Allele-specific transcriptional activity of the variable number of tandem repeats of the inducible nitric oxide synthase gene is associated with idiopathic achalasia

Abstract

Background: Polymorphisms of genes involved in the regulation of the immune response are risk factors for achalasia, but their contribution to disease pathogenesis is unknown. Nitric oxide is involved both in immune function and inhibitory neurotransmission. Objective: The objective of this article is to assess the association and the functional relevance of the CCTTT-inducible nitric oxide synthase (NOS2) gene promoter polymorphism in achalasia. Methods: Genomic DNA was isolated from 181 achalasia patients and 220 controls. Genotyping of the (CCTTT)n repeats was performed by PCR and capillary electrophoresis, and data analyzed by considering the frequency of the different alleles. HT29 cells were transfected with iNOS luciferase promoter-reporter plasmids containing different (CCTTT)n. Results: The alleles’ distribution ranged from 7 to 18, with a peak frequency at 12 repeats. Analysis of the allele frequencies revealed that individuals carrying 10 and 13 CCTTT repeats were respectively less and more frequent in achalasia (OR 0.5, 95% CI 0.3–0.5 and OR 1.6, 95% CI 1–2.4, all p < 0.05). Long repeats were also significantly associated with an earlier onset of the disease (OR 1.69, 95% CI 1.13–2.53, p = 0.01). Transfection experiments revealed a similar allele-specific iNOS transcriptional activity. Conclusion: The functional polymorphism (CCTTT) of NOS2 promoter is associated with achalasia, likely by an allele-specific modulation of nitric oxide production.

Full text

Original Article
Allele-specific transcriptional activity of the
variable number of tandem repeats of the
inducible nitric oxide synthase gene is
associated with idiopathic achalasia
Giovanni Sarnelli
1
, Michela Grosso
2
, Ilaria Palumbo
1
, Marcella Pesce
1
,
Alessandra D’Alessandro
1
, Giovanni Zaninotto
3
, Vito Annese
4
,
Raffaella Petruzzelli
2
, Paola Izzo
2
, Rossana Sepulveres
2
, Dario Bruzzese
5
,
Giuseppe Esposito
6
and Rosario Cuomo
1
Abstract
Background: Polymorphisms of genes involved in the regulation of the immune response are risk factors for achalasia, but
their contribution to disease pathogenesis is unknown. Nitric oxide is involved both in immune function and inhibitory
neurotransmission.
Objective: The objective of this article is to assess the association and the functional relevance of the CCTTT-inducible nitric
oxide synthase (NOS2) gene promoter polymorphism in achalasia.
Methods: Genomic DNA was isolated from 181 achalasia patients and 220 controls. Genotyping of the (CCTTT)n repeats was
performed by PCR and capillary electrophoresis, and data analyzed by considering the frequency of the different alleles.
HT29 cells were transfected with iNOS luciferase promoter-reporter plasmids containing different (CCTTT)n.
Results: The alleles’ distribution ranged from 7 to 18, with a peak frequency at 12 repeats. Analysis of the allele frequencies
revealed that individuals carrying 10 and 13 CCTTT repeats were respectively less and more frequent in achalasia (OR 0.5,
95% CI 0.3–0.5 and OR 1.6, 95% CI 1–2.4, all p<0.05). Long repeats were also significantly associated with an earlier onset
of the disease (OR 1.69, 95% CI 1.13–2.53, p¼0.01). Transfection experiments revealed a similar allele-specific iNOS
transcriptional activity.
Conclusion: The functional polymorphism (CCTTT) of NOS2 promoter is associated with achalasia, likely by an allele-specific
modulation of nitric oxide production.
Keywords
Idiopathic achalasia, iNOS, genetic polymorphism, (CCTTT)n pentanucleotide, nitric oxide
Received: 6 January 2016; accepted: 15 April 2016
Introduction
Idiopathic achalasia is a rare esophageal motor dis-
order characterized by aperistalsis and defective relax-
ation of the lower esophageal sphincter (LES), leading
to bolus impaction and symptoms of dysphagia and
regurgitation.
1-3
Although a wealth of evidence points
toward the loss of the nitrergic innervation as the
underlying pathophysiological abnormality of achala-
sia;
4
the mechanism leading to this selective neurode-
generation remains to be elucidated.
Hereditary, neurodegenerative, infectious and auto-
immune mechanisms have all been forwarded as
1
Gastroenterology Unit, Department of Clinical Medicine and Surgery
University Federico II, Naples, Italy
2
Department of Biochemistry and Medical Biotechnology, University
Federico II, Naples, Italy
3
Imperial College-St Mary’s Hospital, Department of Academic Surgery,
London, UK
4
Unit of Gastroenterology SOD2, Azienda Ospedaliera Universitaria,
Careggi, Firenze, Italy
5
Department of Public Health, University Federico II, Naples, Italy
6
Department of Physiology and Pharmacology, ‘‘La Sapienza’’ University of
Rome, Italy
Corresponding author:
Giovanni Sarnelli, Department of Clinical Medicine and Surgery, University
‘‘Federico II’’ of Naples, Via Sergio Pansini, 5 80131 Naples, Italy.
Email: sarnelli@unina.it
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putative pathogenetic hypotheses and to date achalasia
is widely considered a multifactorial disorder. In the
wake of the strong pathogenetic role of T. Cruzii infec-
tion in Chagas disease, it has indeed been suggested
that sporadic achalasia, as well, may be the result of a
self-sustained inflammatory process secondary to acute
gastrointestinal infections and that individual suscepti-
bility of developing achalasia following such an initial
trigger may be genetically determined.
5,6
Achalasia,
albeit rarely inherited, has indeed been associated
with several polymorphisms in genes involved in the
regulation of the immune response
7-12
and the control
of esophageal motility.
13-20
In this complex scenario,
nitric oxide (NO) represents a unique molecule since,
depending on its concentration, it is involved in either
inhibitory neurotransmission, or defense against
infections.
21-23
NO is constitutively produced by endothelial (eNOS
or NOS3) or neuronal (nNOS or NOS1) NO synthases
and, at higher concentrations, by the inducible form of
NO synthase (iNOS or NOS2),
24
under stimulation of a
variety of proinflammatory cytokines.
20-22
Despite its
antitumoral and antimicrobial activities,
25,26
aberrant
iNOS expression may have detrimental consequences
as excessive NO production has been proved to exert
neurotoxic effects, particularly for nitrergic neurons.
NO release mediated by iNOS isoform may, indeed,
induce transcriptional downregulation of nNOS,
thus eventually leading to impaired nitrergic
innervation.
27-29
iNOS-dependent NO release is genetically deter-
mined and different iNOS gene promoter polymorph-
isms have been involved in individual responses to
infection-induced immune activation.
30
The highly
polymorphic pentanucleotide (CCTTT)n repeat located
in the iNOS gene promoter region may be functionally
relevant for the regulation of iNOS gene transcrip-
tion.
31
The distribution of pentanucleotide microsatel-
lite (CCTTT)n alleles has been studied in different
ethnic groups and it has been associated with predis-
position to infectious and autoimmune diseases.
32-35
Based on this background, we aimed to examine
whether the polymorphic pentanucleotide (CCTTT)n
of the iNOS gene promoter is involved in the suscepti-
bility to suffer from idiopathic achalasia and to inves-
tigate the functional role of this genetic polymorphism.
Materials and methods
Study participants
A total of 181 consecutive adult unrelated Caucasian
Italian achalasia patients (male 97, mean age 56 18
years) were recruited from October 2008 until
November 2010. Diagnosis of achalasia was based on
standard clinical, radiological, endoscopic tests and
confirmed by esophageal manometry according to
international criteria.
36
None of the patients had a
family history of achalasia so all were considered as
sporadic cases; furthermore 12 patients with comorbid
autoimmune disorders (five patients with diabetes mel-
litus type I, six with rheumatoid arthritis, one with pri-
mary biliary cirrhosis) were excluded from the study.
A group of 220 healthy white, unrelated individuals
(130 males, mean age 50 13 years) without symptoms
of or a history of gastrointestinal disease were included
as ethnically matched controls. The control group con-
sisted mainly of blood donors and ethnically matched
hospital employees. All individuals gave their consent
to participate in the protocol and the study was
approved by the University Ethics Commitee.
Genotyping
Total DNA was extracted from peripheral blood leuko-
cytes using the Nucleon BACC Genomic DNA
Extraction Kit (GE Healthcare Europe GmbH, 79111
Freiburg, Germany). The iNOS pentanucleotide alleles
were analyzed after polymerase chain reaction (PCR)
amplification with the following set of primers: forward
50-FAM ACCCCTGGAAGCCTACAACTGCAT-30
and reverse 50-CCACTGCACCCTAGCCTGTCTCA-
30. The size of the labeled PCR products was analyzed
by capillary electrophoresis on an ABI PRISM 3130
sequencer with a GeneScan 500LIZ size standard.
Constructions of iNOS luciferase promoter-
reporter plasmids containing different numbers
of (CCTTT)n repeats
PCR was used to obtain a 1.2 Kb fragment immediately
upstream of the transcription start site of the human
iNOS gene (pINOS). The forward primer 50-CAAAGT
GTTGGTACCGTGAGATCA-30is located –1183 bp
from the transcription start site and the reverse
primer 50-CTTCGGGACTCTCGAGAACTGCCCA
G-30is located þ122 bp.
The PCR product was cloned into a pGL4 vector
(Promega Madison, WI, USA), which contains the pro-
moter without firefly luciferase reporter.
The (CCTTT)n pentanucleotide repeat region was
cloned into the pGL4 construct using a pair of primers,
50-ATGGAGGTACCATGGCATCCTGATTATCTC
CA-30(forward) and 50-TTCCAAGATCTAAGCAGG
AATGAGGCTGAGT-30(reverse), by directional PCR
from human genomic DNA obtained from individuals
with different repeats. We obtained constructs with 9,
10, 11, 12, 13, 14, 15 and 16 repeats. All the constructs
were also sequenced to confirm the authenticity of the
PCR products.
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Cell cultures, transient transfections, cell
induction and luciferase assays
Human colon adenocarcinoma grade II cell line, HT29
(Sigma, Milan, Italy), was maintained in Dulbecco’s
Modified Eagle’s Medium (Sigma) supplemented with
10% (v/v) heat-inactivated fetal bovine serum at 37C
in a humidified 5% CO
2
-containing atmosphere. Cell
cultures were kept sub-confluent and transiently trans-
fected for luciferase assays.
Transfection of HT29 was performed with
Lipofectamine 2000 (Invitrogen Inc, USA). In brief,
the day before transfection, cells were plated into
Falcon 12 well plates at a density of 1.5 10
5
/ml in
Optimem medium (Invitrogen Inc, USA). Cells were
transiently transfected with 0.25 mg of each construct.
To normalize the luciferase assay, 0.025 mg of the pRL-
CMV vector (Promega) coding for the Renilla luciferase
was transiently co-transfected. The pGL4-null was used
as a negative control, whereas the pCMVluc (0.05 mg)
was the positive control for the assay. After transfec-
tion, cells were treated for four hours with a mixture
containing bacterial lipopolysaccharide (LPS) (10 mg/
ml) (Sigma), interferon gamma (IFNg) (100 units/ml)
(R&D Systems, Minneapolis, MN, USA) and tumor
necrosis factor alpha (TNFa) (2 ng/ml) (Sigma) for
iNOS induction. Cell extracts were prepared 24 hours
after induction, and 40 ml of lysate was used for the
determination of luciferase activity using the Dual-
Luciferase Reporter Assay System (Promega) on a
20/20
n
luminometer (Turner Biosystems, Sunnyvale,
CA, USA), according to the manufacturers’ protocols.
Statistical analysis
Results are given as number of cases and percentages
for categorical data, and as mean standard deviation
for quantitative variables. Data were analyzed by use of
t-test for independent samples in case of quantitative
variables and with the Fisher exact test in case of cat-
egorical variables. Association among iNOS CCTTT
polymorphism and the presence of achalasia was quan-
tified through the use of crude and stratified odds
ratio (OR).
The statistical significance level was set at 5%
(a¼0.05), and two-tailed tests were used throughout.
Confidence intervals (CIs) are based on 95% CI. All the
statistical analyses have been realized using R
version 3.01.
Results
Patients’ demographic and clinical features
Demographic and clinical features of participants
included in our analysis are shown in Table 1.
The mean age of patients was 56 18 years, 97 were
males and 84 females. Age at diagnosis was 49 17
years and on average patients had a history of symp-
toms duration of 7.3 6.9 years. Gender did not affect
the disease’s duration, nor was age at diagnosis signifi-
cantly different between males and females. The major-
ity of the patients reported dysphagia as their prevalent
and most bothersome symptom (100%), but food
regurgitation and chest pain were also frequently
reported by 70% and 50%, respectively. At barium
esophagogram a dilated body of the esophagus, a
tapered beaklike narrowing of the distal esophagus
adjacent to the gastroesophageal junction, or both
were observed in 5%, 35% and 45% of patients,
respectively. As far as esophageal manometry param-
eters the LES basal pressure and the mean amplitude
wave were 47 13 and 38 14 mmHg, respectively.
Impaired swallow-induced LES relaxation was
observed in 100% of the patients, while aperistalsis in
the distal two-thirds of the esophagus and simultaneous
contraction (>40 mmHg) were respectively observed in
80% and 20% of participants, allowing us to subclas-
sify patients as having classic or vigorous achalasia
(n¼145 and 36, respectively).
Association between iNOS CCTTT gene
polymorphisms and achalasia
The distribution of alleles having various repeat num-
bers ranged from 7 to 18 CCTTT and is shown in
Figure 1. In patients as well as controls the number
of (CCTTT)n repeats showed a central distribution
and a peak frequency at 12 repeats. This pattern was
similar to that reported previously for the white
Caucasian population.
32
Analysis of the allele frequencies revealed that indi-
viduals carrying the allele 10 had a significantly lower
risk of having achalasia (OR 0.55, 95% CI 0.35–0.90,
p¼0.02), while no significant differences were observed
for the other (CCTTT)n repeats (see Table 2).
When data were stratified by gender, it emerged that
among the females, those with 10 and 13 CCTTT
repeats had a reduced and increased risk of achalasia,
respectively (OR 0.39, 95% CI 0.19–0.80, p¼0.009 and
Table 1. Demographics and clinical features of achalasia (181)
and control participants (220)
Patients Controls
Male/Female 97/84 130/120
Age 56 18 50 13
Duration (years) 7.3 6.9 _
Age at diagnosis (years) 49 17 _
Sarnelli et al. 3
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OR 2.14, CI 95% 1.11–4.12, p¼0.022). In males, only
those with 11 CCTTT repeats had a significant reduc-
tion in achalasia susceptibility (OR 0.52, 95% CI 0.29–
0.93, p¼0.026).
Previous studies classified CCTTT alleles into short
(7–11) and long (12–18) forms, according to the
number of repeats.
37-39
Thus, by sorting our cohort
into long and short alleles, we found that individuals
with CCTTT>11 had a significantly increased cumula-
tive risk for achalasia (OR 1.69, 95% CI 1.13–2.53,
p¼0.01); Figure 2 shows that gender likely influences
such an effect, as this association was significant
in males (OR 2.01, 95% CI 1.16–3.46, p¼0.012),
but not in females (OR 1.42, 95% CI 0.77–2.62,
p¼0.261).
Effect of iNOS CCTTT polymorphisms and age
of onset of achalasia
To evaluate whether the iNOS CCTTT polymorphism
could represent a risk factor making some individuals
more susceptible to the development of achalasia, we
evaluated the effect of (CCTTT)n on achalasia onset.
We failed to find any significant association between
single different CCTTT repeats and age or the duration
of the disease (data not shown). However, when we
computed the analysis by considering the short and
50%
40%
30%
20%
10%
0% 7 8 9 10 1112 13 14 151617 18
Controls
Achalasia
p=0.016
Figure 1. Allelic distribution of (CCTTT)n in achalasia and control participants. The allele distribution ranged from 7 to 18 with a peak
frequency at 12 repeats in patients as well as controls. Individuals carrying 10 (CCTTT) repeats showed a reduced risk of developing
achalasia, while no significant differences were observed in the allelic distribution of the other (CCTTT) repeats.
Gender
Female Male
40.0% Controls
Achalasia
Controls
Achalasia
30.0% p=0.261 p=0.012
20.0%
10.0%
0.0%
Short Long Long
(CCTTT)n polymorphism
Short
Figure 2. Allelic distribution of long (7–11) and short (12–18)
(CCTTT) repeats by gender in achalasia and healthy individuals.
Males carrying the long alleles form had an increased risk of
having achalasia (odds ratio (OR) 2.01, 95% confidence interval (CI)
1.16–3.46, p¼0.012).
Table 2. Frequencies of (CCTTT)n alleles in achalasia and healthy
individuals
Alleles Controls Achalasia p
70 (0) 2 (1.1) 0.20
8 3 (1.4) 3 (1.7) 1.00
9 22 (10) 20 (11) 0.72
10 63 (28.5) 33 (18.2) 0.02
11 79 (35.7) 49 (27.1) 0.06
12 107 (48.4) 89 (49.2) 0.88
13 72 (32.6) 74 (40.9) 0.09
14 41 (18.6) 31 (17.1) 0.71
15 19 (8.6) 25 (13.8) 0.10
16 8 (3.6) 6 (3.3) 0.87
17 0 (0) 1 (0.6) 0.45
18 2 (0.9) 0 (0) 0.50
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long CCTTT alleles forms, we found that patients car-
rying a longer number of CCTTT repeats (i.e. >11)
develop the disease significantly earlier than those
with short alleles (42 18 vs. 51 17 years, p¼0.01).
Effects of different CCTTT polymorphisms on
transcriptional activity of the iNOS gene
To determine whether variable numbers of the CCTTT
repeats were associated with the modulation of gene
expression, we evaluated promoter activities using a
variety of iNOS promoter-luciferase constructs in tran-
sient transfection experiments.
As shown in Figure 3, luciferase activity was almost
silent in cells transfected with the empty vector; in con-
trast, in cells transfected with vectors containing the
promoter region of the iNOS gene, luciferase activity
was significantly increased. More interestingly, stimula-
tion with LPS, IFNgand TNFaresulted in a gradual
and significant increase of luciferase activity, along with
the increasing number of CCTTT repeats, with a peak
occurring for constructs bearing 12 and 13 (CCTTT)
repeats, and the lowest transcriptional activity for 10
(CCTTT).
Conclusions
Idiopathic achalasia is the best characterized esopha-
geal motor disorder, nonetheless its pathogenesis is
not yet resolved. The occurrence of familial achalasia
and its association with well-defined genetic syndromes
suggests the involvement of genetic factors.
40-44
To date, several genetic association studies have
been reported in achalasia and some of these studies
focused on candidate genes possibly linked to achalasia
through their involvement in particular cell pathways,
above all the regulation of immune response and the
inhibitory neurotransmission.
6-11,16
Although the genetic contribution to achalasia has
been strongly supported,
10,11
the clinical relevance of
the reports are hampered either by the low number
of studied patients or by the weak pathophysio-
logical translation of the studied genes. NO may rep-
resent an ideal candidate to explain inhibitory nerve
degeneration occurring in achalasia patients because
it is involved both in defense against infections and
inhibitory neurotransmission, and excessive concen-
trations of NO have been demonstrated in vitro to
be neurotoxic, particularly for NOS-expressing
neurons.
29,45,46
Several single-nucleotide (SNP) or microsatellite
(STR) polymorphisms have been described in the
iNOS promoter region and many attempts have been
made so far to investigate their possible functional sig-
nificance in modulation of iNOS expression.
27-29
Although iNOS activity can be regulated by post-
transcriptional mechanisms, the human iNOS gene is
regulated predominantly at the transcriptional level by
a complex cytokine combination including IFNg, inter-
leukin (IL)-1band TNFa.
Unstimulated
LPS/IFNg/TNFa
16
15
14
13
12
11
10
(CCTTT)n
9
50 100 150
Relative luciferase activity
plNOS (–1183/+122)
pGL4 empty vector
* ° #
* ° #
* ° #
* ° #
* ° ##
* °
* °
*
*
Figure 3. Effects of varying numbers of (CCTTT)n repeats on NOS2 gene transcription. The CCTTT repeat sequences enhance the minimal
NOS2 promoter induction in response to LPS, IFNgand TNFastimulation, with a significant increase in luciferase activity (*p<0.0001 vs.
unstimulated). Analysis of the difference among the different (CCTTT)n within the stimulated group revealed that constructs containing 12
repeats produced a significantly greater induction of luciferase as compared to all the other constructs (p<0.01), whereas the 13-repeat
construct produced significantly greater luciferase activity than the 9, 10, 14, 15 and 16-repeat constructs (#p<0.01 and ## p<0.05,
respectively). Conversely, the 10-repeat construct was associated with a significantly lower transcriptional activity than 11, 12 and 13
constructs (all p<0.01). Data are the mean of six determinations and expressed as mean SEM. Data analysis was performed by ANOVA
with Bonferroni post-test. NOS2: nitric oxide synthase; LPS: lipopolysaccharide; IFNg: interferon gamma; TNFa: tumor necrosis factor
alpha; ANOVA: analysis of variance.
Sarnelli et al. 5
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In the present study we investigated the role of a
longer polymorphic pentanucleotide repeat in the
iNOS gene promoter region that has already been
linked to predisposition to different immune-mediated
conditions like infectious or degenerative diseases.
46,47
The distribution of (CCTTT)n pentanucleotide in our
population reproduced that observed in the Caucasian
population in previous studies with a peak frequency at
12 repeats.
35
As reported from others association stu-
dies,
40,48
in the subgroup of female participants, a sig-
nificantly reduced or increased risk of developing
achalasia was observed in individuals carrying 10 and
13 (CCTTT) repeats, respectively. Sorting into long
(12–18) and short (7–11) alleles in our cohort, we
found that long alleles were more frequent in achalasia
patients than in the control group, and, although this
association is significant in male patients but not in
females, it is likely that this may be a result of the
small analyzed sample size. In addition we also provide
evidence that among achalasia patients, those with
longer (CCTTT)n have a significant risk of developing
an earlier onset achalasia as compared to those with the
shorter alleles form, further indicating that such a gen-
etic background, if present, may account for a more
premature disease onset.
Few studies have tried to assess whether polymorph-
isms in the nNOS,iNOS or eNOS genes were associated
with achalasia yielding contrasting results.
14-16
Some of
these studies failed to produce any conclusive associ-
ation because no significant differences in genotypes
and allele distribution were found, likely excluding a
causative role of these polymorphisms.
14,15
In a more
recent study from India the iNOS22GA and
nNOS29TT genotypes were identified as risk factors
for achalasia, respectively.
16
However, it is of note
that though the SNP of iNOS gene explored by
Mearin et al.
14
is an exonic polymorphism, it does not
determine a change in the amino acid sequence, and its
functionality in iNOS expression is still unclear. On the
contrary, the (CCTTT)n microsatellite is a polymorph-
ism that has already been linked to several
autoimmune and degenerative disorders and has a
well-established functional significance in the regula-
tion of iNOS gene expression. Longer repeat numbers
of this polymorphism had indeed been related with a
higher iNOS transcriptional rate induced by IL-1b.
33
In
our research, the luciferase activities of cells transfected
with vectors containing the promoter region of the
iNOS gene gradually increased along with the increas-
ing number of CCTTT repeats, until the constructs
with 12 and 13 (CCTTT) repeats, thus pointing out
the functional relevance of this polymorphism in regu-
lating the expression of the iNOS gene, and likely
reflecting an increased production of NO. Similarly,
the observation that constructs with 10 repeats was
associated with a lower transcriptional activity seems
to suggest that the reduced NO production is protective
and is in line with the reduced risk of achalasia in indi-
viduals carrying such an allele.
Since there is evidence suggesting that iNOS-
dependent NO production could induce downregula-
tion of nNOS expression,
28
one can speculate that,
under proinflammatory stimuli, high levels of NO
may contribute to impair the normal functioning of
nitrergic neurons thus leading to the highly selective
neurodegeneration observed in achalasia patients. In
this context, our study is limited because we did not
study the nNOS in our patients and thus our pathogen-
etic hypothesis remains speculative. However, a more
detailed knowledge of cellular responses in vivo would
be warranted to identify the complex interplay between
iNOS and nNOS isoforms and since several post-tran-
scriptional modifications in NOS genes have been
described so far, this cannot be obtained by studying
isolated genetic polymorphisms on peripheral blood
mononuclear cells (PBMCs).
49
Here we provide evidence that genetic variations in
the promoter region of the iNOS gene are associated
with the susceptibility to achalasia. Furthermore, we
demonstrated that patients carrying longer alleles
have a significant risk of developing an earlier onset
of achalasia, possibly as a result of increased iNOS
gene expression. Although limited by the low number
of the studied population, our data provide a plausible
pathophysiological mechanism to explain the selective
neurodegeneration and the reduced nNOS expression
occurring in the myenteric plexus of achalasia patients.
Therefore, larger multicentric studies aimed at under-
standing molecular mechanisms regulating iNOS gene
expression could help to pave the way to novel thera-
peutic tools able to control excessive NO production
and also to identify genetic factors determining the sus-
ceptibility to achalasia.
Declaration of conflicting interests
The authors declared no potential conflicts of interest with
respect to the research, authorship, and/or publication of this
article.
Funding
This study was partially funded by Regione Campania
(L.R. 5 2008) to G.S.
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