Download transepithelial nasal potential difference npd measurements in cystic

Developmental Period Medicine, 2013, XVII,131
© IMiD, Wydawnictwo Aluna
E DI TOR I A L
TRANSEPITHELIAL NASAL POTENTIAL DIFFERENCE
NPD MEASUREMENTS IN CYSTIC FIBROSIS CF
Dorota Sands
Cystic Fibrosis Centre,
Institute of Mother and Child, Warsaw, Poland
Abstract
The main underlying physiologic abnormality in cystic fibrosis (CF) is dysfunction of the CF transmembrane conductance
regulator (CFTR), which results in abnormal transport of sodium and chloride across epithelial surfaces. CFTR function
could be tested in vivo using measurements of nasal transepithelial potential difference (PD). Nasal measurements show
characteristic features of CF epithelia, including hyperpolarized baseline readings (basal PD), excessive depolarization in
response to sodium channel inhibitors, such as amiloride (∆Amiloride), and little or no chloride (Cl–) secretion in response to
isoproterenol in a chloride-free solution (∆Cl– free-isoproterenol). PD test is applied for CF diagnosis and monitoring of new
therapeutic modulations and corrections.
Key words: cystic fibrosis, nasal transepithelial potential difference (PD)
DEV. PERIOD MED., 2013, XVII, 1, 1317
1. BACKGROUND
Cystic fibrosis (CF) is the most common severe
autosomal recessive disorder among Caucasians.
CF results from mutations in the gene that encodes
a protein-the cystic fibrosis transmembrane regulator
(CFTR). CFTR is a chloride (Cl–) channel located at the
apical membrane of exocrine epithelial cells (1). As an ion
transporter, it promotes chloride efflux and secondarily
inhibits sodium influx via the epithelial sodium channel
(ENaC). Its dysfunction in CF leads to increased sodium
absorption and impaired chloride secretion. The resulting
mucus dehydration compromises mucociliary clearance. CF
airways become obstructed, more vulnerable to infection
and inflammation ultimately leading to their occlusion
and destruction. Obstruction of pancreatic ducts, the
biliary tract, and the vas deferens can occur. CF patients
typically develop persistent pulmonary infections and often
have pancreatic insufficiency, diminished weight, chronic
hepatobiliary inflammation, and male infertility. Respiratory
failure is the most common cause of death.
At present, treatment of CF is mainly symptomatic,
aimed at improving mucociliary clearance and treating
infection (1, 2).
2. CF DIAGNOSIS
The diagnosis of cystic fibrosis (CF) is usually made
by clinical findings or newborn screening, sweat chloride
concentration and genetic analysis.
There is great heterogeneity in the clinical manifestations
of cystic fibrosis (CF). Some patients may have all the
classical manifestations of CF from infancy and have
a relatively poor prognosis, while others have much
milder disease manifestations. According to the European
Diagnostic Working Group terminology patients are
diagnosed with classic or typical CF if they have one or
more phenotypic characteristics and a sweat chloride
concentration of >60 mmol/l (3). Patients with classic CF
can have exocrine pancreatic insufficiency or pancreatic
sufficiency. The disease can have a severe course with
rapid progression of symptoms or a milder course
with very little deterioration over time. Patients with
non-classic or atypical CF have a CF phenotype in at
least one organ system and a normal (<30 mmol/l) or
borderline (30-60 mmol/l) sweat chloride level. In these
patients confirmation of the diagnosis of CF requires
detection of two disease causing mutations or direct
quantification of CFTR dysfunction by nasal potential
difference measurement (PD). Non-classic CF includes
patients with multiorgan or single organ involvement.
Most of these patients have exocrine pancreatic sufficiency
and milder lung disease.
Most men with CF are infertile due to obstructive
azoospermia, which in its severest form presents as congenital
bilateral absence of the vas deferens (CBAVD) (4, 5). CBAVD
also occurs in 1 to 2% of infertile males who are otherwise
healthy, the majority of whom carry CFTR gene mutations
on one or both alleles (6, 7). Higher frequency of CFTR gene
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mutations have been also observed in patients with other
single organ diseases, like idiopathic chronic pancreatitis,
chronic rhinosinusitis, disseminated bronchiectasis (8).
These are clinical entities associated with CFTR dysfunction
named CFTR-related disorders (CFTR-RD), where the
diagnosis of CF cannot be unambiguously established.
They are all listed on WHO diagnostic list for single organ
disease phenotypes associated with CFTR mutations (9).
It is likely that this classification will need further revision
in the future as our knowledge and understanding of these
conditions increase.
Muta!ons analysis
The heterogeneity of cystic fibrosis (CF) disease is partially
explained by this high number of different mutations in the
CFTR gene (10). More than 1900 sequence variations have
been reported in the CFTR gene including missense, small
deletion or insertion, frameshift, and nonsense mutations,
often with geographic or ethnic variations in frequency
(2). The classification of CFTR gene mutations in five
classes according to their functional effects on CFTR
protein production and function is based on functional
studies: severe vs non-severe.
When patients who are homozygous, or compound
heterozygous for a class I-III CFTR mutation, are compared
to patients who carry at least one class IV-V CFTR mutation,
the latter group tends to have less severe disease (11).
However, it is only partly predictive of individual
outcomes, because many CFTR mutations have different
functional consequences and cannot be assigned to one
particular class and only a limited number of mutations have
been studied. The CF phenotype is affected by the CFTR
genotype as well as by other genetic and environmental
factors.
Extensive mutation analysis cannot guarantee detection
of the two disease-causing mutations in all patients; 1-5%
of alleles remain undetermined in CF patients with the
classical form and even more in patients with atypical
presentations.
The limitations of genetic testing include an inability
to detect mutations in the noncoding and promoter
regions of CFTR.
Mutation analysis is not the answer to every diagnostic
dilemma.
The sweat test has been used for more than 50 years
for the diagnosis of cystic fibrosis (CF) and remains an
important diagnostic test in the genomic era. It has been
documented that sweat chloride is related to the abnormal
function of CFTR and shows good discrimination for
the diagnosis of CF (12). However, most studies did not
include a ‘healthy’ control group, were performed prior
to the availability of the CFTR gene mutation analysis
and did not comply with the currently accepted sweat
test method. There is no definitive study that quantitates
the sweat chloride and sodium concentration in a truly
normal population. Further work is required to re-establish
sweat electrolyte reference intervals, using the current
standardized sweat test, CFTR gene mutation analysis
to exclude carriers, and correct statistical analyses in
healthy participants with ages spanning from infancy
to adulthood.
Intestinal current measurements (ICM) is one of
the alternative methods for demonstration of defective
ion transport. It can be carried out on suction biopsy
tissue obtained from the colonic mucosa. Biopsy tissue
is mounted in an Ussing chamber, the transepithelial
electrical activity, expressed as the short-circuit current
(SCC), measured and the response to secretagogues
tested. The normal intestine generates a rise in SCC,
a reflection of increased Cl– secretion, when secretagogues
are added, but this response is impaired in CF tissues
(13, 14).
Nasal potential difference (NPD) measurement
is an electrophysiological test that was developed by
Knowles et al. (15) and is a part of the criteria for CF
diagnosis according to the CFF consensus statement ,
also in European consensus cited above (16). This test
assesses CFTR activity by the change in the transepithelial
potential difference as a reaction to superinfusion of the
nasal mucosa by several solutions (17).
It is used to demonstrate abnormal function of the
CFTR protein, establish a diagnosis of CF in patients
with atypical presentation and as a surrogate outcome
measure for new therapies that correct the airway ion
transport defect. CF nose and lower airway demonstrate
similar abnormalities of ion transport.
3. PRINCIPLES OF THE PD
MEASUREMENTS
Patients with CF have a characteristic pattern of
bioelectric properties which reflects defective cAMPmediated (CFTR) Cl– secretion and accelerated rate of Na+
transport across ENaC. Increased sodium absorption is
reflected by a basal hyperpolarization of the nasal mucosa
(a large negative PD) together with an increased response
to amiloride, which blocks ENaC. The defect in chloride
transport is demonstrated by a reduced or abolished
repolarization in response to both an electrochemical
gradient favorable to chloride efflux during perfusion
of the mucosa with chloride free solution and addition
of isoprenaline, a cAMP agonist (18).
Five aspects of bioelectric properties in the nasal
epithelium usually tested include: 1) the basal PD; 2)
the inhibition of PD with perfusion of amiloride; 3) the
basal chloride conductance, as indexed by the chloride
diffusion PD in response to perfusion of solution containing
amiloride but no chloride; 4) the increase in the chloride
diffusion PD with the addition of isoproterenol to the
perfusate; and 5) the hyperpolarization associated with
the addition of ATP to the perfusate (19).
Technical aspects
Nasal potential difference can be measured by a high
impedance voltmeter between two electrodes: an exploring
electrode placed on the surface of the epithelium and a
reference electrode (subcutaneous needle or abrasion)
in contact with interstitial milieu.
A fluid-filled bridge or a solid agar bridge were used,
referenced to either a subcutaneous electrode or an
abraded skin electrode system. However, recent studies
showed superior reliability of the agar nasal catheter
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approach compared to the perfusion nasal catheter
method. Measurements under the inferior turbinate
and the floor of the nasal cavity are valid, repeatabilities
of both methods are similar (20). The region under the
inferior turbinate or on the floor is explored for the site of
most negative voltage and the probe is stabilized at that
point, manually or physically with tape. The solutions
are then sequentially perfused over the nasal mucosa
usually through the second lumen of a double-barreled
catheter and changed after a minimum of 3 min. With
each solution, there is an attempt to obtain a steady,
noise-free voltage plateau for the terminal 30 sec.
Results
Baseline voltage is -18.2±8.3 mV (mean ± SD) for
normals, and -45.3±11.4 mV for CF patients. There is a lack
of response to perfusion with zero chloride +isoproterenol
(+3.2 ±3.5 mV) vs that in normals (-23.7±10.2 mV)
(17).
Perfusion with agents that increase cAMP (like
isoproterenol) are then likely to induce a greater degree of
Cl– secretion. Consequently, the larger changes in PD may
then allow clearer quantification of the CF bioelectric defects
(18). Taken together, the net response to zero-chloride
plus isoproterenol provides the clearest discrimination
between CF and normal subjects; the Cl– conductance
is absent (or very low) in CF patients.
There is overlap of the baseline values and in the responses
to amiloride between CF and normal subjects.
Method limita!ons
There are many variations between the protocols used
in different centers, like: warming of the solutions, type
of catheter, perfusion speed, composition of solutions.
Method is time consuming and need experience
operator, which limits larger use of this awaited insight
into CFTR function. Variability of NPD measurements can
also reduce the power of studies using NPD as outcome
measures. It might be caused by the following: the material
used (catheters, electrodes); the offset of the electrodes;
the positioning and fixation of the catheter; stability
of the measure, duration of the experiment. However,
when performed in a single center, NPD could be a
reproducible test for CF patients.
The basal PD can be lowered by nonspecific damage
to the tissue, such as abrasion or inflammation, f.ex.
rhinitis.
4. AREA OF PD APPLICATION
Airway ion transport abnormalities in CF can be
assessed in vivo by measuring the transepithelial nasal
potential difference (PD) (21).
This technique may be useful in assisting in the diagnosis
of cystic fibrosis, as well as for monitoring the effect of
pharmacological agents and gene transfer approaches to
correct the abnormalities of ion transport (22).
NPD and pathophysiology of CF
The relation between the number and severity of
CFTR gene mutations and the degree of CFTR-mediated
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dysfunction of transepithelial transport was evaluated
(23). This comprehensive, prospective study of healthy
males and men with a variety of CFTR-associated
disorders demonstrated a wide spectrum of CFTRmediated abnormalities of transepithelial transport,
which showed a relation to the number and functional
severity of CFTR gene mutations. The men with CBAVD
showed a wide range of transepithelial measurements,
which correlated with the number of identified CFTR
mutations and overlapped with those in the healthy
control subjects.
A number of different studies have examined the
relationship between the extent of ion transport abnormality
and the severity of disease in CF (21, 24, 25).
Studies of Fajac and Leal showed a correlation of
NPD with clinical picture.
Fajac et al. study has shown that in adult CF patients
with basal nasal PD in the normal range, respiratory
function was less impaired than in CF patients with more
negative nasal PD (24). Moreover, among CF patients with
two “severe” mutations, a similar relationship between
nasal PD and respiratory function was found: in patients
with normal nasal PD, respiratory function was less
impaired than in patients with higher nasal PD. Nasal
PD in a given patient had good reproducibility.
However, there was no significant relationship between
nasal potential difference and the severity of the genotype.
Nasal epithelial ion transport in cystic fibrosis was linked
to the clinical expression of the disease. The pancreatic
status appeared to be mostly related to the defect in
epithelial chloride secretion whereas the respiratory
status was mostly related to abnormal sodium transport
(24).
Leal et al. data also indicated that nutritional status,
lung function, age, and chronic P. aeruginosa infection
were co-dependent variables related to both regulatory
(sodium channel) and primary (chloride channel) CFTR
function (21). Multiple inter-relationships between ion
transport defects and clinical variables were confirmed
in this study.
From a practical point of view these findings encourage
the measurement of nasal PD not only for diagnostic
purposes in difficult cases but also to gain more insight
into the degree of residual chloride conductance and
of sodium abnormality in correlation with severity of
the disease.
NPD as diagnos!c technique
The nasal PD test is a delicate in vivo procedure used
to help diagnosis of CF in the presence of atypical clinical
features and equivocal sweat test results.
It has already established position as a part of the
criteria for CF diagnosis according to the American and
European consensus statements (16).
It is used to demonstrate abnormal function of the CFTR
protein, establish a diagnosis of CF in patients with atypical
presentation and in asymptomatic patients suspected to
have CF in CF Newborn Screening Programmes (25,
26, 27).
The test is not diagnostic on its own, but is a very
helpful indicative tool.
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Therapeu!c modula!ons and correc!on
monitoring
Using nasal PD measurements researchers showed , that
distinct components of the CF ion transport profile were
associated with characteristic phenotypic expression. A better
understanding of the mechanism of dysfunction had direct
relevance to therapeutic strategies aimed at improving the CF
phenotype by either restoring reduced/absent chloride transport
or by normalizing exacerbated sodium transport.
The test has been used as a surrogate endpoint for clinical
trials on therapeutic modulation of CF basic defects.
Gene therapy clinical trials in CF patient included
viral and non-viral gene transfer to both the nasal and
bronchial airway epithelium (28).
Most early trials focused on the nasal epithelium as a
surrogate for the lung to allow for easy access and sampling,
and, importantly, to ensure safety. Once an acceptable
safety profile had been established, gene transfer agents
were administered directly into the lung (29).
PD was one of the endpoint assays in the assessment
of gene transfer. Partial (approximately 20% of non-CF)
correction of chloride transport but not of the sodium,
has been reported in some studies. However, it is still
unclear, if gene transfer efficiency using currently available
vectors is sufficient to change more clinically relevant
endpoint assays such as lung function, inflammation or
bacterial colonisation (29).
CFTR nonsense mutations are mutations causing a
premature termination signal resulting in the formation of
truncated or unstable protein and subsequently producing
little or no CFTR chloride channels (30). The aminoglycoside
antibiotics, in addition to their antimicrobial activity, can
increase the frequency of a readthrough of the premature
stop codon, thereby permitting protein translation to
continue to the normal end of the gene (31).
The aim of Wilschanski and coworkers study was to
determine if gentamicin can, in vivo, induce expression
of CFTR in CF patients carrying stop mutations (30).
They used the nasal potential difference (PD) to measure
sodium and chloride transport before and after topical
application of gentamicin drops on the nasal epithelium.
Correction of the abnormal PD suggested that gentamicin
can correct the primary defect among patients carrying
stop mutations. However, the inconvenience of parenteral
administration and the potential for serious toxic effects
preclude long-term systemic use of gentamicin for
supression of nonsense mutations.
Clancy and coworkers study demonstrated that not
all premature stop mutations are equally sensitive to
suppression in vivo (32).These results with first-generation
suppressive agents suggested the need for improved drug
delivery methods and/or more potent suppressors of
nonsense mutations to confer CFTR correction in subjects
with CF heterozygous for nonsense mutations.
PTC124 – Ataluren is an orally bioavailable,
nonaminoglycoside compound that was developed in
a high throughput screening to induce ribosomes to
read through premature stop codons, but not normal
stop codons (33).
This phase II prospective trial recruited adults with
cystic fibrosis who had at least one nonsense mutation
in the CFTR gene. The primary outcome had three
components: change in CFTR-mediated total chloride
transport; proportion of patients who responded to
treatment; and normalization of chloride transport.
Nasal PD was used to assess whether PTC124 could
overcome the effects of a nonsense mutation by restoring
the functional activity of CFTR and increasing total
chloride transport. The results showed that patients
responded to treatment with PTC124, as assessed by
an increase in total chloride transport, indicated by a
change of − 5 mV or more electrically negative and other
clinical improvements.
In many patients, PTC124 shifted total chloride
transport into the normal range.
These trials exemplified the concept of direct assessment
of protein function in vivo and a use of nasal PD as a
sensitive method to assess the activity of full-length,
functional CFTR protein on epithelial cell surfaces, and
CFTR-mediated chloride ion transport in nasal mucosa
as a surrogate for lower airway epithelium. The link
between changes in NPD towards normal values and
clinical improvement still need to be demonstrated.
4. SUMMARY
Thanks to NPD is possible to observe pathophysiological
events leading from CFTR mutation to CF phenotype.
It correlates to some extent with genetic abnormalities
and disease severity.
It is helpful as diagnostic test and as an endpoint of
new disease modifying therapies.
There are still a lot of practical issues to be solved, like
standardization and large variability of measurements (34).
There is an unresolved question of correction which is needed
for clinical effect as the biggest challenge for this interesting
technique still remains our understanding of the impact of
potential changes on clinical course of the disease.
Acknowledgements
Dr François Vermeulen (Catholic University of Leuven,
Belgium) for manuscript revisions and consultations.
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Conflict of interest:
The Authors declare no conflict of interest
Received: 05.02.2013 r.
Accepted: 08.02.2013 r.
Published online
Address for correspondence:
Dorota Sands
Institute of Mother and Child
Kasprzaka 17a, 01-211 Warszawa, Poland
tel.: (+48 22) 32-77-190
fax: (+48 22) 32-77-043
e-mail: [email protected]