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



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.
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
, Michela Grosso
, Ilaria Palumbo
, Marcella Pesce
Alessandra D’Alessandro
, Giovanni Zaninotto
, Vito Annese
Raffaella Petruzzelli
, Paola Izzo
, Rossana Sepulveres
, Dario Bruzzese
Giuseppe Esposito
and Rosario Cuomo
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
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.
Idiopathic achalasia, iNOS, genetic polymorphism, (CCTTT)n pentanucleotide, nitric oxide
Received: 6 January 2016; accepted: 15 April 2016
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
Although a wealth of evidence points
toward the loss of the nitrergic innervation as the
underlying pathophysiological abnormality of achala-
the mechanism leading to this selective neurode-
generation remains to be elucidated.
Hereditary, neurodegenerative, infectious and auto-
immune mechanisms have all been forwarded as
Gastroenterology Unit, Department of Clinical Medicine and Surgery
University Federico II, Naples, Italy
Department of Biochemistry and Medical Biotechnology, University
Federico II, Naples, Italy
Imperial College-St Mary’s Hospital, Department of Academic Surgery,
London, UK
Unit of Gastroenterology SOD2, Azienda Ospedaliera Universitaria,
Careggi, Firenze, Italy
Department of Public Health, University Federico II, Naples, Italy
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.
United European Gastroenterology Journal
0(0) 1–8
!Author(s) 2016
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DOI: 10.1177/2050640616648870
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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.
albeit rarely inherited, has indeed been associated
with several polymorphisms in genes involved in the
regulation of the immune response
and the control
of esophageal motility.
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
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),
under stimulation of a
variety of proinflammatory cytokines.
Despite its
antitumoral and antimicrobial activities,
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
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.
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-
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.
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.
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.
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
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
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,
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 37C
in a humidified 5% CO
-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
/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
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.
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.
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.
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,
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
0% 7 8 9 10 1112 13 14 151617 18
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.
Female Male
40.0% Controls
30.0% p=0.261 p=0.012
Short Long Long
(CCTTT)n polymorphism
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
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
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.
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.
Although the genetic contribution to achalasia has
been strongly supported,
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
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.
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.
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.
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.
As reported from others association stu-
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.
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.
In a more
recent study from India the iNOS22GA and
nNOS29TT genotypes were identified as risk factors
for achalasia, respectively.
However, it is of note
that though the SNP of iNOS gene explored by
Mearin et al.
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.
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,
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).
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
This study was partially funded by Regione Campania
(L.R. 5 2008) to G.S.
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... For transfection experiments, freeze-dried PNAs were dissolved in RNase-free water and trifluoroacetic acid (TFA). Jurkat cells were plated in 12-well plates at a density of 4 × 10 5 cells/ well and transiently transfected with either 1 μM or 2.5 μM PNA or scrambled PNA as an aspecific negative control, using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) as transfection reagent as previously reported [27,28]. Also, CLL PBMCs were plated in 12-well plates at a density of 1 × 10 6 cells/well and transiently transfected with 1 μM PNA or scrambled PNA. ...
Full-text available
We herein report an innovative antisense approach based on Peptide Nucleic Acids (PNAs) to down-modulate CD5 expression levels in chronic lymphocytic leukemia (CLL). Using bioinformatics tools, we selected a 12-mer tract of the CD5 mRNA as the molecular target and synthesized the complementary and control PNA strands bearing a serine phosphate dipeptide tail to enhance their water solubility and bioavailability. The specific recognition of the 12-mer DNA strand, corresponding to the target mRNA sequence by the complementary PNA strand, was confirmed by non-denaturing polyacrylamide gel electrophoresis, thermal difference spectroscopy, circular dichroism (CD), and CD melting studies. Cytofluorimetric assays and real-time PCR analysis demonstrated the downregulation of CD5 expression due to incubation with the anti-CD5 PNA at RNA and protein levels in Jurkat cell line and peripheral blood mononuclear cells from B-CLL patients. Interestingly, we also observed that transfection with the anti-CD5 PNA increases apoptotic response induced by fludarabine in B-CLL cells. The herein reported results suggest that PNAs could represent a potential candidate for the development of antisense therapeutic agents in CLL.
... В работе G. Sarnelli и соавт. [41] исследовали взаимосвязь полиморфизма CCTTT-индуцибельной синтазы оксида азота (NOS2) с ахалазией. ДНК-анализ был проведен 181 пациентам с ахалазией и 220 пациентам группы контроля. ...
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Achalasia is a chronic neuromuscular disorder of esophagus with supposed autoimmunity, genetic disturbancies and viral infection. The disease is characterized by poor relaxation of lower esophageal sphincter and absent of body peristalsis. The inflammation, eosinophilic and lymphocytic infiltration may play role in loss of inhibitory ganglion in the myenteric plexus. High resolution manometry is a gold standard for diagnosis of achalasia, because it distinguish three types of disorder. The aim of treatment of achalasia is to reduce symptoms, such as dysphagia. The most effective modality of surgical treatment are Heller myotomy and peroral endoscopy myotomy. The pneumodilation is less preferable because of short-term effect and high rate of relapse. The relapse after surgical treatment can be present and correlates increase with the time after intervention
... K.M. Warpeha и соавт. обнаружили, что повторы 9, 12 и 14 NOS2A(CCTTT)n давали прогрессивное увели-чение экспрессии генов [12,17,21]. ...
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The article presents the results of a study of the associations of NO synthase and arginase genes in children with bronchial asthma with clinical, laboratory and functional parameters depending on the level of nitrogen oxide in exhaled air (FeNO). We examined 107 children with bronchial asthma, they were divided into 2 groups depending on the level of FeNO. We found a number of associations in the group of patients with an elevated level of FeNO (≥20 ppb): carriage of alleles and genotypes containing short tandem repeats of S (9–11) NOS1(AAT)n gene, with an early debut and severe course of the disease, an increased level of total IgE in blood serum; carriage of alleles and genotypes containing long tandem repeats L (12–16) of the NOS2A(CCTTT) n gene, with a moderate course of the disease, with an increased level of IgE; carriage of the allele *A of the ARGII gene (rs3742879) with a moderate course of the disease; carriage of the *G allele and heterozygous *AG genotype of the ARGII gene (rs3742879) with a decreased level of FEV1/FVC; carriage of L alleles and a combination of the SL and LL genotypes of the NOS1(AAT)n gene with elevated blood eosinophils (eosinophilia); a combination of S/L + L/L genotypes of the NOS1(AAT)n gene with fungal sensitization. The authors established the correlations between disease severity and NOS1(AAT)n; the age of the manifestation of the disease and NOS1(AAT)n; FEV1/FVC and ARGII (rs3742879); feedback between blood eosinophilia and NOS1(AAT)n. The authors also determined a number of associations in the group of patients with low level of FeNO (<20 ppb): carriage of alleles and genotypes containing short tandem repeats of S (9–11) gene NOS1(AAT)n, with fungal sensitization; carriage of alleles and genotypes containing long tandem repeats of L (12–16) gene NOS2A(CCTTT)n, with reduced FEV1 and FEV1/FVC; carriage of the homozygous genotype of *GG gene ARGII(rs3742879) with epidermal sensitization. With a reduced level of FeNO, the study determined a relationship between the severity of bronchial asthma and NOS1(AAT)n; degree of effectiveness of anti-inflammatory basic therapy and NOS1(AAT)n; fungal sensitization and NOS1(AAT)n; feedback between FEV1 and NOS2(CCTTT)n; FEV1/FVC and NOS2(CCTTT)n.
... Nonetheless, recent evidence suggests a possible role of an autoimmune reaction triggered by a viral infection that leads to an inflammatory process and consequent disruption of inhibitory neurons within the myenteric plexus, releasing nitric oxide. 1,2 Achalasia is a rare disease with incidence rate of 1.63/ 100,000 population, and prevalence of 10/100.000 population, 3 it is generally diagnosed between the ages of 30 and 60 years, and both genders appear to be equally affected. ...
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Achalasia is a chronic idiopathic disease characterized by the absence of esophageal body peristalsis and by defective lower esophageal sphincter (LES) relaxation. The incidence rate ranges from 1.07 to up to 2.8 new cases per year per 100,000 population. Presenting symptoms include dysphagia, regurgitation, vomiting, and weight loss. The diagnosis of achalasia has undergone a revolution in the last decade due to the advent of high-resolution manometry (HRM) and the consequent development of the Chicago Classification. Recent progress has allowed achalasia to be more precisely diagnosed and to be categorized into three subtypes, based on the prevalent manometric features of the esophageal peristalsis. Treatment options are pharmacotherapy, endoscopic management (Botox injection or pneumatic dilation), and surgery, e.g. laparoscopic Heller myotomy (LHM). More recently, a new endoscopic technique, per oral endoscopic myotomy (POEM), has developed as a less invasive approach alternative to the traditional LHM. Since the first POEM procedure was performed in 2008, increasing evidence is accumulating regarding its efficacy and safety profiles. Currently, POEM is being introduced as a reasonable therapeutic option, though Achalasia: Modern Diagnosis and Management Rambam Maimonides Medical Journal 2 January 2019  Volume 10  Issue 1  e0008 randomized controlled trails are still lacking. The current review sheds light onto the diagnosis and management of achalasia, with special focus on the recent advances of HRM and POEM.
... 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) [66]. ...
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Idiopathic achalasia is a relatively infrequent esophageal motor disorder for which major histo-compatibility complex (MHC) genes are well-identified risk factors. However, no information about HLA-achalasia susceptibility in Mexicans has previously been reported. We studied a group of 91 patients diagnosed with achalasia and 234 healthy controls with Mexican admixed ancestry. HLA alleles and conserved extended haplotypes were analyzed using high-resolution HLA typing based on Sanger and next-generation sequencing technologies. Admixture estimates were determined using HLA-B and short tandem repeats. Results were analyzed by non-parametric statistical analysis and Bonferroni correction. P-values < 0.05 were considered significant. Patients with achalasia had 56.7% Native American genes, 24.7% European genes, 16.5% African genes and 2.0% Asian genes, which was comparable with the estimates in the controls. Significant increases in the frequencies of alleles DRB1*14:54 and DQB1*05:03 and the extended haplotypes DRB1*14:54-DQB1*05:03 and DRB1*11:01-DQB1*03:01, even after Bonferroni correction (pC<0.05), were found in the achalasia group compared to those in the controls. Concluding, the HLA class II alleles HLA-DRB1*14:54:01 and DQB1*05:03:01 and the extended haplotype are risk factors for achalasia in mixed-ancestry Mexican individuals. These results also suggest that the HLA-DRB1*14:54-DQB1*05:03 haplotype was introduced by admixture with European and/or Asian populations.
... 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) [66]. ...
Conference Paper
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Backround: Idiopathic achalasia is an esophageal motor disorder that is relatively infrequent. Major histocompatibility genes (MHC) are well-identified risk factors in achalasia. However, no information about HLA-achalasia susceptibility in Mexicans has been previously reported. Objective: The aim of the present study is to describe the distribution of HLA class I and class II blocks and CEHs and their most probably ancestral origin using high resolution HLA typing in a group of Mexican mixed ancestry individuals with achalasia. Materials and methods: We studied a group of 97 patients with diagnosis of achalasia and 234 healthy controls from Mexican admixed ancestry. HLA alleles and conserved extended haplotypes were analysed by using high resolution HLA typing based on Sanger sequencing and next generation sequencing. Admixture estimations were determined by using HLA-B and short tandem repeats; and results were analysed by nonparametric statistics and Bonferroni correction, p values < 0.05 were considered significant. Results: We found that patients with achalasia have 56.7% of Native American genes, 24.7% of European, 16.5% of African and 2.0% of Asian genes that were comparable with the estimations in controls. A significant increase in the frequency of the alleles DRB1*14:54 and DQB1*05:03, and the extended haplotypes DRB1*14:54-DQB1*05:03 and DRB1*11:01-DQB1*03:01, even after Bonferroni correction (pCorr <0.05), were found in the achalasia group compared to controls. Conclusion: In conclusion, the HLA class II alleles HLA-DRB1*14:54:01 and DQB1*05:03:01 and the extended haplotype are risk factors for achalasia in mixed ancestry Mexican individuals. These results also suggest that the HLA-DRB1*14:54DQB1*05:03 haplotype was introduced by admixture with European populations. Funding: This work did not receive any type of financing.
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Coffee contains human health-related molecules, namely polyphenols that possess a wide range of pharmacological functions, and their intake is associated with reduced colon cancer risk. This study aimed to assess the changes in the anti-inflammatory and antioxidant activity of coffee after simulated gastrointestinal digestion. The evaluation of intracellular reactive oxygen species (ROS) levels in the HT-29 human colon cancer cell line and three in vitro spectrophotometric assays were performed to determine the antioxidant activity of the samples. Characterization of coffee composition was also assessed through a Q-Orbitrap high-resolution mass spectrometry analysis. The results highlighted that the levels of polyphenols in the digested coffee brews were higher than those of the non-digested ones. All assayed samples decreased the levels of intracellular ROS when compared to untreated cells, while digested coffee samples exhibited higher antioxidant capacity and total phenolic content than not-digested coffee samples. Digested coffee samples showed a higher reduction in interleukin-6 levels than the not-digested samples in lipopolysaccharide-stimulated HT-29 cells treated for 48 h and fewer cytotoxic effects in the MTT assay. Overall, our findings suggest that coffee may exert antioxidant and anti-inflammatory properties, and the digestion process may be able to release compounds with higher bioactivity.
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GATA-1 is a key regulator of hematopoiesis. A balanced ratio of its two isoforms, GATA-1FL and GATA-1S, contributes to normal hematopoiesis, whereas aberrant expression of GATA-1S alters the differentiation/proliferation potential of hematopoietic precursors and represents a poor prognostic factor in myeloid leukemia. We previously reported that GATA-1S over-expression correlates with high levels of the succinate dehydrogenase subunit C (SDHC). Alternative splicing variants of the SDHC transcript are over-expressed in several tumors and act as potent dominant negative inhibitors of SDH activity. With this in mind, we investigated the levels of SDHC variants and the oxidative mitochondrial metabolism in myeloid leukemia K562 cells over-expressing GATA-1 isoforms. Over-expression of SDHC variants accompanied by decreased SDH complex II activity and oxidative phosphorylation (OXPHOS) efficiency was found associated only with GATA-1S. Given the tumor suppressor role of SDH and the effects of OXPHOS limitations in leukemogenesis, identification of a link between GATA-1S and impaired complex II activity unveils novel pro-leukemic mechanisms triggered by GATA-1S. Abnormal levels of GATA-1S and SDHC variants were also found in an acute myeloid leukemia patient, thus supporting in vitro results. A better understanding of these mechanisms can contribute to identify novel promising therapeutic targets in myeloid leukemia.
Background There is evidence that idiopathic achalasia has an autoimmune component and a significant association with several autoimmune comorbidities has been described. However, data regarding the prevalence of autoimmune diseases in achalasia are not well established, and few studies have explored this association. Objective Our primary aim was to prospectively investigate the type and frequency of autoimmune comorbidities in a large cohort of consecutive achalasia patients. Our secondary aim was to investigate the effects of autoimmune comorbidities on achalasia phenotype (clinical features and manometric pattern). Methods The study population consisted of 375 consecutive patients (215 females—median age 55 ± 17 years), referred at our tertiary referral center from January 2008 to January 2018, with clinical and instrumental (EGDS, barium esophagogram, and manometry) diagnosis of idiopathic achalasia. Gender- and age-matched subjects undergoing manometry and pH-impedance monitoring for typical gastroesophageal reflux (GERD) complaints served as controls. In all patients a detailed history taking was carried out, recording the presence and type of autoimmune comorbidities. Results The overall prevalence of autoimmune comorbidities was two times higher in achalasia than in control patients (12.3 vs. 5%, respectively). The presence of comorbidities did not significantly affect disease’s phenotype, as the age of disease onset was similar in achalasia patients with and without comorbidities (50.13 ± 14.47 and 48.3 ± 18.71, respectively, P = NS). Conclusions Although larger epidemiologic studies are needed to confirm our data, our findings likely suggest that achalasia has a complex multifactorial pathophysiology with an autoimmune component.
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Background: Achalasia is a primary oesophageal motility disorder. Although aetiology remains mainly unknown, a genetic risk variant, rs28688207 in HLA-DQB1, showed strong achalasia association suggesting involvement of immune-mediated processes in the pathogenesis. High-resolution manometry recognises three types of achalasia. The aim of our study was to perform the first genotype-phenotype analysis investigating the frequency of rs28688207 across the high-resolution manometry subtypes. Methods: This was a cross-sectional retrospective study. Achalasia patients from tertiary centres in the Czech Republic (n = 163), Germany (n = 114), Greece (n = 70) and controls were enrolled. All subjects were genotyped for the rs28688207 insertion. The Kruskal-Wallis test was used for the genotype-phenotype analysis. Results: A total of 347 achalasia patients (type I - 89, II - 210, III - 48) were included. The overall frequency of the rs28688207 was 10.3%. The distribution of the insertion was significantly different across the high-resolution manometry subtypes (p = 0.038), being most prevalent in type I (14.6%), followed by type II (9.5%) and III (6.3%). Conclusion: The frequency of the HLA-DQB1 insertion differs among high-resolution manometry achalasia subtypes. The insertion is most prevalent in type I, suggesting that immune-mediated mechanisms triggered by the insertion may play a more prominent role in the pathogenesis of this subtype.
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Achalasia is known to result from degeneration of inhibitory neurons, which are mostly nitrinergic. Characteristic features of achalasia include incomplete lower esophageal sphincter (LES) relaxation and esophageal aperistalsis. Nitric oxide (NO), produced by NO synthase (NOS), plays an important role in peristalsis and LES relaxation. Therefore, we evaluated genetic polymorphisms of NOS gene isoforms (endothelial NOS [eNOS], inducible NOS [iNOS], and neuronal NOS [nNOS]) in patients with achalasia and healthy subjects (HS). Consecutive patients with achalasia (diagnosed using esophageal manometry) and HS were genotyped for 27-base pair (bp) eNOS variable number of tandem repeats (VNTR), iNOS22G/A (rs1060826), nNOS C/T (rs2682826) polymorphisms by polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP), respectively. Among 183 patients (118 [64.5%] male, age 39.5 ± 13.0 years) with achalasia and 366 HS (254 [69.4%] male, age 40.8 ± 11.0 years), eNOS4a4a genotype of 27-bp VNTR was more common among achalasia than HS (20 [10.9%] vs 13 [3.6%]; P < 0.001; OR, 3.72; 95% CI, 1.8-7.7). Patients with achalasia had iNOS22GA genotypes more often than HS (95 [51.9%] vs 93 [25.4%]; P < 0.001; OR, 3.0; 95% CI, 2.1-4.4). Frequency of genotypes GA + AA was higher in patients than HS (97 [53%] vs 107 [29.2%]; P < 0.001; OR, 2.7; 95% CI, 1.8-3.9). Also, nNOS29TT variant genotype in rs2682826 was commoner among patients compared to HS (14 [7.7%] vs 6 [1.6%]; P < 0.001; OR, 5.91; 95% CI, 2.2-15.8). Achalasia is associated with eNOS4a4a, iNOS22GA, and nNOS29TT genotypes. This may suggest that polymorphisms of eNOS, iNOS, and nNOS genes are risk factors for achalasia.
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Science can move ahead by questioning established or canonical views and, so it may be with the enzymes, nitric oxide synthases (NOS). Nitric oxide (NO) is generated by NOS isoforms that are often described by their tissue-specific expression patterns. NOS1 (nNOS) is abundant in neural tissue, NOS2 is upregulated in activated macrophages and known as inducible NOS (iNOS), and NOS3 (eNOS) is abundant in endothelium where it regulates vascular tone. These isoforms are described as constitutive or inducible, but in this perspective we question the broad application of these labels. Are there instances where "constitutive" NOS (NOS1 and NOS3) are inducibly expressed; conversely, are there instances where NOS2 is constitutively expressed? NOS1 and NOS3 inducibility may be linked to post-translational regulation, making their actual patterns activity much more difficult to detect. Constitutive NOS2 expression has been observed in several tissues, especially the human pulmonary epithelium where it may regulate airway tone. These data suggest that expression of the three NOS enzymes may include non-established patterns. Such information should be useful in designing strategies to modulate these important enzymes in different disease states.
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Idiopathic achalasia is a rare motor disorder of the oesophagus characterised by neuronal loss at the lower oesophageal sphincter. Achalasia is generally accepted as a multifactorial disorder with various genetic and environmental factors being risk-associated. Since genetic factors predisposing to achalasia have been poorly documented, we assessed whether single nucleotide polymorphisms (SNPs) in genes mediating immune response and neuronal function contribute to achalasia susceptibility. 391 SNPs covering 190 immune and 67 neuronal genes were genotyped in an exploratory cohort from Central Europe (589 achalasia patients, 794 healthy volunteers (HVs)). 24 SNPs (p<0.05) were validated in an Italian (160 achalasia patients, 278 HVs) and Spanish cohort (281 achalasia patients, 296 HVs). 16 SNPs in linkage disequilibrium (LD) with rs1799724 (r(2)>0.2) were genotyped in the exploratory cohort. Genotype distributions of patients (1030) and HVs (1368) were compared using Cochran-Armitage trend test. The rs1799724 SNP located between the lymphotoxin-α (LTA) and tumour necrosis factor-α (TNFα) genes was significantly associated with achalasia and withstood correction for testing multiple SNPs (p=1.17E-4, OR=1.41 (1.18 to 1.67)). SNPs in high LD with rs1799724 were associated with achalasia. Three SNPs located in myosin-5B, adrenergic receptor-β-2 and interleukin-13 (IL13) showed nominally significant association to achalasia that was strengthened by replication. Our study provides evidence for rs1799724 at the LTA/TNFα locus as a susceptibility factor for idiopathic achalasia. Additional studies are needed to dissect which genetic variants in the LTA/TNFα locus are disease-causing and confirm other variants as potential susceptibility factors for achalasia.
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Despite increasing understanding of the pathophysiology of achalasia, the etiology of this esophageal motility disorder remains largely unknown. However, the occurrence of familial achalasia and its association with well-defined genetic syndromes suggest the involvement of genetic factors. Mutant mouse models display gastrointestinal disturbances that are similar to those observed in achalasia patients. The candidate gene approach has revealed some promising results; however, it has not established conclusive links to specific genes so far. The aim of this review was to summarize current knowledge of the genetics of achalasia. We also discuss the extent to which our understanding of achalasia is likely to be enhanced through future molecular genetic research.
Achalasia significantly affects patients' quality of life and can be difficult to diagnose and treat. To review the diagnosis and management of achalasia, with a focus on phenotypic classification pertinent to therapeutic outcomes. Literature review and MEDLINE search of articles from January 2004 to February 2015. A total of 93 articles were included in the final literature review addressing facets of achalasia epidemiology, pathophysiology, diagnosis, treatment, and outcomes. Nine randomized controlled trials focusing on endoscopic or surgical therapy for achalasia were included (734 total patients). A diagnosis of achalasia should be considered when patients present with dysphagia, chest pain, and refractory reflux symptoms after an endoscopy does not reveal a mechanical obstruction or an inflammatory cause of esophageal symptoms. Manometry should be performed if achalasia is suspected. Randomized controlled trials support treatments focused on disrupting the lower esophageal sphincter with pneumatic dilation (70%-90% effective) or laparoscopic myotomy (88%-95% effective). Patients with achalasia have a variable prognosis after endoscopic or surgical myotomy based on subtypes, with type II (absent peristalsis with abnormal pan-esophageal high-pressure patterns) having a very favorable outcome (96%) and type I (absent peristalsis without abnormal pressure) having an intermediate prognosis (81%) that is inversely associated with the degree of esophageal dilatation. In contrast, type III (absent peristalsis with distal esophageal spastic contractions) is a spastic variant with less favorable outcomes (66%) after treatment of the lower esophageal sphincter. Achalasia should be considered when dysphagia is present and not explained by an obstruction or inflammatory process. Responses to treatment vary based on which achalasia subtype is present.
Thirty years after the discovery of its production by activated macrophages, our appreciation of the diverse roles of nitric oxide (NO) continues to grow. Recent findings have not only expanded our understanding of the mechanisms controlling the expression of NO synthases (NOS) in innate and adaptive immune cells, but have also revealed new functions and modes of action of NO in the control and escape of infectious pathogens, in T and B cell differentiation, and in tumor defense. I discuss these findings, in the context of a comprehensive overview of the various sources and multiple reaction partners of NO, and of the regulation of NOS2 by micromilieu factors, antisense RNAs, and 'unexpected' cytokines. Copyright © 2015 Elsevier Ltd. All rights reserved.
Cancer metastasis is the spread and growth of tumor cells from the original neoplasm to further organs. This review analyzes the role of nitric oxide (NO(•)), a signaling molecule, in the regulation of cancer formation, progression, and metastasis. The action of NO(•) on cancer relies on multiple factors including cell type, metastasis stage, and organs involved. Various chemotherapy drugs cause cells to release NO(•), which in turn induces cytotoxic death of breast, liver, and skin tumors. However, NO(•) has also been clinically connected to a poor cancer prognosis because of its role in angiogenesis and intravasation. This supports the claim that NO(•) can be characterized as both pro-metastatic and anti-metastatic, depending on specific factors. The inhibition of cell proliferation and anti-apoptosis pathways by NO(•) donors has been proposed as a novel therapy to various cancers. Studies suggest that NO(•)-releasing non-steroidal anti-inflammatory drugs act on cancer cells in several ways that may make them ideal for cancer therapy. This review summarizes the biological significance of NO(•) in each step of cancer metastasis, its controversial effects for cancer progression, and its therapeutic potential.
Idiopathic achalasia is characterized by a failure of the lower esophageal sphincter to relax due to a loss of neurons in the myenteric plexus. This ultimately leads to massive dilatation and an irreversibly impaired megaesophagus. We performed a genetic association study in 1,068 achalasia cases and 4,242 controls and fine-mapped a strong MHC association signal by imputing classical HLA haplotypes and amino acid polymorphisms. An eight-residue insertion at position 227-234 in the cytoplasmic tail of HLA-DQ$beta$1 (encoded by HLA-DQB1*05:03 and HLA-DQB1*06:01) confers the strongest risk for achalasia (P = 1.73 × 10(-19)). In addition, two amino acid substitutions in the extracellular domain of HLA-DQ$alpha$1 at position 41 (lysine encoded by HLA-DQA1*01:03; P = 5.60 × 10(-10)) and of HLA-DQ$beta$1 at position 45 (glutamic acid encoded by HLA-DQB1*03:01 and HLA-DQB1*03:04; P = 1.20 × 10(-9)) independently confer achalasia risk. Our study implies that immune-mediated processes are involved in the pathophysiology of achalasia.
Esophageal achalasia is a well-known pathology with an autosomal recessive pattern of inherence described in the familiar cases. Its principal symptom is dysphagia, secondary to the poor relaxation of the lower esophageal sphincter. Chagas disease is one of the many causes involved in the development of this disease, being of great importance in our country because of the high prevalence of the vector. Various syndromes include achalasia in their symptomatology, such as the triple A syndrome or Allgrove syndrome (Addisonianism, achalasia, and alacrimia). We reported a family with a classical autosomal pattern of inherence with six affected members, four men and two women, with achalasia diagnosis as well as esophagus cancer in two of them, secondary to the disease, and no other findings.