Genetic variants in CDC42 and NXPH1 as susceptibility factors for constipation and

diarrhea predominant irritable bowel syndrome

Mira M Wouters1, Diether Lambrechts2,3, Michael Knapp4, Isabelle Cleynen1, Peter

Whorwell5, Lars Agréus6, Aldona Dlugosz7, Peter Thelin Schmidt7, Jonas Halfvarson8,

Magnus Simrén9, Bodil Ohlsson10, Pontus Karling11, Sander Van Wanrooy1, Stéphanie

Mondelaers1, Severine Vermeire1, Greger Lindberg12, Robin Spiller13, George Dukes14, Mauro

D’Amato15, Guy Boeckxstaens1

1 Translational Research Center for Gastrointestinal Disorders, Leuven University, Leuven,

Belgium 2 Vesalius Research Center, VIB, Leuven, Belgium, 3 Laboratory for Translational

Genetics, Department of Oncology, Leuven University, Leuven, Belgium 4 Institute for

Medical Biometry, Informatics and Epidemiology, University of Bonn, Germany, 5 Dept of

Medicine, University of Manchester, Manchester, UK, 6 Centre for Family Medicine,

Karolinska Institutet, Stockholm, Sweden, 7 Department of Medicine, Karolinska Institutet,

Stockholm, Sweden, 8 Department of Internal Medicine, Örebro University Hospital, Örebro,

Sweden, 9 Department of Internal Medicine, Gothenburg University, Gothenburg, Sweden, 10

Department of Clinical Sciences, Skånes University Hospital, Malmoe, Sweden, 11

Department of Medicine, Umeå University, Umeå, Sweden, 12 Department of Medicine,

Karolinska Institutet, Stockholm, Sweden, 13 Queen's Medical Centre, Nottingham, UK, 14

GlaxoSmithKline, North Carolina, USA, 15 Department of Biosciences and Nutrition,

Karolinska Institutet, Stockholm, Sweden

Corresponding author:Mira WoutersTranslational Research Center for Gastrointestinal DisordersHerestraat 49 bus 7013000 Leuven, BelgiumTel (32) 16 33 08 37Fax (32) 16 33 07 23Email: mira.wouters@med.kuleuven.be

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Keywords: irritable bowel syndrome (IBS), genetic variant, IL-13, NXPH1, CDC42

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Abstract

Objective: The complex genetic aetiology underlying irritable bowel syndrome (IBS) needs to be assessed in large-scale genetic studies. Two independent IBS cohorts were genotyped to assess whether genetic variability in immune, neuronal and barrier integrity genes is associated with IBS.

Design: 384 SNPs covering 270 genes were genotyped in an exploratory cohort (935 IBS patients, 639 controls). 33 SNPs with Puncorrected<0.05 were validated in an independent set of 497 patients and 887 controls. Genotype distributions of single SNPs were assessed using an additive genetic model in IBS and clinical subtypes, IBS-C and IBS-D, both in individual and combined cohorts. Trait anxiety (N=614 patients, 533 controls), lifetime depression (N=654 patients, 533 controls) and mRNA expression in rectal biopsies (N=22 patients, 29 controls) were correlated with SNP genotypes.

Results: Two SNPs associated independently in the exploratory and validation cohort: rs17837965-CDC42 with IBS-C (ORexploratory=1.59[1.05-1.76]; ORvalidation=1.76[1.03-3.01]) and rs2349775-NXPH1 with IBS-D (ORexploratory=1.28[1.06-1.56]; ORvalidation=1.42[1.08-1.88]). When combining both cohorts, the association of rs2349775 withstood post-hoc correction for multiple testing in the IBS-D subgroup. Additionally, 3 SNPs in immune-related genes (rs1464510-LPP, rs1881457-IL13, rs2104286-IL2RA), one SNP in a neuronal gene (rs2349775-NXPH1) and 2 SNPs in epithelial genes (rs245051-SLC26A2, rs17837965-CDC42) were weakly associated with total-IBS (Puncorrected<0.05). At the functional level, rs1881457 increased IL13 mRNA levels, whereas anxiety and depression scores did not correlate with rs2349775-NXPH1.

Conclusion: Rs2349775 (NXPH1) and rs17837965 (CDC42) were associated with IBS-D and IBS-C, respectively, in two independent cohorts. Further studies are warranted to validate our findings and to determine the mechanisms underlying IBS pathophysiology.

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Significance of the study

What is already known about this subject?

Previous studies showed that IBS exhibits the typical features of a complex

multifactorial disorder influenced by both environmental and genetic factors

Defects in mucosal barrier integrity, inflammation and immune interactions with

bacteria in the gut, neuronal sensitization (altered nociceptor expression, increased

anxiety and depression) and altered motor function are emerging as candidate genes

contributing to the pathogenesis of IBS

Although TNFSF15 variants have repeatedly been associated with IBS, genetic studies

in IBS have been largely inconclusive due to small sample sizes.

What are the new findings?

Two SNPs, rs17837965 in CDC42 and rs2349775 in NXPH1, were associated with

IBS-C and IBS-D respectively, in two independent cohorts of 1432 IBS patients and

1526 controls

The rs2349775 variant in NXPH1 withstood post-hoc correction for multiple testing in

the combined IBS-D subgroup, suggesting that it represents a true susceptibility factor

for IBS

How might it impact on clinical practice in the foreseeable future?

Our data may contribute to the identification of important targets in IBS which may

ultimately result in improved clinical management

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Background

Irritable bowel syndrome (IBS) is a multifactorial disorder, which is more common in women

and presents with a variety of symptoms, including altered bowel habits, bloating and

increased abdominal pain sensation (1). As IBS diagnosis is based on symptoms, it consists of

several clinical subtypes including constipation predominant IBS (IBS-C), diarrhea

predominant IBS (IBS-D) or alternating constipation and diarrhea IBS (IBS-A) with

potentially different underlying pathophysiological pathways. Diet, psychological distress,

infections, low-grade inflammation and immunological alterations, dysregulated interactions

with potentially altered microbiota, sensitization/upregulation of nociceptors and changes in

intestinal motility and altered gut-brain communication have all been proposed as contributing

factors (2-4).

Epidemiological studies of familial aggregation (5;6) and twins (7-11) suggest that

genetic predisposition may explain up to 22-48% of the incidence of IBS. However, these

data are conflicting as Mohammed et al demonstrated that social learning has an equal or

greater influence (12). Moreover, environmental risk factors such as psychological stress,

smoking and the use of antibiotics (8) significantly contribute to IBS susceptibility as well.

Overall, IBS exhibits typical features of a complex genetic disorder influenced by both

environmental and genetic factors. Although our current knowledge on genetic predisposition

to IBS is very limited, a number of studies already assessed the risk effects of single

nucleotide polymorphisms (SNPs) in candidate genes. One study reported that SNPs in TLR9,

IL6 and CDH1, which are involved in immune activation and epithelial barrier integrity, were

nominally associated with post-infectious IBS (PI-IBS) (13). Another study focused on

functional gastrointestinal disorders and correlated colonic transit rate and pain sensation with

polymorphisms in the neuropeptide S receptor gene (NPSR1) (14), a gene involved in

inflammation, anxiety and nociception. In the largest genetic study of IBS so far, a strong

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association was reported between rs4263839 in TNFSF15 (15), which is involved in Th17

immune response, and IBS (particularly IBS-C) in two independent cohorts from Sweden and

USA. Although with a different subtype (IBS-D) and with another SNP, this association was

recently replicated in UK individuals (16). Additional smaller studies identified SNPs in

serotonergic genes (17-21) and inflammatory genes (22-24) as susceptibility SNPs for IBS.

All genetic variants previously associated with IBS have been reviewed elsewhere (25-27).

In view of the diversity of IBS aetiologies and phenotypic subtypes (IBS-A, IBS-C,

IBS-D, PI-IBS), there is a great need for much larger studies systematically evaluating genetic

variants in IBS. We therefore evaluated genetic predisposition to IBS, using a hypothesis-

driven genetic association approach with validation in an independent cohort (except for the

genes that were already reported in this validation cohort). In particular, 384 variants located

in 270 genes involved in immune modulation, inflammation, motor function, pain processing,

nociceptor sensitization, altered neuronal function (centrally and peripherally) and epithelial

barrier integrity were genotyped in a cohort of 1432 IBS patients.

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Subjects and Methods

Study populations

Two independent cohorts of IBS patients and controls from UK/US/Canada and

Sweden were included in this study. Informed consent was obtained from all participants and

local ethics committees approved the study protocol. The demographics and clinical

characteristics of both cohorts are reported in Table 1. The IBS patients were divided into

subgroups based on the predominant bowel habit according to the Rome II criteria (1).

Exploratory cohort: This cohort consisted of 935 IBS patients and 639 controls from the UK,

US or Canada. All patients confirmed Caucasian ethnicity. IBS patients were included if they

had a history of IBS for at least 6 months according to Rome II criteria. IBS patients had a

colonoscopy/barium enema during the last 12 months with normal results, supporting an IBS

diagnosis. Controls were included if they had no previous IBS diagnosis (based on physician

assessment and negative Rome II criteria) and no former history of chronic GI diseases.

Controls were matched to IBS patients according to age (+/- 5 years), gender, ethnicity and

referring physician, where feasible. All samples from the exploratory cohort were kindly

provided by Glaxo Smith Kline (UK).

Validation cohort: 497 IBS patients were recruited from primary, secondary and tertiary care

centres and clinics in Stockholm, Gothenburg, Malmoe and Kalix-Haparanda in Sweden. The

diagnosis of IBS was based on Rome II criteria and symptom phenotypes, consensus criteria

and validated questionnaires of gastrointestinal and somatic symptoms. 887 Controls were

healthy blood donors free of gastrointestinal complaints and inflammatory disease.

Genotyping

To select genetic variants in genes involved in immune modulation, inflammation,

motor function, pain processing, nociceptor sensitization, altered neuronal function (centrally

and peripherally), we performed a systematic review using the public database PubMed: with

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highest priority, we included SNPs previously characterized and potentially associated with

IBS in serotonergic pathways, immune responses, and intestinal epithelial barrier and motor

function (13-15;17-22;28-30) (keywords “Irritable bowel syndrome” and “SNP” or “genetic

variant”). Next, using the same overall approach, we selected SNPs that were previously

identified as susceptibility variants in independent association studies of diseases with

potentially comparable underlying pathophysiological mechanisms (keywords were “SNP” or

“genetic variant” and the following disease: diseases mediated by aberrant immune activation

and/or inflammation: Crohn’s disease, ulcerative colitis, type 1 diabetes, rheumatoid arthritis,

multiple sclerosis, chronic obstructive pulmonary disease, ankylosing spondylitis, hyper IgE

syndrome, barrier dysfunction related diseases: celiac disease and diseases due to altered

neuronal function: anxiety, major depression, somatic pain sensation). Finally, we also

selected SNPs based on a HAPMAP tagging approach (www.hapmap.org) in tight-junction

genes, nociceptors such as TRP genes, Toll-like receptors, NOD-like receptors and cytokines

(see list of selected SNPs in supplementary Table 1). In total, 443 SNPs were selected,

evaluated and scored by the Illumina Technical Support Service for potential genotyping

using Illumina Golden Gate. SNPs with a GGGT score lower than 0.6 were excluded.

Likewise, we excluded tagging SNPs if more than 4 tagging SNPs per gene were selected. Of

the remaining 384 SNPs, 318 SNPs had a GGGT score above 0.8 and 66 SNPs had a GGGT

score between 0.6 and 0.8.

The final 384 SNPs included 25 SNPs that were previously associated with IBS (13-

15;17-22;28-31), 265 SNPs that were previously identified as susceptibility variants for

immune mediated diseases, genes involved in barrier dysfunction related diseases and

neuropathologies and 94 SNPs were selected based on the SNP tagging approach. Out of the

384 SNPs, 19 SNPs had a minor allele frequency <0.05 in the CEPH population (Utah

residents with Northern and Western European Ancestry, CEU).

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Samples from the exploratory cohort were genotyped with custom-designed oligo

chips using Illumina Golden Gate (Vesalius Research Center, KULeuven). Based on the

results of this exploratory study, SNPs nominally associated with total IBS, IBS-C or IBS-D

with Puncorrected<0.05 were selected for validation in the Swedish cohort by Sequenom®

MassARRAY (Fig. 1). Quality control (QC) criteria for Golden Gate and

Sequenom®MassARRAY genotyping included: a SNP call rate of >0.95 in patients and

controls, a sample call rate >0.95, a minor allele frequency (MAF) of >0.01 and a Hardy-

Weinberg equilibrium- (HWE-) p-value of >0.0001 in controls and >0.00001 in cases.

The STAI Trait anxiety and depression questionnaires and the mRNA expression

analysis in colonic biopsies of the exploratory cohort are described in supplementary Material

and Methods.

Statistical analyses

Fisher's exact tests were used to test for Hardy-Weinberg Equilibrium (HWE). For the

analysis of individual SNPs, 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/). Furthermore, we performed a Cochran-Mantel-

Haenszel meta-analysis in the combined cohort using the Statistical Analysis Software (SAS)

(http://www.sas.com). Analyses were performed for all IBS patients and two IBS subgroups

(IBS-C and IBS-D). The 140 IBS-A samples in our exploratory cohort did not provide enough

power to detect statistical significance. SNPs with p<0.05 in the IBS-A subgroup were

therefore not further validated. SNPs with a Puncorrected<0.05 in the exploratory cohort were

selected for follow-up analysis in the validation cohort. To correct for multiple testing (33

SNPs in 3 IBS groups, i.e., total IBS, IBS-C and IBS-D), a p-value <5E-04 was considered

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significant. In addition, False Discovery Rate (FDR) correction was applied for the number of

SNPs per IBS subgroup (Figure 2-4, supplemental Table 1-3) (R project software v2.15;

http://www.r-project.org/). Linkage disequilibrium (LD) between markers in the two studied

populations was based on HapMap data using Haploview 4.2 software

(http://www.broadinstitute.org/haploview). The CaTS Power Calculator for Two Stage

Association Studies was used for power calculations. RT-qPCR results, trait anxiety and

depression scores were analyzed with Graphpad Prism software (GraphPad Software, Inc., La

Jolla, CA) using the Mann–Whitney U-test for unpaired samples.

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Results

Genetic variants associated with IBS

Out of 384 SNPs, 21 SNPs failed our quality control due to low call rates, while

another 6 SNPs were excluded because they were monomorphic. All remaining 357 SNPs

were subsequently assessed for association with IBS and its different clinical subtypes (935

IBS patients, of which 430 had IBS-D and 325 had IBS-C, and 639 controls) in our

exploratory cohort. Genotype distributions and association results of all SNPs are reported in

Supplemental Tables 4-7.

Total IBS

In total, eighteen SNPs were weakly associated (Puncorrected<0.05) with total IBS in the

discovery cohort (supplemental Table 5). The rs744166 variant in STAT3 was previously

reported not to be associated with IBS in the validation cohort (15;28) and was therefore not

replicated. Overall, 3 SNPs failed genotyping in the validation cohort: rs1758241 (unknown

gene) was not further studied, rs3024505 (IL10) and rs1800925 (IL13) failed but nearby

located SNPs rs1800896 (R2=0.253) and rs1881457 (R2=0.747) respectively - also identified

in the exploratory cohort - were successfully genotyped. Only one SNP, rs4986791 near

TLR4, was weakly associated (Puncorrected<0.05) with IBS in the exploratory and validation

cohorts, but this SNP had opposite allelic effects in the two cohorts (ORExploratory=3.38 [1.53-

7.44] and ORvalidation=0.47 [0.31-0.7]) (Fig. 2, sTable 1). Next, we combined the exploratory

and validation cohorts and we assessed using a Cochran-Mantel-Haenszel meta-analysis

whether SNPs associated significantly with total IBS (Fig. 2, sTable 1). Although none of the

validated SNPs was resistant for multiple testing (P<5E-04), 6 SNPs were weakly associated

at the uncorrected threshold (Puncorrected<0.05), namely rs1464510 in LPP, rs1881457 in IL13,

rs245051 in SLC26A2, rs2349775 in NXPH1, rs2104286 in IL2RA and rs17837965 near

CDC42 (Fig. 2, sTable 1, sFig. 1).

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IBS subphenotypes

All SNPs with a Puncorrected<0.05 in the IBS-C or IBS-D subgroup of the exploratory

cohort were selected for validation. One SNP, namely rs17837965 in CDC42, was associated

with IBS-C in both independent cohorts (PCMH IBS-C=2.88E-03; ORexpl=1.59 [1.05-1.76] and

ORvalidation=1.76 [1.03-3.01]) (Fig. 3, sTable 2) and may represent a true association signal.

Also for IBS-D, only 1 SNP, namely SNP rs2349775 (NXPH1), was associated with IBS-D in

both cohorts. Moreover, rs2349775 was the only SNP that withstood post-hoc correction for

multiple testing (PCMH IBS-D=4.62E-04; ORexpl =1.28 [1.06-1.56] and ORvalidation =1.42 [1.08-

1.88]) (Fig. 4, sTable 3) and that may therefore represent a true association signal.

Functional analysis of the newly identified candidate SNPs

Next, in an attempt to provide functional evidence that the 6 SNPs weakly associated

with total IBS (Puncorrected<0.05) were functionally implicated in IBS pathophysiology, we

correlated SNP genotypes with expression of their target genes. Because the function of SNP

rs2104286 in IL2RA has already been reported in literature (i.e. rs2104286 modulates soluble

IL2RA serum levels in healthy controls, MS and T1D patients (32)), this SNPs was not

studied at the functional level. The neuronal gene NXPH1 was not detected by qPCR in rectal

biopsies or PBMCs of neither IBS nor controls (n=3, data not shown). This SNP was

therefore correlated with IBS comorbidity symptoms, i.e. anxiety and depression.

1. Association of NXPH1 with anxiety and depression

NXPH1 (rs2349775) was previously associated with neuroticism (33) and was

preferentially associated with IBS-D in our study. Trait anxiety was slightly higher in IBS

compared to controls (IBS (n=614): 45 [IQR 42-48] vs controls (n=533): 44 [IQR 42-47],

P=0.0002). There was no correlation between the rs2349775 (NXPH1) genotype and trait

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anxiety (data not shown). Besides anxiety, IBS patients had higher life time depression scores

compared to controls (IBS (n=654): 7, IQR 0-9 vs controls (n=533): 3 [IQR 0-8], P<0.0001).

However, in IBS and controls, depression scores were not affected by the genotype of

rs2349775 (NXPH1) (GG (n=79): 4 [IQR 0-9]; AG (n=448): 6 [IQR 0-9]; AA (n=586): 6

[IQR 0-9]).

2. Functional effect of rs1881457 on IL13 mRNA expression in rectal biopsies

The rs1881457 SNP in IL13 is located on chromosome 5q31 in a genomic region

containing a cluster of Th2 cytokine genes (IL13, IL4 and IL5). IL13 mRNA expression was

quantified in rectal biopsies of 22 IBS patients and 29 controls where we compared the

extreme phenotypes, wild type versus homozygous risk allele carriers. IL13 mRNA

expression was significantly higher in the rs1881457 AA than in the CC genotype carriers

(PMann Whitney U <0.0001; Fig. 5A). This effect was seen in IBS patients (PMann Whitney U =0.048; Fig.

5B) and controls (PMann Whitney U =0.0004; Fig. 5B).

3. Expression profiles in rectal biopsies of nominally significant SNPs in epithelial genes

Although rs245051 in SLC26A2 is also located on chromosome 5q31, this SNP occurs

independently of rs1881457 (IL13) (r2=0). There was no difference in SLC26A2 mRNA

expression between IBS patients and controls (data not shown). Moreover, rs245051 did not

affect SLC26A2 mRNA levels in IBS (data not shown). Finally, we were not able to assess the

effect of rs17837965 on CDC42 mRNA expression due to a lack of biopsies exhibiting the

less frequent GG genotype.

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Discussion

We demonstrated that genetic variants in CDC42 and NXPH1 were associated with

IBS-C and IBS-D, respectively, in two independent cohorts. Only 1 SNP, namely rs2349775

(NXPH1), withstood post-hoc correction for multiple testing. Moreover, six novel potential

candidate risk loci were weakly associated with IBS (Puncorrected<0.05) in the combined cohort,

namely 3 SNPs in immune related genes IL2RA, IL13 and LPP, 1 SNP in the neuronal

expressed gene NXPH1, and 2 SNPs in epithelial expressed genes SLC26A2 and CDC42.

SNPs located in immune-related genes

Immune activation or dysregulation has been proposed as underlying mechanism of

IBS, especially following an episode of infectious gastroenteritis. Besides increased numbers

of T cells and enteroendocrine cells in post-infectious IBS rectal biopsies (34), much attention

has been given to the role of mast cells, monocytes and T cells in other IBS subtypes (2). We

potentially identified three novel SNPs associated with IBS in genes with an immune

function, namely rs2104286 in interleukin 2 receptor alpha (IL2RA), rs1464510 in Lipoma-

preferred partner (LPP) and rs1881457 in interleukin 13 (IL13).

Rs1881457 in IL13 is located at chromosome 5q31 in the promotor region of IL13, a

cluster of genes involved in Th2 immune response. Kiesler et al reported that the rs1881457 C

allele, which is associated with allergic asthma, results in enhanced IL13 expression in vitro

(35). In contrast, we found that the A allele was weakly associated with IBS and

rs1881457*AA biopsies expressed more IL13 mRNA compared to rs1881457*CC biopsies,

an effect seen in both IBS patients and controls. The A risk allele was present in 82 and 80%

of IBS patients compared to 79 and 77% of controls in the exploratory and validation cohort

respectively. Our cohort provided 62% power to detect a positive association (Puncorrected<0,05)

for rs1881457. Altogether, our results indicate that IBS patients carry more A alleles

compared to healthy individuals and carriers of this allele express more IL13 mRNA

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compared to C allele carriers. Because rs1881457 is located in a dense, highly correlated

region, we cannot deduct which SNP is causal or reflects other causal genetic variants.

Nevertheless, we speculate that in a minority of IBS patients, genotypic variants in the Th2

cytokine cluster at 5q31 may contribute to IBS pathogenesis by increasing IL13 expression,

possibly even more pronounced in response to pathogens or intraluminal antigens.

Of note, although in yet another subset of patients (IBS-A) compared to the original

observations (all IBS subtypes and IBS-C (15), or IBS-D (16)), we detected association with

the TNFSF15 gene in the exploratory cohort (for SNP rs4263839: P trend=4.26E-02; SNP

rs6478108: Ptrend=4.09E-02, supplemental Table 8). All IBS-A patients in the exploratory

cohort derived from Canada or USA sites and did not include UK samples (16), hence this is

truly a repeat. Genetic variation at this locus has now been repeatedly associated to IBS and

its subtypes in four different case-control cohorts, namely from Sweden (15), USA (15), UK

(16) and now in a US/Canada cohort, which strongly suggests this may be in fact the first true

causative locus identified in IBS.

SNPs in genes involved in neuronal functioning

IBS patients have more psychopathology, particularly anxiety disorders and

depression, compared to the general population (36). Rs2349775 in NXPH1 was previously

associated with neuroticism (33). In two independent cohorts, rs2349775 in NXPH1 was

associated with IBS-D (75% of patients vs 70% of controls in exploratory cohort, 77% of

patients vs 70% of controls in validation cohort). This is the only SNP that withstood

correction for multiple testing. In the IBS-D cohort, we had 79% statistical power to detect a

positive association for this SNP (Puncorrected<0,05). In line with the available literature, lifetime

depression (37) was increased in our exploratory IBS cohort compared to controls. However,

rs2349775 in NXPH1 had no effect on depression or trait anxiety levels. Besides having

central effects, secreted NXPH1 inhibits the proliferation of hematopoietic progenitor cells

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involved in the immune response (38). We speculate that genetic variants in NXPH1 may alter

the immune response to a gastrointestinal insult, resulting in low grade inflammation and

diarrhea. However, the functional role of genetic variations in NXPH1 on IBS susceptibility

and diarrhea needs to be further explored.

SNPs in epithelial genes

The epithelial lining of the intestine is crucial in maintaining tolerance and controls

antigen exposure to the mucosal immune system. One of the mechanisms proposed to

contribute to the pathophysiology of IBS is impaired intestinal barrier function (39). In the

present study, we found a consistent association between rs17837965 in cell division cycle 42

(CDC42) and IBS-C in two independent cohorts (G allele in 6.5% patients vs 4.2% controls in

exploratory cohort, 7.2% of patients vs 4.2% controls in validation cohort). CDC42 is

associated with schizophrenia (40), is crucial for normal dendritic spine development in the

brain (40) and is also involved in intestinal stem cell differentiation and proliferation (41).

Intestinal stem cells lacking CDC42 undergo defective cell division, abnormal

morphogenesis, elevated apoptosis, and failed Paneth cell differentiation (41;42), indicating

that genetic variants in CDC42 may alter epithelial barrier function. Moreover, the loss of

absorptive microvilli from the surface of small intestinal enterocytes (Caco-2 cells) in

response to E. coli or its toxins was linked to CDC42 functionality (43). Additionally, CDC42

regulates epithelial membrane transport and secretion (41;44). Altogether, we speculate that

genetic variants of CDC42 may alter secretion and/or barrier function, resulting in

constipation. Rs17837965 GG carriers are rather rare (4-7%) making it difficult to perform

functional studies aiming to dissect the functional effect of genetic variants in CDC42.

Finally, we identified a weak association between SLC26A2 and IBS(-D). SLC26a2, is

an ubiquitously expressed electroneutral SO(4)(2-) transporter with high expression levels in

colonic epithelial lining (45;46). SLC26A2 is downregulated in Crohn’s disease (47) but there

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was no difference in SLC26A2 mRNA expression between IBS and controls. Again, little is

known about SLC26A2 and how genetic variants of SLC26A2 contribute to IBS remains to be

evaluated.

In summary, our data indicate that genetic variants in genes involved in neuronal

processing (NXPH1), and epithelial barrier function (CDC42) might contribute to IBS

pathogenesis. However, although our results are encouraging, it needs to be stressed that the

SNPs identified in our study are weakly associated (Puncorrected<0.05) and despite

biological plausibility may represent false positive associations. Hence, our findings are

hypothesis-generating and still need to be confirmed in independent studies. We would like to

emphasize, however, that they were identified in a much larger IBS population than normally

is the case in the IBS field. We therefore believe that our studies may serve as a very valuable

source to select and confirm candidate susceptibility SNPs or genes involved in IBS.

Nevertheless, our study is limited by the fact that SNPs were often selected based on

GWA studies focussing on diseases with potentially comparable underlying

pathophysiological mechanisms as IBS. Therefore, our selection contained several intronic

SNPs as well as SNPs with an unknown function or SNPs with an (yet) unclear contribution

to IBS pathogenesis. Our current sample size provides 79% power to detect associations with

a P value <5E-4 and a risk effect size of 1.25 and MAF of 0.40 (supplemental Table 10). To

reach genome wide significance (α=10E-7), a two-stage approach with 5000 IBS patients and

5000 healthy controls would, for instance, be required to have 95% power to detect

associations with SNPs having a MAF of 0.20 and risk of 1.25 with (genome wide

significance). Such high number of patients was not available, and therefore an extensive

hypothesis driven genetic association approach was used instead. As a result, none of the

selected SNPs could be used as population-based markers to assess potential population

stratification. Additionally, IBS diagnosis is very common (15%) in the general population;

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IBS risk variants may therefore also be present in a significant proportion of controls.

However, since we used controls that are free of IBS diagnosis, our results could sustain this

bias and may become even more significant when comparing IBS patients to an ideal set of

controls, which would consist of healthy volunteers devoid from any of the IBS inclusion

criteria. Finally, in the future, the discovery of IBS disease genes may be complemented by

studies focussing on endophenotypes underlying IBS, such as colonic transit and visceral

pain.

In conclusion, genetic variants in CDC42 and NXPH1 were associated with IBS-C and

IBS-D respectively in two independent cohorts. Further studies are warranted to validate our

findings and to determine the mechanisms by which these variants underlie IBS

pathophysiology.

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Table 1. Demographic and clinical characteristics of IBS patients and controls

UK/US/Canadian cohort Sweden cohort Combined cohortControl IBS Control IBS Control IBS

N 639 935 887 497 1526 1432Mean age, years 42.55±14.44 42.76±14.35 45.67±12.30 44.28±13.35

Men/women 158/481 179/756 553/334 107/390Alternating (IBS-A) 140 218 358

Constipation (IBS-C) 325 118 443Diarrhoea (IBS-D) 430 161 591

Post infectious (IBS-PI) 40 40

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Figure legends

Fig. 1. Study design

Fig. 2. Association of genetic variants with total IBS

Shown from left to right are: Nearest gene with NCBI dbSNP database accession numbers (rs number) in the respective cohort (exploratory cohort (UK), validation cohort (SWE), and combined cohort (COMB), Armitage's Trend test (P_Trend), Cochran-Mantel-Haenszel meta-analysis (P_CMH), false positive report P_CMH (FDR P_CMH), allelic odds ratio (OR) and lower and upper bound of the corresponding 95% confidence interval (CI (95%)) * SNP was not associated with total IBS but with C-IBS in the exploratory cohort

Fig. 3. Association results of genetic variants with constipation predominant IBS

Shown from left to right are: Nearest gene with NCBI dbSNP database accession numbers (rs number) in the respective cohort (exploratory cohort (UK), validation cohort (SWE), and combined cohort (COMB), Armitage's Trend test (P_Trend), Cochran-Mantel-Haenszel meta-analysis (P_CMH), false positive report P_CMH (FDR P_CMH), allelic odds ratio (OR) and lower and upper bound of the corresponding 95% confidence interval (CI (95%))

Fig. 4. Association results of genetic variants with diarrhea predominant IBS

Shown from left to right are: Nearest gene with NCBI dbSNP database accession numbers (rs number) in the respective cohort (exploratory cohort (UK), validation cohort (SWE), and combined cohort (COMB), Armitage's Trend test (P_Trend), Cochran-Mantel-Haenszel meta-analysis (P_CMH), false positive report P_CMH (FDR P_CMH), allelic odds ratio (OR) and lower and upper bound of the corresponding 95% confidence interval (CI (95%))

Fig. 5. IL13 mRNA expression is dependent on the rs1881457 genotype

IL13 mRNA expression was decreased in the rs1881457 CC genotype compared to the AA genotype (A). In both IBS and controls, the protective C alleles resulted in decreased IL13 mRNA expression compared to the A risk alleles (B). There was no difference in IL13 mRNA expression in controls (ctrls) compared to IBS (B). Each dot represents an individual, the line represents the median.

Author contributions:

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

MK: statistical analysis

IC: analysis and interpretation of data, critical revision of the manuscript for important intellectual content

LA, AD, PTS, JH, BO, PK, SVW, SM: acquisition of samples and characterization of patients

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PW, MS, RS: acquisition of samples and characterization of patients, critical revision of the manuscript for important intellectual content

SV: critical revision of the manuscript for important intellectual content

GL: provided funding for validation phase, acquisition of samples

MD: acquisition of data, critical revision of the manuscript for important intellectual content

GD: obtained funding, acquisition of samples and data, critical revision of the manuscript for important intellectual content

GEB: study supervision, obtained funding, critical revision of the manuscript for important intellectual content

Grant support:

GlaxoSmithKline Pharmaceuticals has supported the data collection and the provision of samples and data from the UK/US/Canadian cohort. This work was financially supported by research grant G.0699.10N from the Fund for Scientific Research (FWO) Flanders, Belgium. Mira Wouters and Isabelle Cleynen are postdoctoral researchers and Séverine Vermeire is a senior clinical investigator of the FWO. Guy Boeckxstaens received research funding by a grant from the Flemish government (Odysseus Program, FWO). Research activities in Sweden were supported by funds from the Swedish Research Council to Mauro D’Amato. Magnus Simrén was supported by the Swedish Medical Research Council (grants 13409, 21691 and 21692) and The Marianne and Marcus Wallenberg Foundation. Magnus Simrén has received unrestricted research grants from Danone and AstraZeneca, and served asa Consultant/Advisory Board member for AstraZeneca, Danone, Novartis, Almirall, and Shire/Movetis.

Acknowledgements

The authors would like to thank Dr. Theadore Ptak, Renee Henry, Ellen Goldstein, Cindy Lee, Deborah Roach, Jacqueline Rabuzin and Elizabeth Crosland (Toronto Digestive Disease Associates (TDDA Inc.), Toronto, Ontario, Canada), Dr. Mark Silverberg and Lori Baladjay (Mount Sinai Hospital, Toronto, Ontario, Canada), Dr. Yehuda Ringel, Dr. Robert Sandler, Alesia N. Aileo, Sarah Causey and Sarah Yeskel (University of North Carolina at Chapel Hill, NC, USA) for collecting samples and acquiring data and Dr. Rachel Gibson (GlaxoSmithKline) for logistic support. We thank all of the patients and the supporting staff at each site.

Disclosures:

The authors report no conflict of interest

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