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) Translational Research Center for Gastrointestinal Disorders (TARGID), University of Leuven, Belgium

(2) Vesalius Research Center, VIB, Leuven, Belgium (3) Laboratory for Translational Genetics, University of Leuven, Belgium(4) Department of Genomics, Life & Brain Center, University of Bonn, Germany (5) Institute of Human Genetics, University of Bonn, Germany (6) Immunology and Gastroenterology Departments, Instituto de Investigacion Sanitaria del

Hospital Clínico San Carlos (IdISSC), Madrid, Spain (7) Department of Gastroenterology and Hepatology, Academic Medical Centre, the

Netherlands(8) Division of Gastroenterology, IRCCS "Casa Sollievo della Sofferenza" Hospital, San

Giovanni Rotondo, Italy (9) Unit of Gastroenterology SOD2, Azienda Ospedaliera Universitaria, Careggi, Firenze, Italy(10) Institute for Genomic Mathematics, University of Bonn, Germany(11) Department of Biostatistics, Harvard School of Public Health, Boston, USA (12) German Clinic of Diagnostics, Wiesbaden, Germany (13) Department of General, Visceral and Transplant Surgery, University Medical Center of

Mainz, Germany(14) Department of Gastroenterology, Humanitas Clinical and Research Center - Istituto Clinico

Humanitas IRCCS, Milan (15) Department of Surgery, Oncology and Gastroenterology, University of Padova, Italy(16) Gastroenterology Unit, Department of Clinical and Experimental Medicine, Federico II

University, Napoli, Italy (17) Institute for Medical Biometry, Informatics and Epidemiology, University of Bonn,

Germany

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Corresponding author:Mira WoutersTranslational Research Center for Gastrointestinal DisordersHerestraat 49, O&NI, box 701B-3000 Leuven, BelgiumTel (32) 16 33 08 37Fax (32) 16 33 07 23Email: 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.

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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

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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.

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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 immune-

mediated or neuropsychiatric diseases. Potential achalasia-related phenotypes included immune-

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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) p-

value >0.0001 in HVs.

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Statistical analyses

Fisher's exact tests were used to test for HWE. For the association analysis in each case-

control 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 meta-

analysis 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.

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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

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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 [0.78-1.04]).

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=1.58E-

5). 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).

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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).

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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 necrosis-

factor-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=4.38E-

4, 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 immune-

mediated 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 TNF-

superfamily 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

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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 (r 2=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 (P trend_exploratory=2.44E-4; Table 2). The

rs3135388 SNP is not in LD with rs1799724 (r2=0), but is nearly synonymous with the HLA-

DRB1*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

Central European exploratory cohort (1)

Italianvalidation cohort (2)

Spanishvalidation cohort (3)

achalasia HVs achalasia HVs achalasia HVsfemale/male 281/308 408/386 74/86 24/248 125/155 133/162unknown 6 1 1age at onset in years 41.76±18.25 unknown 45.12±16.8

1total number 589 794 160 278 281 296

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 MAF achalasia

MAF HV P_trend P_CMH OR CI (95%)

rs2476601 1 114179091 PTPN22 A/G Bel_NL_G 0.13 0.10 3.23E-02 1.29 1.02-1.64Italy 0.02 0.05 2.19E-02 0.4 0.18-0.89Spain 0.09 0.06 1.33E-01 1.4 0.90-2.18

combined 6.97E-02 1.2 0.99-1.47rs10494217 1 119270711 TBX15 T/G Bel_NL_G 0.14 0.11 8.87E-04 1.4 1.16-1.70

Italy 0.12 0.13 8.24E-01 0.96 0.63-1.45Spain 0.14 0.15 7.48E-01 0.95 0.68-1.31

combined 1.27E-02 1.22 1.04-1.42rs2274910 1 159118670 ITLN1 C/T Bel_NL_G 0.29 0.33 4.44E-02 1.18 1.00-1.40

Italy 0.29 0.29 8.93E-01 1.02 0.75-1.38Spain 0.27 0.29 4.79E-01 1.09 0.85-1.41

combined 5.25E-02 1.13 1.00-1.28rs1800896 1 205013520 IL10 G/A Bel_NL_G 0.51 0.47 3.37E-02 1.17 1.01-1.37

Italy 0.39 0.36 3.58E-01 1.14 0.86-1.51Spain 0.35 0.38 3.15E-01 0.89 0.70-1.13

combined 1.33E-01 1.09 0.97-1.23rs6785049 3 121016423 NR1I2 A/G Bel_NL_G 0.37 0.41 4.58E-02 1.17 1.00-1.37

Italy 0.45 0.38 2.18E-02 0.72 0.55-0.96Spain 0.43 0.43 7.75E-01 0.97 0.76-1.22

combined 6.79E-01 1.02 0.91-1.15rs11707609 3 124986114 MYLK G/A Bel_NL_G 0.42 0.38 4.23E-02 1.16 0.99-1.35

Italy 0.39 0.42 2.96E-01 0.87 0.65-1.14Spain 0.41 0.47 4.70E-02 0.79 0.62-0.99

combined 9.97E-01 1 0.89-1.12rs4143832 5 131890876 unknown C/A Bel_NL_G 0.16 0.20 1.04E-02 1.3 1.06-1.59

Italy 0.17 0.16 5.50E-01 0.89 0.62-1.29Spain 0.17 0.18 6.92E-01 1.06 0.78-1.44

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combined 5.43E-02 1.16 1.00-1.35rs1800925 5 132020708 IL13 C/T Bel_NL_G 0.18 0.22 1.37E-02 1.26 1.04-1.53

Italy 0.20 0.19 7.49E-01 0.95 0.67-1.33Spain 0.17 0.21 9.92E-02 1.28 0.95-1.72

combined 1.20E-02 1.2 1.04-1.39rs2569190 5 139993100 CD14 A/G Bel_NL_G 0.49 0.45 2.56E-02 1.19 1.02-1.38

Italy 0.51 0.52 7.13E-01 0.95 0.72-1.25Spain 0.50 0.53 3.56E-01 0.9 0.71-1.13

combined 2.85E-01 1.06 0.95-1.19rs12654778 5 148185934 ADRB2 A/G Bel_NL_G 0.40 0.36 3.69E-02 1.18 1.01-1.37

Italy 0.40 0.37 3.23E-01 1.15 0.87-1.53Spain 0.40 0.38 3.45E-01 1.12 0.89-1.42

combined 1.44E-02 1.16 1.03-1.30rs1799724 6 31650461 LTA / TNFα T/C Bel_NL_G 0.13 0.10 1.69E-03 1.46 1.16-1.85

Italy 0.18 0.19 7.52E-01 0.94 0.66-1.34Spain 0.15 0.08 4.38E-04 2.02 1.38-2.96

combined 1.17E-04 1.41 1.18-1.67rs3135388 6 32521029 HLA-DRA C/T Bel_NL_G 0.09 0.15 1.01E-04 1.63 1.28-2.07

Italy 0.04 0.06 3.33E-01 1.36 0.72-2.59Spain 0.06 0.06 9.16E-01 0.97 0.61-1.57

combined 2.44E-04 1.46 1.19-1.78rs550014 6 154476586 OPRM1 A/G Bel_NL_G 0.22 0.27 1.95E-03 1.32 1.10-1.57

Italy 0.28 0.23 9.01E-02 0.75 0.55-1.02Spain 0.21 0.20 8.12E-01 0.97 0.73-1.29

combined 1.38E-01 1.11 0.97-1.27rs975537 7 30663882 CRHR2 A/T Bel_NL_G 0.20 0.24 1.02E-02 1.27 1.06-1.53

Italy 0.19 0.20 6.91E-01 1.07 0.76-1.52Spain 0.20 0.18 3.73E-01 0.88 0.65-1.18

combined 8.42E-02 1.13 0.98-1.31rs1468412 7 86271387 GRM3 A/T Bel_NL_G 0.25 0.29 8.22E-03 1.26 1.06-1.49

Italy 0.28 0.28 8.00E-01 1.04 0.77-1.41Spain 0.26 0.28 4.44E-01 1.11 0.86-1.44

combined 1.31E-02 1.18 1.03-1.34rs11195419 10 112829358 ADRA2A A/C Bel_NL_G 0.14 0.10 4.47E-04 1.51 1.20-1.91

Italy 0.13 0.11 3.86E-01 1.21 0.79-1.85

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Spain 0.11 0.12 5.52E-01 0.89 0.62-1.29combined 6.43E-03 1.28 1.07-1.53

rs929270 12 46398644 P11 G/A Bel_NL_G 0.29 0.26 4.89E-02 1.19 1.01-1.41Italy 0.27 0.27 8.67E-01 1.03 0.75-1.40Spain 0.22 0.22 9.72E-01 1 0.75-1.32

combined 1.06E-01 1.11 0.98-1.27rs708486 14 51810721 PTGDR T/C Bel_NL_G 0.42 0.46 2.96E-02 1.18 1.02-1.38

Italy 0.50 0.48 5.10E-01 0.91 0.69-1.20Spain 0.44 0.45 7.82E-01 1.03 0.82-1.30

combined 1.32E-01 1.09 0.97-1.23rs1014531 16 9763295 GRIN2A A/G Bel_NL_G 0.42 0.39 3.53E-02 1.18 1.01-1.37

Italy 0.37 0.43 1.13E-01 0.8 0.60-1.06Spain 0.43 0.40 3.16E-01 1.13 0.89-1.44

combined 1.47E-01 1.09 0.97-1.23rs1801275 16 27281901 IL4R A/G Bel_NL_G 0.18 0.22 1.59E-02 1.25 1.04-1.52

Italy 0.12 0.15 2.11E-01 1.28 0.85-1.93Spain 0.22 0.19 2.04E-01 0.83 0.63-1.11

combined 1.08E-01 1.13 0.97-1.31rs2236754 17 68675676 SSTR2 G/A Bel_NL_G 0.25 0.21 1.26E-02 1.25 1.04-1.49

Italy NA NA NA NA NASpain 0.25 0.23 4.46E-01 1.11 0.85-1.45

combined 1.66E-02 1.20 1.03-1.40rs11078827 17 9926566 GAS7 C/T Bel_NL_G 0.52 0.48 2.95E-02 1.18 1.02-1.37

Italy 0.59 0.59 9.54E-01 1.01 0.76-1.33Spain 0.55 0.58 2.79E-01 0.88 0.70-1.11

combined 2.56E-01 1.07 0.95-1.20rs173365 17 41256855 CRHR1 T/C Bel_NL_G 0.47 0.43 4.64E-02 1.17 1.00-1.36

Italy 0.45 0.49 2.84E-01 0.86 0.66-1.13Spain 0.46 0.48 4.70E-01 1.09 0.87-1.38

combined 1.43E-01 1.09 0.97-1.22rs2292382 18 45878935 MYO5B G/T Bel_NL_G 0.11 0.15 1.08E-02 1.34 1.07-1.68

Italy 0.10 0.11 7.12E-01 1.09 0.69-1.72Spain 0.10 0.14 3.20E-02 1.46 1.03-2.09

combined 1.67E-03 1.33 1.11-1.58

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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 MAF achalasia

MAF HV

OR [95% CI]

rs929138 6 31611677 G/A 5,22E-01 0.24 0.28 0.82 [0.45-1.48]rs2239705 6 31621381 T/C 4,31E-02 0.20 0.17 1.22 [1.01-1.49]rs2523500 6 31626333 C/T 4,97E-01 0.36 0.34 1.06 [0.91-1.24]rs6916921 6 31628405 T/C 4,40E-03 0.13 0.10 1.42 [1.12-1.80]

rs116749187 6 31637876 T/C 4,73E-01 0.13 0.17 0.78 [0.40-1.52]rs2857602 6 31641357 C/T 3,19E-01 0.41 0.39 1.08 [0.93-1.26]rs2844484 6 31644203 T/C 3,37E-01 0.41 0.39 1.08 [0.92-1.30]rs2844483 6 31644775 A/C 2,67E-01 0.41 0.48 0.74 [0.44-1.26]rs2071590 6 31647747 T/C 2,37E-01 0.35 0.43 0.72 [0.42-1.23]rs2239704 6 31648120 T/G 3,63E-01 0.41 0.39 1.07 [0.92-1.26]rs769178 6 31655493 A/C 2,06E-03 0.13 0.10 1.46 [1.15-1.85]

rs3132452 6 31671219 T/G 5,04E-01 0.13 0.17 0.77 [0.37-1.62]rs1046089 6 31710946 A/G 7,74E-04 0.29 0.35 0.76 [0.64-0.89]

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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.

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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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