Genetic variation in the lymphotoxin-α- (LTA-)/tumor necrosis-factor-α- (TNFα-) locus as a risk
factor for idiopathic achalasia
Mira M. Wouters (1), Diether Lambrechts (2, 3), Jessica Becker (4, 5), Isabelle Cleynen (1),
Jan Tack (1), Ana G Vigo (6), Antonio Ruiz de León (6), Dr. Elena Urcelay (6), Julio Pérez de la
Serna (6), Wout Rohof (7), Vito Annese (8,9), Anna Latiano (8), Orazio Palmieri (8), Manuel
Mattheisen (4, 5, 10, 11), Michaela Mueller (12), Hauke Lang (13), Uberto Fumagalli (14), Luigi
Laghi (14), Giovanni Zaninotto (15), Rosario Cuomo (16), Giovanni Sarnelli (16),
Markus M. Nöthen (4, 5), Séverine Vermeire (1), Michael Knapp (17), Ines Gockel (13),
Johannes Schumacher (4, 5), Guy E. Boeckxstaens (1)
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
(12)
(13)
(14)
(15)
(16)
(17)
Translational Research Center for Gastrointestinal Disorders (TARGID), University of
Leuven, Belgium
Vesalius Research Center, VIB, Leuven, Belgium
Laboratory for Translational Genetics, University of Leuven, Belgium
Department of Genomics, Life & Brain Center, University of Bonn, Germany
Institute of Human Genetics, University of Bonn, Germany
Immunology and Gastroenterology Departments, Instituto de Investigacion Sanitaria del
Hospital Clínico San Carlos (IdISSC), Madrid, Spain
Department of Gastroenterology and Hepatology, Academic Medical Centre, the
Netherlands
Division of Gastroenterology, IRCCS "Casa Sollievo della Sofferenza" Hospital, San
Giovanni Rotondo, Italy
Unit of Gastroenterology SOD2, Azienda Ospedaliera Universitaria, Careggi, Firenze, Italy
Institute for Genomic Mathematics, University of Bonn, Germany
Department of Biostatistics, Harvard School of Public Health, Boston, USA
German Clinic of Diagnostics, Wiesbaden, Germany
Department of General, Visceral and Transplant Surgery, University Medical Center of
Mainz, Germany
Department of Gastroenterology, Humanitas Clinical and Research Center - Istituto Clinico
Humanitas IRCCS, Milan
Department of Surgery, Oncology and Gastroenterology, University of Padova, Italy
Gastroenterology Unit, Department of Clinical and Experimental Medicine, Federico II
University, Napoli, Italy
Institute for Medical Biometry, Informatics and Epidemiology, University of Bonn,
Germany
1
Corresponding author:
Mira Wouters
Translational Research Center for Gastrointestinal Disorders
Herestraat 49, O&NI, box 701
B-3000 Leuven, Belgium
Tel (32) 16 33 08 37
Fax (32) 16 33 07 23
Email: mira.wouters@med.kuleuven.be
Keywords:
Achalasia, TNFalpha, LTA, genetic variant, susceptibility, achalasia
Word count: 3595
The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of
all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to
the BMJ Publishing Group Ltd and its Licensees to permit this article (if accepted) to be published in
Gut editions and any other BMJPGL products to exploit all subsidiary rights, as set out in our licence.
2
Abstract
Background Idiopathic achalasia is a rare motor disorder of the oesophagus characterized 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.
Methods 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). Sixteen SNPs in linkage disequilibrium (LD) with rs1799724 (r2>0.2) were
genotyped in the exploratory cohort. Genotype distributions of patients (1,030) and HVs (1,368) were
compared using Cochran-Armitage trend test.
Results The rs1799724 SNP located between the lymphotoxin-α (LTA) and tumor necrosis-factor-α
(TNFα) genes was significantly associated with achalasia and withstood correction for testing multiple
SNPs (p=1.17E-4, OR=1.41 [1.18-1.67]). SNPs in high LD with rs1799724 were associated with
achalasia. Three other SNPs located in myosin-5B (MYO5B), adrenergic-receptor-β-2 (ADRB2), and
interleukin-13 (IL13) showed nominally significant association to achalasia across all samples.
Conclusions 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.
3
Significance of the study
What is already known about this subject?

Achalasia is hypothesized to be an (auto-)immune-mediated disease, possibly triggered by a
viral infection such as HSV1, characterized by neuronal loss

Achalasia is a complex disorder with various genetic and environmental factors contributing
to disease susceptibility

Former genetic studies suggested potential associations between achalasia and genetic variants
in genes involved in immune response and neuronal function
What are the new findings?

The rs1799724 SNP, located between lymphotoxin-α (LTA) and tumor necrosis-factor-α
(TNFα) was significantly associated with achalasia and withstood correction for testing 359
SNPs.

Three other SNPs located in myosin-5B, adrenergic-receptor-β-2 and interleukin-13 were
potentially associated with achalasia (puncorrected<0.05) and had the same allelic effect across all
the three study cohorts
How might it impact on clinical practice in the foreseeable future?

Our data may contribute to the identification of important disease targets in achalasia, which
ultimately may result in improved clinical management
4
Background
Achalasia is a rare motor disorder of the oesophagus, characterized by incomplete relaxation
of the lower oesophageal sphincter (LES) and absence of oesophageal peristalsis (1). Histological
examination of resection specimen from achalasia patients has demonstrated a significant decrease in
the number of myenteric neurons in the distal oesophagus and the LES (2). Why these neurons
gradually disappear in achalasia patients remains unclear. In the past decade, accumulating evidence
suggests that achalasia may be an immune-mediated inflammatory disorder. Indeed, resection
specimens show infiltration of myenteric ganglia with CD3/CD8 positive lymphocytes expressing
activation markers (3;4). Notably, when isolated from oesophageal tissue and incubated with Herpes
Simplex Virus 1 (HSV1) antigens, T cells from achalasia patients proliferate and release Th-1 type
cytokines IFNγ and IL-2 (5). In addition, IgM antibodies and evidence of complement activation (6)
were shown within myenteric ganglia. Finally, (auto)antibodies against myenteric neurons have
repeatedly been shown in serum of achalasia patients (7-9), especially in patients carrying specific
human leucocyte antigens (HLA) (10). These findings support the hypothesis that achalasia may be an
immune-mediated disease, possibly triggered by a viral infection such as HSV1.
In the majority of cases, achalasia represents a sporadic disease (isolated achalasia), whereas
in the minority of cases it is a familial disorder (familial achalasia) that in most cases follows a
dominant inheritance pattern (11;12). Genetic studies focusing on isolated achalasia have identified
SNPs in genes involved in (auto-)immune responses and neuronal function. In particular, HLA alleles
(9;10;13-15) and SNPs in PTPN22 (16), IL10 (17) and IL23R (18) have been associated with
idiopathic achalasia. Furthermore, SNPs located in genes involved in lower oesophageal sphincter
relaxation such as vasoactive-intestinal-peptide receptor-1 (VIPR1) (19) and c-Kit (20) were
associated with achalasia. Taken together, these studies provide initial evidence for immune and
neuronal-related mediators as predictors of achalasia. It should be emphasized, however, that the
number of patients evaluated in these studies was small (typically, between 80 and 300 patients were
assessed), potentially leading to a high chance of false-positive associations. Hence, there is a great
need for much larger studies systematically evaluating genetic variability in achalasia.
5
Therefore, the aim of this study was to evaluate genetic variability of immune modulation and
neuronal function as susceptibility factors for achalasia. First, we analyzed 384 SNPs in a large
discovery set of 589 achalasia patients and 794 controls. Second, in an effort to independently
replicate our results, two independent cohorts consisting of 441 achalasia patients and 574 controls
(Spain and Italy) were assessed.
6
Materials and Methods
Study population
Three independent cohorts of achalasia patients and healthy volunteers (HVs) from Central
Europe (Belgium, Netherlands and Germany), Spain and Italy were included in this study. Informed
consent was obtained from all participants and local ethics committees approved the study protocol
and genetic studies. Achalasia patients (all idiopathic cases) were diagnosed by oesophageal
manometry. The demographics and clinical characteristics of all cohorts are reported in Table 1.
Exploratory cohort: This sample consisted of 589 idiopathic achalasia patients from Central
Europe (Belgium, Netherlands and Germany) and 794 German controls. The recruitment strategy of
HVs in this cohort was population-based and, therefore, controls with gastrointestinal complaints
and/or inflammatory diseases could not be excluded. Additionally, to demonstrate that the use of a
single German control population did not affect the analysis in the exploratory cohort, 380 HVs of
self-reported Belgian-Flemish ethnicity for 3 generations were genotyped.
Italian validation cohort: 160 Italian idiopathic achalasia patients from North (Firenze and
Milan) and South Italy (Naples and San Giovanni Rotondo at the IRCCS Hospital ‘Casa Sollievo della
Sofferenza’), and 278 Italian HVs, respectively from Firenze and San Giovanni Rotondo, were
included. The Italian control group consisted of blood donors and healthy individuals without a history
of immune-mediated diseases.
Spanish validation cohort: 281 Spanish idiopathic achalasia patients and 296 Spanish HVs
were included. Spanish controls are ethnicity and gender-matched healthy blood donors consecutively
recruited at the Hospital Clínico San Carlos, Spain.
Genotyping
Overall, 391 SNPs covering 258 genes were selected. All SNPs and the corresponding genes
are presented in Supplemental Table S1. We included 16 SNPs because they have previously been
reported as susceptibility variants for achalasia (15-22) (Supplemental Table S2). The remaining SNPs
were selected because they were identified as susceptibility variants in association studies for immunemediated or neuropsychiatric diseases. Potential achalasia-related phenotypes included immune-
7
mediated diseases (Crohn’s disease, ulcerative colitis, type 1 diabetes, rheumatoid arthritis, multiple
sclerosis, chronic obstructive pulmonary disease, ankylosing spondylitis, hyper IgE syndrome, celiac
disease). Since achalasia is characterized by malfunctioning and gradual loss of enteric neurons, SNPs
previously associated with neurological or neuropsychiatric diseases were also included in our study
design. In particular, we chose to select SNPs associated with (auto-immune) neurodegenerative
diseases such as Alzheimer’s disease (23), multiple sclerosis (24) and psychiatric diseases
characterized by structural changes in neuronal cyto-architecture, such as major depression (25). All
589 achalasia samples from the exploratory cohort were analyzed with custom-designed chips using
the Golden Gate Illumina platform at the Vesalius Research Center, KULeuven, Belgium. These
results were compared to German HVs, which already served as universal controls for various GWAS
(26-28). In the latter sample, 141 SNPs were genotyped using Illumina’s HumanOmniExpress
BeadArrays and 250 were imputed using IMPUTE2 (29) and reference datasets from the 1000
genomes project (30) together with post-quality control genome-wide GWAS data for German HVs.
Finally, our selection of 391 SNPs was expanded with 16 SNPs in weak linkage disequilibrium (LD)
with rs1799724 (based on 1000 Genomes data, LD r2>0.2) located 40 kb up or downstream of
rs1799724, and 2 functional SNPs located in a much larger genomic region encompassing rs1799724,
i.e., rs1046089 and rs9332739 (based on the GWAS catalogue). This set of 18 SNPs was genotyped
in cases from the exploratory cohort using Sequenom Massarray® (operational at the Vesalius
Research Center (31)) and compared to genotyping data that were already available for the German
HVs.
In the exploratory sample, 39 SNPs showed association with achalasia (p<0.05, uncorrected)
and were validated in the Italian and Spanish cohort using Sequenom® MassARRAY. Since
rs2236754 and rs7210080 in SSTR2 are synonymous SNPs, only rs2236754 was genotyped in the
validation cohorts. Genotype data of 4 SNPs failed and 7 SNPs not pass quality control (QC)
assessment and were therefore excluded. Quality control (QC) criteria for Golden Gate and
MassARRAY included an individual SNP call rate of >0.95 in patients and HVs, a sample SNP call
rate of >0.95, a minor allele frequency (MAF) of >0.01 and a Hardy-Weinberg equilibrium (HWE) pvalue >0.0001 in HVs.
8
Statistical analyses
Fisher's exact tests were used to test for HWE. For the association analysis in each casecontrol sample we used the Cochran-Armitage trend test under the assumption of an additive genetic
model and Plink v1.07 software (http://pngu.mgh.harvard.edu/~purcell/plink/). The additive model
assumes that on a log scale, the risk in carriers of 2 copies of the at-risk allele is doubled compared to
carriers of only a single at-risk allele. Furthermore, we performed a Cochran-Mantel-Haenszel metaanalysis across all samples using the Statistical Analysis Software (SAS) (http://www.sas.com). An
uncorrected p-value <0.05 was considered nominally significant, whereas a p-value <1.4E-04
(Bonferroni-corrected for 359 SNPs) was considered significant after correction for multiple testing.
Binary logistic regression was used to assess whether risk-effects were gender-dependent by
considering disease status as a dependent variable and SNP, gender and study as covariates. SPSS was
used for regression analyses.
9
Results
Of all 391 SNPs, 25 SNPs failed QC due to low call rates, while another 7 SNPs were excluded
because they were monomorphic. All remaining 359 SNPs fulfilled QC criteria and were subsequently
assessed for association with achalasia in our exploratory case-control sample consisting of 589 Dutch,
Belgian and German achalasia patients and 794 German HVs. Association results of all SNPs are
listed in Supplemental Table S1.
In total, 39 SNPs (10% of all SNPs) were nominally significantly associated with achalasia in
the exploratory cohort (p-values ranged between 2.85E-05 and 4.89E-02, Supplemental Table S1).
After removing 1 synonymous SNP, all remaining 38 SNPs were selected for genotyping in the Italian
and Spanish replication cohorts using Sequenom MassARRAY (160 Italian patients vs. 278 HVs, 281
Spanish patients vs. 296 HVs). Overall, 4 markers failed genotyping and 7 SNPs did not fulfil QC
criteria and were therefore excluded from the study. Furthermore, 3 SNPs showed genotype
inconsistencies between the MassARRAY and GoldenGate platforms and were therefore also
excluded from the study. These inconsistencies were detected because 480 patients from the
exploratory sample were genotyped for these 38 SNPs as internal controls on Sequenom
MassARRAY. For all other 24 SNPs, a genotype concordance rate of minimum 99% between
Sequenom and GoldenGate assay was observed.
Of all 24 SNPs that were genotyped in the Italian and Spanish cohorts, two SNPs were
significantly replicated in these cohorts, while for another 8 SNPs ORs showed the same association
trend in the Spanish cohort and for 7 SNPs ORs showed the same trend in the Italian cohort (Table 2).
Because the statistical power in the Italian and Spanish validation cohorts was only moderate (between
16.8% and 76% with p<0.05 based on results obtained in the exploratory cohort; Genetic Power
Calculator (32)), we performed a Cochran-Mantel-Haenszel test using all 24 SNP-markers and
assessed genotype distributions simultaneously in all three cohorts (Table 2). One SNP, rs1799724, on
chromosome 6p21 nearby lymphotoxin-α (LTA) and tumor necrosis-factor-α (TNFα) was significantly
associated with achalasia (Table 2). Notably, rs179724 withstood correction for testing all 353 SNPs
in the exploratory study (pmeta-analysis=1.17E-04, OR=1.41 [1.18-1.67]). This SNP showed association in
10
the Central European and Spanish cohorts (p=1.69E-03 and p=4.38E-04, respectively), whereby the
minor T-allele represents the at-risk allele that was 3% and 7% more common in achalasia patients
compared to controls, resulting in an increased risk for achalasia of OR=1.46 [1.16-1.85] and 2.02
[1.38-2.96] (Table 2). However, rs1799724 was not significantly associated in the Italian cohort
(OR=0.94 [0.66-1.34], Table 2, Fig. 1A). However, when comparing Belgian and Dutch achalasia
patients to an additional control population of 380 matched Belgian HVs, the association of rs1799724
with achalasia remained significant (P=5.60E-5, OR=1.50 [1.23-1.82]) (Supplemental Table S3),
indicating that the use of the German control population in the exploratory cohort did not introduce
any spurious association signal (Supplemental Table S4). Unfortunately, since rs1799724 was not a
tagging SNP, we could not assess the potential effects of rs1799724 on LTA or TNFα expression using
eQTL databases. A more detailed overview of this risk locus is given in Figure 2.
Additionally, 3 other SNPs were significantly associated with achalasia in the meta-analysis
(Table 2). SNP rs2292382 is located on chromosome 18q21 in myosin-5b (MYO5B) and all three
populations showed the same association trend (Fig. 1B, pmeta-analysis=1.67E-03, OR=1.33 [1.11-1.58]).
Also rs12654778 on chromosome 5q33 near the adrenergic-receptor-β-2 (ADRB2) was significantly
associated in the meta-analysis (Fig. 1C, pmeta-analysis=1.44E-02, OR=1.16 [1.03-1.30]). Furthermore
rs1800925 on chromosome 5q31 near interleukin-13 (IL13) showed significant association in the
meta-analysis (Fig. 1D, pmeta-analysis=1.20E-02, OR=1.20 [1.04-1.39]). Comparison of Belgian and
Dutch achalasia patients to an additional control population of 380 matched HVs confirmed the initial
association observed for rs2292382 (pmeta-analysis =1.7E-02, OR=1.15 [1.03-1.30]) and rs12654778
(pmeta-analysis=1.3E-03, OR=0.75 [0.62-0.89]), but not rs1800925 (pmeta-analysis=0.14, OR=0.90 [0.781.04]).
Based on previously reported sex-specific genetic associations with auto-immune diseases,
including idiopathic achalasia (16), we determined the association for each of the 23 SNPs after
stratification for gender. Our most significant SNP, rs1799724 at LTA/TNFα, showed no sex-specific
association, as revealed by a logistic regression considering gender as a covariate (OR=1.53; P=1.58E5). Also two other SNPs significant in the meta-analysis showed no sex-specific association
(rs2292382 in MYO5B: OR=0.75 and P=1.1E-03, rs12654778 in ADRB2: OR=1.15 and P=1.5E-02).
11
For none of the other SNPs significant in the exploratory cohort, we observed a sex-specific
association (Supplemental Table S5 and S6).
Finally, to gain more insights into the potential functional effects of rs1799724, which is
located in the 5’UTR region of TNFα, or any other SNP linked to rs1799724, we genotyped 16 SNPs
in weak LD (r2>0.2) with rs1799724 in the exploratory cohort (Fig. 2, supplemental Table S7) and 2
functional SNPs located in a much larger genomic region around rs1799724, i.e., rs1046089 and
rs9332739. Three SNPs failed and 2 SNPs did not pass quality control leaving us with 13 successfully
genotyped markers (Table 3). Two SNPs nearly synonymous to rs1799724, i.e., rs6916921 and
rs769178, were also associated with achalasia in the exploratory cohort (PTrend= 2.06E-3 and 4.40E-3),
whereas SNPs in weaker LD were not associated with achalasia. Intriguingly, we identified a
functional SNP in the HLA region, i.e., rs1046089, which was not in LD with rs1799724 (r2=0), but
was significantly associated with achalasia (OR=0.76 and PTrend=7.74E-04).
12
Discussion
In a case-control study of 1,030 achalasia patients and 1,368 HVs, the strongest association
signal was identified for rs1799724, a SNP located between lymphotoxin-α (LTA) and tumor necrosisfactor-α (TNFα) (p=1.17E-4, OR=1.41). Besides its association with achalasia in the exploratory
cohort (p=1.69E-3, OR=1.46), this SNP was independently replicated in the Spanish cohort (p=4.38E4, OR=2.02). A meta-analysis across all 3 independent cohorts also revealed that rs1799724 was still
associated with achalasia after a conservative Bonferroni correction for all 353 SNPs tested.
It has been hypothesized that the loss of neurons in achalasia results from an aberrant immunemediated inflammatory process triggered by a (latent) infection with herpes simplex virus 1 (HSV-1)
(5) in patients with a particular immunogenetic background (33). Our most significant SNP,
rs1799724, is located on chromosome 6p21, 382bp downstream of LTA and 857bp upstream of TNFα.
Of interest, both genes are involved in immunological processes, i.e. lymphotoxin (LTA) is a TNFsuperfamily member that is crucial for the development and orchestration of robust immune responses
(34), particularly in antiviral responses (35-37). Tumor necrosis factor-alpha (TNFα) on the other
hand, is a pro-inflammatory cytokine involved in the pathogenesis of many (auto-immune)
inflammatory diseases, including Crohn's disease (38), rheumatoid (39), psoriatic arthritis (40) and
inflammatory bowel disease (38). In line herewith, rs1799724 has been implicated as a risk factor for
immunological diseases, such as inflammatory bowel disease (IBD) (41;42) and chronic obstructive
pulmonary disease (43;44). Of interest, patients with at least one rs1799724 at-risk variant had a
nearly 2-fold increased risk (HR=1.97, [1.10–3.50]) to develop oesophagitis following radiation
treatment (45). Moreover, in the context of genome-wide association (GWA) studies assessing genetic
risk for neonatal lupus (46), associations surpassing the threshold of genome-wide significance have
been reported both for variants in LTA and TNFα. However, these studies did not report on the
association with rs1799724 specifically, as this SNP did not belong to the top GWAS-findings. Of
note, two studies reported that rs1799724 also increases the risk for Alzheimer’s disease, thereby
providing further evidence for the relevance of the immune system in neurodegeneration (47). Finally,
rs1799724 has also been associated with outcome and severity of viral infections such as respiratory
syncytial virus (RSV), the onset of asthma (43) and hepatitis B virus infections (HBV (48)). Of
13
interest, TNFα polymorphisms were also associated with AIDS-progression (49). Since recent data
suggest that a viral infection may trigger an aberrant immune response against myenteric neurons in
the oesophagus, one may speculate that the association of rs1799724 with achalasia indirectly supports
this hypothesis.
Since there are no functional data reporting how rs1799724 could potentially affect expression
of LTA or TNFα, it is impossible to assign risk effects of rs1799724 to the altered expression of either
LTA, TNFα or both. Most studies so far considered TNFα as the culprit disease gene. Of note,
rs1800629 in TNFα, which is only 549 bp apart from rs1799724 and was previously found as a
susceptibility factor for rheumatoid arthritis (50) and systemic lupus erythematosus (51), did not show
any association with achalasia (Supplemental Table S1). However, this can be explained by the low
degree of LD between both variants (r2=0.01).
Genotyping of SNPs that were in LD with rs1799724 revealed that only those SNPs that are in
high LD, namely rs6916921 (r2=1) and rs769178 (r2=0.964), were associated with achalasia
(Supplemental Fig. 1). There are however no direct functional data available on these SNPs. We can
speculate that CR082032, also referred to as rs1800610 and in high LD with rs1799724 (r2=0.895)
may be the causal SNP. Rs1800610 is located in the 3’-downstream mRNA-UTR region of TNFα and
has previously been associated with breast cancer (52) and upper aerodigestive tract malignant tumors
(53). The functional consequences of rs1800610 have, however, also not been studied (53). In
addition, we identified another association signal in the HLA region, i.e., for rs1046089 (OR=0.76 and
P=7,74E-04). Although this SNP is not in LD with rs1799724 (r2=0), it was selected for validation
because it represents a missense mutation (Arg1740His) and is located in relatively close proximity to
rs1799724 (~60 kb). Rs1046089 is located in exon 22 of BAT2 and was previously associated with
malaria disease (54) and rheumatoid arthritis (55). Expression data for rs1046089 demonstrated that
the polymorphism is associated with altered expression of HLA-DRB4 in monocytes and HLA-DQA1
in lymphoblastoid cell lines. Interestingly, rs1046089 is synonymous to rs3135388, a SNP that is
significantly associated with achalasia in the exploratory cohort (Ptrend_exploratory=2.44E-4; Table 2). The
rs3135388 SNP is not in LD with rs1799724 (r2=0), but is nearly synonymous with the HLADRB1*1501 variant (54). In particular, the rs3135388 at-risk T-allele is associated with a 3- to 6-fold
14
increased risk of developing multiple sclerosis (54), whereas it has also been associated with other
autoimmune diseases, such as systemic lupus erythematodes (SLE) (54). However, rs3135388 was not
associated with achalasia in the Italian or Spanish replication cohorts, and as a result it did not
withstand Bonferroni correction for multiple testing in the meta-analysis. We therefore cannot
formally consider rs3135388 as an established susceptibility locus for achalasia. Based on our results,
fine-mapping studies assessing a larger set of SNPs that systematically cover all of the genetic
variability in chromosomal region 6p21 are needed, to pinpoint the causal risk variant of this complex
locus that possibly contains two independent at-risk signals. Additionally, our data encourage efforts
devoted at identifying additional SNPs underlying the risk to develop achalasia. In particular, other
risk variants that have already been linked to other autoimmune disorders or that are located in genes
involved in mediating auto-immunity could be tested for association with achalasia.
In contrast to the robust association with the LTA/TNFα locus, our findings for MYO5B,
ADRB2 and IL13 are more difficult to interpret at the functional level. The markers in these genes
were nominally significantly associated with achalasia in the meta-analysis and although replication in
validation cohorts strengthened the observed association, they did not survive correction for multiple
testing. ADRB2 is expressed in several neuronal populations and either amplifies or reduces neuronal
damage depending on the context and the nature of the toxic insult (56). MYO5B, the unconventional
type Vb myosin motor protein, is predominantly found in intestinal tissues (57). MYO5B mutations are
associated with disrupted epithelial cell polarity and dysregulation of intracellular protein trafficking
(58). IL13 mediates a wide variety of immune-relevant processes (59-62). The functional effect of
these variants on the expression of their respective genes is not yet understood. Moreover, the
significance level for the latter SNP is only moderate and these findings should therefore be
considered as preliminary.
A number of SNPs were only nominally significantly associated with achalasia in the
exploratory cohort, but showed no association signal in the validation cohorts. Although these SNPs
are more likely to represent false-positive findings, they may still provide mechanistic hints to the
pathogenesis of achalasia. Two of these SNPs were previously associated with auto-immune disease:
namely rs6679793 SNP in FCRL5 was associated with autoimmune thyroid disease (63), whereas
15
rs2303138 in tensin 1 (TNS1) was associated with ankylosing spondylitis (63). Other SNPs are
characterized by altered neuronal function or neurotoxicity and were located in the alpha-2
adrenoceptor (ADRA2A) (64;65), the transcription factor TBX15 (63), the µ opioid receptor gene,
OPRM1 (66) and somatostatin receptor 2 (SSTR2). However, their (functional) contribution to
neurodegeneration in achalasia is not known and needs further confirmation. Indeed, these markers
represent very interesting candidates for replication studies on independent cohorts of achalasia
patients. For instance, to reach genome-wide significance (α=10E-7), 4000 achalasia patients and 4000
healthy controls would be required to have 94% power to detect associations with SNPs having a
MAF of 0.20 and risk of 1.3. Finally, we observed that none of the variants, for which an association
with achalasia was previously reported (20) (Supplemental Table S2), significantly replicated in the
exploratory cohort (Supplemental Table S1).
In conclusion, in line with previous associations suggesting rs1799724 as a risk factor for
other neurodegenerative and immune-mediated diseases, our findings provide evidence that
LTA/TNFα represents a susceptibility locus for achalasia. Since it remains unclear whether rs1799724
or another variant in this locus represents the true risk conferring variant, fine-mapping association
studies using dense marker sets across LTA and TNFα are needed.
16
Table 1. Demographic and clinical characteristics of achalasia patients and healthy volunteers
female/male
unknown
age at onset in years
total number
Central European
exploratory cohort (1)
achalasia
HVs
281/308
408/386
41.76±18.25
589
Italian
validation cohort (2)
achalasia
HVs
74/86
24/248
6
unknown
794
160
278
Spanish
validation cohort (3)
achalasia
HVs
125/155
133/162
1
1
45.12±16.8
1
281
296
1 The central European exploratory cohort includes 139 patients from the Translational Research
Center for Gastrointestinal Disorders (TARGID), University of Leuven, Belgium; 147 patients
from the AMC, University of Leuven, the Netherlands and 303 patients from the Department of
General, Visceral and Transplant Surgery, University Medical Center of Mainz, Germany. All
controls are from the Institute of Human Genetics and Department of Genomics, Life & Brain
Center, University of Bonn, Germany
2 The Italian validation cohort includes 37 patients from the Instituto Clinico Humanitas, Milan, 72
patients from the IRCCS Casa Sollievo della Sofferenza Hospital, San Giovanni Rotondo and
Azienda Ospedaliero Universitaria, Firenze and 51 patients from the Frederico II University
Hospital, Napoli. All controls for the IRCCS Casa Sollievo della Sofferenza Hospital, San
Giovanni Rotondo and Azienda Ospedaliero Universitaria, Firenze.
3 In the Spanish validation cohort, all patients and controls are from the Immunology and
Gastroenterology Department of Hospital Clínico S. Carlos, Madrid, Spain
17
Table 2. Association of genetic variants in LTA/TNFα, MYO5B, ADRB2 and IL13 with ideopathic achalasia. Shown from left to right are: NCBI dbSNP
database accession numbers (SNP), chromosome and chromosomal position (Chr, Position), nearest gene (Gene), two nucleotides of the SNP, first allele is the
risk allele (Allele), frequency of the minor allele in patients (MAF achalasia) and healthy volunteers (MAF HV) (MA of Bel_NL_G cohort taken as MA in
Italian and Spanish cohort), Armitage's trend test (P_trend), Cochran-Mantel-Haenszel meta-analysis (P_CMH), allelic odds ratio (OR) and lower and upper
bound of the corresponding 95% confidence interval (CI)
SNP
Chr
Position
Nearest gene
Allele
Samples
rs2476601
1
114179091
PTPN22
A/G
Bel_NL_G
Italy
Spain
combined
Bel_NL_G
Italy
Spain
combined
Bel_NL_G
Italy
Spain
combined
Bel_NL_G
Italy
Spain
combined
Bel_NL_G
Italy
Spain
combined
Bel_NL_G
Italy
Spain
combined
Bel_NL_G
Italy
Spain
rs10494217
1
119270711
TBX15
T/G
rs2274910
1
159118670
ITLN1
C/T
rs1800896
1
205013520
IL10
G/A
rs6785049
3
121016423
NR1I2
A/G
rs11707609
3
124986114
MYLK
G/A
rs4143832
5
131890876
unknown
C/A
MAF
achalasia
0.13
0.02
0.09
MAF
HV
0.10
0.05
0.06
P_trend
P_CMH
3.23E-02
2.19E-02
1.33E-01
6.97E-02
0.14
0.12
0.14
0.11
0.13
0.15
8.87E-04
8.24E-01
7.48E-01
0.29
0.29
0.27
0.33
0.29
0.29
4.44E-02
8.93E-01
4.79E-01
0.51
0.39
0.35
0.47
0.36
0.38
3.37E-02
3.58E-01
3.15E-01
1.27E-02
5.25E-02
1.33E-01
0.37
0.45
0.43
0.41
0.38
0.43
4.58E-02
2.18E-02
7.75E-01
0.42
0.39
0.41
0.38
0.42
0.47
4.23E-02
2.96E-01
4.70E-02
0.16
0.17
0.17
0.20
0.16
0.18
1.04E-02
5.50E-01
6.92E-01
6.79E-01
9.97E-01
18
OR
CI (95%)
1.29
0.4
1.4
1.2
1.4
0.96
0.95
1.22
1.18
1.02
1.09
1.13
1.17
1.14
0.89
1.09
1.17
0.72
0.97
1.02
1.16
0.87
0.79
1
1.3
0.89
1.06
1.02-1.64
0.18-0.89
0.90-2.18
0.99-1.47
1.16-1.70
0.63-1.45
0.68-1.31
1.04-1.42
1.00-1.40
0.75-1.38
0.85-1.41
1.00-1.28
1.01-1.37
0.86-1.51
0.70-1.13
0.97-1.23
1.00-1.37
0.55-0.96
0.76-1.22
0.91-1.15
0.99-1.35
0.65-1.14
0.62-0.99
0.89-1.12
1.06-1.59
0.62-1.29
0.78-1.44
rs1800925
5
132020708
IL13
C/T
rs2569190
5
139993100
CD14
A/G
rs12654778
5
148185934
ADRB2
A/G
rs1799724
6
31650461
LTA / TNFα
T/C
rs3135388
6
32521029
HLA-DRA
C/T
rs550014
6
154476586
OPRM1
A/G
rs975537
7
30663882
CRHR2
A/T
rs1468412
7
86271387
GRM3
A/T
rs11195419
10
112829358
ADRA2A
A/C
combined
Bel_NL_G
Italy
Spain
combined
Bel_NL_G
Italy
Spain
combined
Bel_NL_G
Italy
Spain
combined
Bel_NL_G
Italy
Spain
combined
Bel_NL_G
Italy
Spain
combined
Bel_NL_G
Italy
Spain
combined
Bel_NL_G
Italy
Spain
combined
Bel_NL_G
Italy
Spain
combined
Bel_NL_G
Italy
5.43E-02
0.18
0.20
0.17
0.22
0.19
0.21
1.37E-02
7.49E-01
9.92E-02
1.20E-02
0.49
0.51
0.50
0.45
0.52
0.53
2.56E-02
7.13E-01
3.56E-01
0.40
0.40
0.40
0.36
0.37
0.38
3.69E-02
3.23E-01
3.45E-01
0.13
0.18
0.15
0.10
0.19
0.08
1.69E-03
7.52E-01
4.38E-04
0.09
0.04
0.06
0.15
0.06
0.06
1.01E-04
3.33E-01
9.16E-01
0.22
0.28
0.21
0.27
0.23
0.20
1.95E-03
9.01E-02
8.12E-01
0.20
0.19
0.20
0.24
0.20
0.18
1.02E-02
6.91E-01
3.73E-01
0.25
0.28
0.26
0.29
0.28
0.28
8.22E-03
8.00E-01
4.44E-01
0.14
0.13
0.10
0.11
4.47E-04
3.86E-01
2.85E-01
1.44E-02
1.17E-04
2.44E-04
1.38E-01
8.42E-02
1.31E-02
19
1.16
1.26
0.95
1.28
1.2
1.19
0.95
0.9
1.06
1.18
1.15
1.12
1.16
1.46
0.94
2.02
1.41
1.63
1.36
0.97
1.46
1.32
0.75
0.97
1.11
1.27
1.07
0.88
1.13
1.26
1.04
1.11
1.18
1.51
1.21
1.00-1.35
1.04-1.53
0.67-1.33
0.95-1.72
1.04-1.39
1.02-1.38
0.72-1.25
0.71-1.13
0.95-1.19
1.01-1.37
0.87-1.53
0.89-1.42
1.03-1.30
1.16-1.85
0.66-1.34
1.38-2.96
1.18-1.67
1.28-2.07
0.72-2.59
0.61-1.57
1.19-1.78
1.10-1.57
0.55-1.02
0.73-1.29
0.97-1.27
1.06-1.53
0.76-1.52
0.65-1.18
0.98-1.31
1.06-1.49
0.77-1.41
0.86-1.44
1.03-1.34
1.20-1.91
0.79-1.85
rs929270
12
46398644
P11
G/A
rs708486
14
51810721
PTGDR
T/C
rs1014531
16
9763295
GRIN2A
A/G
rs1801275
16
27281901
IL4R
A/G
rs2236754
17
68675676
SSTR2
G/A
rs11078827
17
9926566
GAS7
C/T
rs173365
17
41256855
CRHR1
T/C
rs2292382
18
45878935
MYO5B
G/T
Spain
combined
Bel_NL_G
Italy
Spain
combined
Bel_NL_G
Italy
Spain
combined
Bel_NL_G
Italy
Spain
combined
Bel_NL_G
Italy
Spain
combined
Bel_NL_G
Italy
Spain
combined
Bel_NL_G
Italy
Spain
combined
Bel_NL_G
Italy
Spain
combined
Bel_NL_G
Italy
Spain
combined
0.11
0.12
5.52E-01
6.43E-03
0.29
0.27
0.22
0.26
0.27
0.22
4.89E-02
8.67E-01
9.72E-01
0.42
0.50
0.44
0.46
0.48
0.45
2.96E-02
5.10E-01
7.82E-01
0.42
0.37
0.43
0.39
0.43
0.40
3.53E-02
1.13E-01
3.16E-01
0.18
0.12
0.22
0.22
0.15
0.19
1.59E-02
2.11E-01
2.04E-01
1.06E-01
1.32E-01
1.47E-01
1.08E-01
0.25
NA
0.25
0.21
NA
0.23
1.26E-02
NA
4.46E-01
0.52
0.59
0.55
0.48
0.59
0.58
2.95E-02
9.54E-01
2.79E-01
0.47
0.45
0.46
0.43
0.49
0.48
4.64E-02
2.84E-01
4.70E-01
1.66E-02
2.56E-01
1.43E-01
0.11
0.10
0.10
0.15
0.11
0.14
1.08E-02
7.12E-01
3.20E-02
1.67E-03
20
0.89
1.28
1.19
1.03
1
1.11
1.18
0.91
1.03
1.09
1.18
0.8
1.13
1.09
1.25
1.28
0.83
1.13
1.25
NA
1.11
1.20
1.18
1.01
0.88
1.07
1.17
0.86
1.09
1.09
1.34
1.09
1.46
1.33
0.62-1.29
1.07-1.53
1.01-1.41
0.75-1.40
0.75-1.32
0.98-1.27
1.02-1.38
0.69-1.20
0.82-1.30
0.97-1.23
1.01-1.37
0.60-1.06
0.89-1.44
0.97-1.23
1.04-1.52
0.85-1.93
0.63-1.11
0.97-1.31
1.04-1.49
NA
0.85-1.45
1.03-1.40
1.02-1.37
0.76-1.33
0.70-1.11
0.95-1.20
1.00-1.36
0.66-1.13
0.87-1.38
0.97-1.22
1.07-1.68
0.69-1.72
1.03-2.09
1.11-1.58
Table 3. Association results of SNPs that are in weak LD (r2>0.2) with rs1797724 within 40 kb distance, and of rs1046089, the nearest functional SNP,
with achalasia in the central European cohort.
Shown from left to right are: NCBI dbSNP database accession numbers (SNP), chromosome and chromosomal position (Chrom, Position), two nucleotides of
the SNP, first allele is the minor allele (Allele), Armitage's trend test (P_trend), frequency of the minor allele in achalasia patients (MAF achalasia) and
healthy volunteers (MAF HV), odds ratio and 95% confidence interval (OR CI 95%)
SNP
Chr
Position
Allele
P_trend
rs929138
rs2239705
rs2523500
rs6916921
rs116749187
rs2857602
rs2844484
rs2844483
rs2071590
rs2239704
rs769178
rs3132452
rs1046089
6
6
6
6
6
6
6
6
6
6
6
6
6
31611677
31621381
31626333
31628405
31637876
31641357
31644203
31644775
31647747
31648120
31655493
31671219
31710946
G/A
T/C
C/T
T/C
T/C
C/T
T/C
A/C
T/C
T/G
A/C
T/G
A/G
5,22E-01
4,31E-02
4,97E-01
4,40E-03
4,73E-01
3,19E-01
3,37E-01
2,67E-01
2,37E-01
3,63E-01
2,06E-03
5,04E-01
7,74E-04
MAF
achalasia
0.24
0.20
0.36
0.13
0.13
0.41
0.41
0.41
0.35
0.41
0.13
0.13
0.29
MAF
HV
0.28
0.17
0.34
0.10
0.17
0.39
0.39
0.48
0.43
0.39
0.10
0.17
0.35
21
OR [95% CI]
0.82 [0.45-1.48]
1.22 [1.01-1.49]
1.06 [0.91-1.24]
1.42 [1.12-1.80]
0.78 [0.40-1.52]
1.08 [0.93-1.26]
1.08 [0.92-1.30]
0.74 [0.44-1.26]
0.72 [0.42-1.23]
1.07 [0.92-1.26]
1.46 [1.15-1.85]
0.77 [0.37-1.62]
0.76 [0.64-0.89]
Figure Legends
Fig. 1. Forrest plot showing the overall effect of the risk allele on the susceptibility to achalasia.
OR, odds ratio with 95% confidence interval.
Fig. 2. Linkage disequilibrium plot of SNPs (r2>0.2) with rs1799724 within 40 kb based on the
1000 Genomes Browser. Five SNPs were in strong linkage disequilibrium (LD r2>0.8). CR082032,
also known as rs1800610, is located 3’ downstream of LTA and TNFalpha intronic and is associated
with upper aerodigestive tract malignant tumours. The functional consequence of rs1800610 is not
known. The synonymous SNP rs6916921 is located in an intron of NFKbIL1, a region that has been
repeatedly associated with various auto-immune diseases. There are no publication data on the other
synonymous SNP, rs116749187.
22
Disclosures:
The authors report no conflict of interest
Grant support:
Mira Wouters and Isabelle Cleynen are postdoctoral researchers and Séverine Vermeire is a senior
clinical investigator of the Fund for Scientific Research (FWO) Flanders, Belgium. Guy Boeckxstaens
received research funding by a grant from the Flemish government (Odysseus Program, FWO).
Acknowledgements
The authors would like to thank the patients and the supporting staff at each site.
Contributors:
All authors read and approved the final version of the manuscript.
MMW: study concept and design, acquisition of data, analysis and interpretation of data; drafting of
the manuscript. DL: technical and material support, critical revision of the manuscript for important
intellectual content. JB: acquisition of data. IC: analysis and interpretation of data. JT, AGV, ARDL,
EU, JPDLS, WR, VA, AL, OP, MM, MM, HL, UF, LL, GZ, RC, GS, MMN, IG: acquisition of
samples and characterization of patients. SV: critical revision of the manuscript for important
intellectual content. MK: statistical analysis. JS: acquisition and interpretation of data, critical revision
of the manuscript for important intellectual content. GEB: study supervision, obtained funding, critical
revision of the manuscript for important intellectual content.
23
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