (33). This treatment type has also been approved for other genetic diseases also caused
by nonsense mutations, such as Duchenne muscular dystrophy and Hurler syndrome (22). There
is an increase in research attempting to prevent or remedy the initial defect causing CF and many
of them are showing promising potential for individualized treatment for CF and other genetic
diseases.
Respiratory Treatments While the basic defect treatments attempt to resolve the cause of cystic fibrosis, the rest
of the treatments here focus on treating the symptoms that lead to the morbidity and mortality
associated with CF. The main therapies focus on treating the lung symptoms, as those cause the
20 most complications for patients. These complications arise from the basic defect, in which the
lack of functional chloride channel in the submucosal glands of the lungs lead to a lack of
hydration in the lungs. This causes thick, difficult to move, mucus and an increase likelihood of
respiratory infection and disease.
Airway Clearance Therapy The first therapy is part of the daily routine of CF patients: chest physical therapy. This
process commonly utilizes percussion and postural drainage. This has clinically been shown to
be beneficial for most patients, however, it requires assistance and therefore adult patients often
seek more independent solutions (19). This has led to chest physiotherapy vest clearance systems
that work similarly to loosen mucus so that it can be better cleared by the mucocilliary escalator.
This is completed by the patient wearing a vest that compresses the chest and can be used during
simultaneous nebulized medication administration, allowing for a more complete treatment (44).
These physical therapies, in the form most suitable for the individual patient, are a crucial daily
part of every CF patients treatment. While there are few completed clinical trials about chest
physiotherapy, the clinical anecdotal evidence is overwhelming and is considered standard of
care (29).
Another therapy used for airway clearance is positive expiratory pressure (PEP). A PEP
device is placed on the patient and he/she respires for 12-15 breaths, then the device is removed
and the patient performs 2-3 forced expiratory breaths. The device provides back pressure into
the patient’s lungs, causing the build-up of gas. This leads to increased clearance of mucus and
functional residual volume over the 12-15 breaths. The force expiration assists in the movement
of mucus. This method has shown similar results to other chest percussion therapies (20). There
is still research being done on the effectiveness of each the individual airway clearance therapies,
but it is evident that they are an important part of CF treatment.
21 Prevention & Treatment of Respiratory Disease The primary cause of mortality and in CF is due to obstructive respiratory disease and
therefore eradication of respiratory infection is vital. Certain types of bacteria, such as
Pseudomonas, are particularly opportunistic and dangerous for CF patients (29). The treatment of
these infections involve the identification of the bacteria via sputum cultures or diagnosis by
symptoms and administering the proper antibiotic or specifically anti-pseudomona (44). Another
issue that arises, is that CF patients often contract antibiotic resistant strains. In this case,
combinations of antibiotics may be administered in order to treat the infection.
In some cases, chronic suppressive antibiotic therapies are employed in order to curb or
control chronic infection. This treatment is more aggressive and can involve administering
aerosolized antibiotic, such as tobramycin (TOBI). Results have shown an increase in FEV1 in
CF patients, but there is concern that this type of treatment with any aerosolized medication
could lead to antimicrobial resistance. While there is no evidence of this yet, further longitudinal
research is heavily anticipated (44).
Nutrition The unique nutritional necessities originates from pancreatic insufficiency that occurs in
90% of CF patients (29). Nutritional requirements are highly individualized and regulated, as the
variety of mutation combinations is responsible for the large variation in the pancreas’ ability to
produce the proper enzymes. Similar to in the lungs, the lack of or presence of nonfunctional
CFTR causes thick secretions and in the pancreas this may result in insufficient enzyme
secretion. This presents as failure to thrive in infants and is remedied by daily enzyme
supplements. This is part of the standard of care for CF patients (29). Similarly, Vitamin A and E
are often depleted in untreated CF patients. Other deficiencies may include vitamin D, and K. All
vitamin deficiencies are corrected through dietary supplementation through daily multivitamins.
22 Other dietary issues may arise and therefore continuous monitoring of patients nutritional status
is important. This will also allow for early detection and correction of potentially dangerous
malnutrition or dyslipidemias (44).
23 Conclusion Cystic fibrosis is a superb example of how focused motivation can produce great results.
Within the last 20 years, the advancements in the knowledge of the basic defect of CF and the
possibilities for treatment have grown astronomically. This has lead to an increase in the life
expectancy of CF patients of over 15 years and this is just one success that CF research has
provided. The research done for CF is now applicable to multiple types of genetic diseases. The
entire CF community, the researchers, physicians, patients, caretakers, consistently demonstrate
perseverance that has resulted in targeted drugs that act on the specific defect in order to reverse
or prevent the issues that arise from the defect.
Acknowledgements Throughout the thesis project, I have had an incredible team of mentors that I would like
to sincerely thank. First, Dr. Doug Keen for consistently being available and encouraging
throughout the thesis writing process. His feedback has been invaluable and allowed me to grow
as a critical thinker. Next, I would like to thank Dr. Mark Brown for his insight into cystic
fibrosis. He has been able to support me in my research by providing clinical applications and
knowledge. Lastly, Dr. Cindy Rankin has provided unquantifiable support throughout my last
year as an undergraduate. Thank you to all of my mentors!
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