Genetic Variants in CDC42 and NXPH1 As Susceptibility Factors for Constipation and Diarrhea

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 Wouters Translational Research Center for Gastrointestinal Disorders Herestraat 49 bus 701 3000 Leuven, Belgium Tel (32) 16 33 08 37 Fax (32) 16 33 07 23 Email: [email protected]

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

Word count: 3791 words

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2 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.

3 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

4 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

5 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.

6 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

7 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).

8 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

9 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.

10 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).

11 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

12 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.

13 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

14 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: Ptrend=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

15 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

16 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;

17 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.

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

UK/US/ Canadia n cohort Sweden cohort Combined cohort Control IBS Control IBS Control IBS N 639 935 887 497 1526 1432 42.55±1 Mean age, years 4.44 42.76±14.35 45.67±12.30 44.28±13.35 Men/women 158/481 179/756 553/334 107/390 Alternating (IBS-A) 140 218 358 Constipation (IBS-C) 325 118 443 Diarrhoea (IBS-D) 430 161 591 Post infectious (IBS-PI) 40 40

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

20 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 as a 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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