Genetic Variation in the Lymphotoxin-Α- (LTA-)/Tumornecrosis-Factor-Α- (Tnfα-) Locus As

Genetic variation in the lymphotoxin-α- (LTA-)/tumornecrosis-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 delHospital 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

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:

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.

Abstract

BackgroundIdiopathic achalasia is a rare motor disorder of the oesophagus characterized by neuronal loss at the lower oesophageal sphincter.Achalasia is generally accepted asa multifactorial disorder with various genetic and environmental factors being risk-associated. Since genetic factors predisposing to achalasia have been poorly documented, weassessed whethersingle nucleotide polymorphisms (SNPs)in genes mediating immune response and neuronal functioncontribute to achalasia susceptibility.

Methods391 SNPs covering 190 immune and 67 neuronal genes were genotyped in an exploratory cohort from Central Europe (589 achalasia patients, 794 healthy volunteers (HVs)). 24SNPs (p<0.05) were validated in an Italian (160 achalasia patients, 278 HVs) and Spanish cohort (281 achalasiapatients, 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.

ResultsThe rs1799724 SNP located between the lymphotoxin-α (LTA) and tumornecrosis-factor-α (TNFα) genes was significantly associated with achalasia and withstoodcorrection for testing multipleSNPs (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 achalasiaacross all samples.

ConclusionsOur 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 theLTA/TNFαlocus are disease-causing and confirm other variants as potential susceptibility factors for achalasia.

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 tumornecrosis-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

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 releaseTh-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 neuronshave 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 representsa 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 studiesfocusing on isolated achalasia have identified SNPs ingenes involved in (auto-)immune responses and neuronal function. In particular, HLA alleles(9;10;13-15)and SNPs inPTPN22(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-peptidereceptor-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 wassmall (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.

Therefore, the aim of this study wasto evaluate genetic variability ofimmune modulation and neuronal functionas 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 441achalasia patients and 574 controls (Spain and Italy) were assessed.

Materials and Methods

Study population

Three independent cohorts of achalasiapatients 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 allcohorts are reported in Table 1.

Exploratory cohort: This sample consisted of 589idiopathic achalasia patients from Central Europe (Belgium, Netherlands and Germany) and794 German controls. The recruitment strategy ofHVs in this cohort was population-based and, therefore, controls with gastrointestinal complaints and/or inflammatory diseasescould 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:160Italian idiopathic achalasia patientsfrom North (Firenze and Milan) and South Italy (Naples and San Giovanni Rotondoat the IRCCS Hospital ‘Casa Sollievo della Sofferenza’), and 278Italian 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 theyhave previously been reported as susceptibility variants for achalasia (15-22)(Supplemental Table S2). The remaining SNPs were selected becausetheywereidentified as susceptibility variants in association studies for immune-mediated or neuropsychiatric diseases. Potential achalasia-related phenotypes included immune-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 toGerman 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 18SNPs 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 theexploratory sample,39SNPs showed association with achalasia (p<0.05, uncorrected)and were validatedin 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 of4SNPs 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.

Statistical analyses

Fisher's exact testswere 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 modelandPlink v1.07 software ( additive model assumes that on a log scale, the risk in carriers of 2 copies of the at-risk allele is doubledcompared tocarriers of only a single at-risk allele. Furthermore, we performed a Cochran-Mantel-Haenszel meta-analysis across all samplesusing the Statistical Analysis Software (SAS) ( An uncorrectedp-value <0.05 was considered nominally significant, whereas ap-value1.4E-04 (Bonferroni-corrected for 359SNPs) wasconsidered 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.

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 resultsof all SNPs are listed in Supplemental TableS1.

In total,39 SNPs (10% of all SNPs) were nominally significantlyassociated 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 SNPswereselected for genotyping in the Italian and Spanishreplication cohorts using Sequenom MassARRAY (160 Italian patients vs. 278 HVs, 281 Spanish patients vs. 296 HVs).Overall,4 markers failed genotyping and 7 SNPsdid not fulfil QC criteria and were therefore excluded from the study. Furthermore, 3 SNPs showed genotype inconsistencies between the MassARRAY and GoldenGate platformsand were therefore also excluded from the study. These inconsistencies were detected because480 patients from the exploratory sample were genotypedfor 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 thesecohorts, while for another 8 SNPs ORs showed the same association trend in the Spanish cohort and for 7 SNPs ORs showed the same trendin 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 p0.05 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 simultaneouslyin all three cohorts (Table 2). One SNP, rs1799724, on chromosome 6p21nearby lymphotoxin-α(LTA) and tumornecrosis-factor-α (TNFα) was significantly associated with achalasia (Table 2).Notably, rs179724 withstoodcorrection for testing all353 SNPs in the exploratory study (pmeta-analysis=1.17E-04, OR=1.41 [1.18-1.67]).This SNP showed association in 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,resultingin 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 rs1799724on LTA or TNFαexpression 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). SNPrs2292382 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 nearthe 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 nearinterleukin-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 determinedthe association for each of the 23 SNPsafter stratificationfor 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).For none of the other SNPs significant in the exploratory cohort, we observed a sex-specific association (Supplemental Table S5 and S6).

Finally, togain more insights into the potential functionaleffects of rs1799724, which is located in the 5’UTR region of TNFα, or any other SNP linked to rs1799724, we genotyped 16SNPs 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 13successfully 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 functionalSNP 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).

Discussion

In acase-control study of 1,030 achalasia patients and 1,368 HVs, the strongest association signal was identified for rs1799724, a SNP locatedbetween lymphotoxin-α (LTA) and tumornecrosis-factor-α (TNFα)(p=1.17E-4, OR=1.41). Besidesitsassociation with achalasiain 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 cohortsalso revealed that rs1799724was still associated with achalasia after a conservativeBonferroni correction for all 353SNPs tested.