REVIEW
URRENT
C
OPINION
Cystic fibrosis in the era of genomic medicine
Carlos E. Milla
Purpose of review
The field of cystic fibrosis (CF) is changing dramatically as the scientific knowledge accumulated since the
cloning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is being translated into
effective therapies to correct the basic defect and provide better disease models and in-depth
understanding of the basic mechanisms of disease.
Recent findings
This review focuses on three main aspects of the recent advances in the field: understanding the lung
disease pathophysiology (in particular, the early events that condition its onset), better definition of the
complex microbiology of the CF airway, and therapeutic developments. Although the most recently
developed therapies, whether approved or under study, do not constitute a definitive cure, the benefit to
patients is already becoming clearly apparent.
Summary
As the field continues to change rapidly and new therapies are being identified, CF has become a
paradigm for the application of concepts such as translational medicine, genomic medicine, and
personalized care, with measurable clinical benefit for the patients affected by this disease.
Keywords
CF airway microbiology, CF correctors and potentiator therapies, early lung disease
INTRODUCTION
The spring of 2013 will mark the 10th anniversary of
the announcement of the completion of the human
genome project [1], an international research effort
to sequence and map all of the genes (together
known as the genome) of humans [2]. It was no
coincidence that this occurred on the date of the
50th anniversary of the publication of the landmark
study by Watson and Crick, who described DNA’s
structure as a double helix [3]. The sequencing of the
human genome was enthusiastically welcomed as
heralding the dawn of a new era in medicine, where
the use of specific genetic knowledge was to inform
the delivery of effective healthcare [4]. In parallel to
this, the development of multiple other ‘-omic’
fields (e.g. proteomics, metabolomics, and transcriptomics) have led to the ability to generate massive amounts of information at the individual level
that could be applied to the detection, understanding, and monitoring of disease states [5]. The field of
cystic fibrosis (CF) has certainly benefited from
these developments. We have witnessed tremendous advances in the diagnostic strategies and our
understanding of the disease pathophysiology, and
gained better insight into the microbiology of CF.
Perhaps of greater importance, we have also
witnessed the success of the first disease-modifying
drugs aimed at the basic defect. This review will
attempt to summarize the recent advances in the
field that have capitalized on the promise of a new
era of ‘genomic medicine’.
LUNG DISEASE PATHOPHYSIOLOGY
Although generally infants with CF are considered
to be free from any lung disease, a substantial body
of evidence has accumulated to suggest that the
pulmonary manifestations of the disease have a very
early onset, if not at birth itself. The earliest systematic descriptions of the lung pathology in young
children with CF pointed to focal changes at the
small airways level, with the presence of mucus
plugging and dilatation of the airway lumen, in
addition to hyperplasia of the bronchial glands
[6]. Several studies conducted in asymptomatic
Correspondence to Carlos E. Milla, MD, Department of Pediatrics,
Center for Excellence in Pulmonary Biology, Stanford University, 770
Welch Road, Suite 350, Palo Alto, CA 94304, USA. Tel: +1 650 736
9824; e-mail: [email protected]
Curr Opin Pediatr 2013, 25:323–328
DOI:10.1097/MOP.0b013e328360dbf5
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Pulmonology
KEY POINTS
Although structurally normal at birth, an inability to
control bacterial infection plays a key role in the early
CF lung disease pathogenesis.
chronic infections in CF patients. Additional studies
with the CF pigs have also provided insight into
another significant respiratory problem in CF
patients: sinus disease. Though free from infection
and inflammatory changes, the sinus cavities of CF
piglets are found to be hypoplastic [20 ]. Infection
becomes established later and adult CF pigs present
all of the features seen in the human disease. An
investigation of the development of chronic Pseudomonas infection in the Danish CF patient cohort
provides further insight by demonstrating that the
paranasal sinuses constitute a niche for early infection and the source of lower airway infection. Of
interest, the investigators also found a number of
phenotypic adaptations of the Pseudomonas colonies
in the sinuses that preceded the lower airway colonization [21]. Taken together, these two studies
provide evidence for an abnormal milieu in the
sinuses that provides an initial niche for the evolution of the Pseudomonas infection.
Early and subtle small airways abnormalities are
unlikely to produce any noticeable symptoms. This
disconnect between the presence of pathological
changes and symptomatology implies that more
sensitive diagnostic methods must be employed to
detect the presence of airway disease to clearly
identify the presence and cause of the earliest pathologic events. Given the challenges posed by young
age and consequent lack of cooperation in this
patient population, innovative methodologies have
been applied to the study of airway disease in infants
with CF. As a result, it is increasingly accepted that
with the application of the appropriate techniques it
is quite feasible to detail even early lung disease in
these infants. Both functional and imaging methodologies have been effectively applied and have
yielded important information in longitudinal
studies. From a functional perspective, multiple studies have clearly established that infants with CF have
lung function abnormalities that result from airway
obstruction and that these abnormalities persist in
the later years of life [22–25]. More recent studies
applying less invasive techniques such as multiple
breath washout methodologies with the use of tracer
gases have provided further insight into these abnormalities [26]. These studies demonstrate that regional
small airway disease results in ventilatory inhomogeneity and this occurs early, well prior to being
detectable by forced expiratory maneuvers [27 ,28].
In addition, the presence of Pseudomonas infection is
associated with abnormalities in Lung Clearance
Index (LCI) [29], a well-validated surrogate measure
of ventilatory homogeneity [30]. Findings from the
functional studies have been corroborated by structural studies with the use of computerized tomography (CT) imaging. If anything, data available
&
The application of novel molecular techniques to
explore the human microbiome has produced
paradigm shifting information on the CF airway
microbiology.
With the approval of the drug Ivacaftor, the concept of
using small molecules as CFTR corrector and potentiator
therapies has been proven as a valid strategy to
produce meaningful clinical benefit.
infants with CF identified by newborn screening
have repeatedly demonstrated the presence of active
inflammation and infection in the lower airway
[7–12], and their association with progression in
the pulmonary disease [13]. The availability of a
large animal model of CF lung disease through
the generation of transgenic pigs with the disease
has deepened our understanding of the earliest
events in CF lungs [14,15]. Piglets with CF present
at birth with severe bowel obstruction and if rescued
from this go on to develop severe lung disease
[14,16]. Although no abnormalities are detected in
their lungs at birth, shortly after CF pigs develop all
the hallmarks of CF lung disease, including mucous
accumulation, inflammation, and infection. Interestingly, although both control and CF pigs have
bacteria detectable in the lower airway shortly
following birth, the CF pigs seem unable to eradicate
the bacteria from their lungs, suggesting a strong
role for unique host–environment interactions at
the genesis of CF lung disease [16]. Then, this animal
model supports the notion that the development of
lung disease has its onset much earlier than anticipated and likely as a consequence of a failure to
eliminate bacteria from the airway. These observations have been further clarified by a recent study
with this pig model of CF identifying a defect in the
ability of the airway mucosa to eradicate the bacteria
[17 ]. Compared with wildtype pigs, airway surface
fluid from CF pigs has a lower pH and reduced
antimicrobial activity. Further, increasing the pH
rescued this defect and restored the antimicrobial
activity of the airway surface fluid. Given that the
cystic fibrosis transmembrane conductance regulator (CFTR) is already recognized as participating in
bicarbonate transport [18,19], the results of this
study provide a direct mechanistic explanation for
the failure to clear infections known to occur in the
CF airway and suggest, perhaps, a potential novel
therapeutic intervention to prevent the serious
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Cystic fibrosis in the era of genomic medicine Milla
from several studies seems to indicate that functional
and structural studies provide complementary information [31,32]. Perhaps the best body of evidence has
come from investigators in Australia, who demonstrated by the use of CT scan imaging methodology
the presence of bronchiectasis at an early age in
patients identified by newborn screening [33]. A
recent follow-up study to their initial observations
on this cohort demonstrated the longitudinal progression of these abnormalities [34 ]. Notable findings in this study included not only the persistence
and progression of bronchiectasis and air trapping,
but also the association of this progression with
worsening of infection and neutrophilic inflammation in the lower airway. Whether functional
studies could be used to screen those patients in
whom additional valuable information could be
obtained with imaging studies has not been explored.
This is an important question as, despite the refinements in technology, imaging studies still involve
radiation exposure. A recent panel convened by the
US National Heart, Lung, and Blood Institute to
identify priority areas for the study of early CF lung
disease highlighted the need to develop novel biomarkers and outcomes measures in order to gain
further insight into the pathophysiology of early lung
disease and, perhaps more importantly, to identify
effective interventions in the youngest patients [35].
&&
CYSTIC FIBROSIS MICROBIOLOGY
Despite the major advances in CF care, progressive
lung disease in association with chronic infections
remains the major cause of morbidity and mortality
in CF [36]. It is also clearly recognized that the
presence of infection in the airways is a dynamic
process that evolves as the patients age [37], likely
a product of multiple factors including adaptation
of the bacteria to the airway environment and
response to the antibiotic therapy [38,39].
Frequently infections become established silently
and even if detected they could persist in spite of
appropriate application of antibiotic therapy. In
addition, even when infections can be clearly
demonstrated, it is also recognized that the clinical
response to the most appropriate antibiotic regimens cannot be fully predicted and tremendous
variability exists from patient to patient [40].
Further, it is a common observation in the clinical
setting that antibiotic therapy confers clinically
detectable benefit even in the context of continuing
infection with antibiotic-resistant organisms [41].
This apparent paradox to the traditional paradigm
of infectious diseases wherein pathogenesis derives
from a primary and identifiable organism that can
be effectively targeted has been difficult to explain.
To reconcile this paradox, a complex multifactorial
process has been proposed in which the conditions
of the airway environment itself, the host inflammatory response, and the characteristics of the
offending microorganisms, including phenotypic
changes with reduced expression of virulence genes
and adaptive mechanisms, determine the disease
severity [42–45]. However, this is still based on a
paradigm of a sole causative, or at least dominant,
microorganism and ignores the possibility of a more
complex microbial community present in the
airway. The availability of nonculture-based microbial identification methodologies has opened the
possibility of performing a more comprehensive
assessment of the microbial ecology in different
niches of the human body [46]. Recently, the application of microbiological identification tools based
on 16S rRNA gene analysis has revealed a highly
complex microbial ecology in the CF lung [47–56].
Additionally, evidence has become available for the
presence in the lower airway of non-CF healthy
individuals of low levels of bacteria found to be
present also in the upper airway [57]. This suggests
that bacteria are not only frequently reaching the
lower airway, but also effectively cleared, a condition that is known to be defective in CF patients.
The assessment of the microbial communities
present in the airways of CF patients has been rapidly growing [44]. A recent comprehensive quantitative analysis of the CF pulmonary microbiome
revealed the presence of a specific signature not seen
in healthy controls [58 ]. The pattern identified
revealed not only the predominance of certain
microbial families, but also the absence or decreased
abundance of several families identified in the
healthy controls. Further, a significant association
between microbial diversity and clinical parameters,
including inflammatory biomarkers, was apparent
among the CF patients. Additional studies have
expanded on this knowledge by starting to reveal
important interactions at the microbial community
level, including the presence of fungal species [59],
spatial heterogeneity in the distribution of the
microbial communities in the lungs [60], loss of
community diversity with disease progression
[61 ], and close longitudinal relationships in the
evolution of the lung and the gut microbiome [62].
Even so, the development of the CF lung microbiome from the time of birth, as well as the local and
systemic host responses triggered as the immune
system develops, has not been explored fully yet.
With the molecular and genomic tools available,
there is a great opportunity at hand to understand
the composition of microbial communities in the
CF airway, how their development influences
the progression of lung disease, and how the
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Pulmonology
composition of these communities has an effect
on the differential responses to therapeutic intervention.
THERAPEUTIC DEVELOPMENTS
Perhaps the most remarkable recent advance in CF
pertains to the approval of the first drug targeting
the basic defect. Ivacaftor (N-[2,4-di-tert-butyl-5hydroxyphenyl]-1,4-dihydro-4oxoquinoline-3-carboxamide) received approval by the Food and Drug
Administration in January 2012 for the treatment of
CF patients carrying at least one copy of the G551D
mutation. The approval of this drug followed from
unprecedented efficacy results in short-term [63]
and long-term [64 ] clinical trials. The drug is a
small molecule identified through high-throughput
screening (HTS) with the use of an in-vitro cell
reporter system [65]. The application of HTS for
drug discovery in CF followed the development of
the methodology to screen in cell cultures compounds with activity on ion channels [66]. The
ability to screen thousands of chemical compounds
rapidly permitted testing the concept that CFTR
function can be restored by rescuing mutated CFTR
protein from degradation and potentiating its
activity in the apical cell membrane [67]. A number
of compounds and chemical classes have been
identified utilizing this strategy and have validated
the use of this approach for drug discovery in CF
[68–71]. As a result, at least two additional compounds are currently far along in clinical trials. The
drug VX-809 has already been demonstrated in vitro
to have activity as a corrector of CFTR with the
F508del mutation [72]. Although the F508del
mutation is already well demonstrated to affect
CFTR folding, thereby preventing its trafficking to
the apical membrane, recent data suggests that even
if rescued the protein will have decreased activity and
perhaps also a low residence time in the membrane.
Not only are the thermodynamics of the protein
significantly affected by the F508del mutation, but
also the interaction of the first nucleotide-binding
domain with adjacent transmembrane spanning
domains is disrupted, which results in defective transport function [73,74 ,75,76]. Thus, it is not surprising
to learn from the results of a recently reported clinical
study that, among patients homozygous for F508del
treated with VX-809, minimal effects in sweat
chloride values and no other significant effects were
noted after 28 days of treatment. Then, a combination therapy approach with the use of a corrector
such as VX-809 and Ivacaftor as a potentiator has
been proposed, and is currently under test in clinical
trials. Preliminary results of an ongoing clinical trial
in CF patients homozygous for the F508del mutation
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have already shown some promising results [77]. A
second corrector agent, VX-661, is also being investigated in clinical trials in combination with Ivacaftor
as a second corrector–potentiator therapeutic regimen (NCT01531673). The results of these studies are
highly anticipated.
An additional therapeutic under development
targets CFTR mutations that result in a premature
termination codon (PTC). The drug Ataluren is
being developed as a suppressor of PTC mutations.
Early small clinical studies with CF patients carrying
at least one copy of a PTC mutation showed promising results, with detectable effects in biomarkers of
CFTR function such as the nasal potential difference
[78–80]. However, a larger international study with
longer-term exposure to the drug did not find a
meaningful effect on pulmonary function or exacerbation rate.
In addition, the application of in-vitro cell assay
platforms has helped to identify other CFTR
mutations in which Ivacaftor could potentially have
a beneficial effect [81 ,82]. This has also presented
the possibility of utilizing in-vitro cell-based assays
to identify patients who could potentially benefit
from novel CF therapies. Given the low frequency of
many mutations known to be associated with CF,
this could represent a more efficient approach
instead of embarking on the traditional path of large
clinical trials, which will be impossible to conduct
for rare mutations.
In addition to the benefits realized by a small
group of patients, the success in the development of
Ivacaftor can also be taken as a successful proof of
concept. This has demonstrated that the CF basic
defect is amenable to targeting for effective restoration of CFTR function and that, as a result, important clinical benefits are realized. As important, the
experience acquired during the Ivacaftor clinical
trials has also helped identify the utility of physiologic biomarkers, such as sweat chloride and nasal
potential difference, in their ability not only to
detect meaningful effects, but also to predict clinical
benefit.
&
CONCLUSION
The field of CF is undergoing dramatic and rapid
change that takes full advantage of the fast pace at
which multiple ‘-omics’ platforms continue to
develop. This has produced not only a more detailed
understanding of the basic mechanisms of disease,
but also the first-in-class drugs directed at the basic
defect. Clearly, the basic defect in this disease can
be effectively targeted for correction with small
molecule drugs, and many of these novel drugs will
have a mutation-specific effect. In addition, our
Volume 25 Number 3 June 2013
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Cystic fibrosis in the era of genomic medicine Milla
understanding of the disease pathogenesis has been
better informed as novel tools to dissect the basic
molecular mechanisms of protein dysfunction and
improved animal models have become available.
The promise of genomic medicine and personalized
care is then being fully realized by patients and
families affected by CF.
Acknowledgements
None.
Conflicts of interest
There are no conflicts of interest.
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human airway microbial ecology reveals a pervasive signature for cystic
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This study provided a comprehensive quantitative characterization of the microbial communities present in the CF airway in comparison to healthy controls and
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Volume 25 Number 3 June 2013
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