University of Groningen Genetic susceptibility for inflammatory bowel disease across ethnicities and diseases van Sommeren, Suzanne

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University of Groningen

Genetic susceptibility for inflammatory bowel disease across ethnicities and diseases

van Sommeren, Suzanne

DOI:

10.33612/diss.100597247

IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from

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Publication date:

2019

Link to publication in University of Groningen/UMCG research database

Citation for published version (APA):

van Sommeren, S. (2019). Genetic susceptibility for inflammatory bowel disease across ethnicities and

diseases. Rijksuniversiteit Groningen. https://doi.org/10.33612/diss.100597247

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Printing of this thesis was financially supported by:

University of Groningen, University Medical Centre Groningen, Nederlandse Vereniging voor Gastroenerologie (NVGE), Norgine BV, Ferring BV, Teva Netherlands BV, Dr. Falk Pharma Benelux BV, Mylan BV, Pfizer BV and Lamepro BV.

ISBN (printed version): 978-94-034-2053-0 ISBN (electronic version): 978-94-034-2052-3 Cover design and layout: Lovebird design.

www.lovebird-design.com

Printing: Eikon +

Copyright © 2019 by Suzanne van Sommeren. All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, without prior permission of the author.

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Genetic Susceptibility for

InflammatoryBowelDiseaseacross

ethnicitiesanddiseases

Proefschrift

ter verkrijging van de graad van doctor aan de

Rijksuniversiteit Groningen

op gezag van de

rector magnificus prof. dr. E. Sterken

en volgens besluit van het College voor Promoties.

De openbare verdediging zal plaatsvinden op

maandag 25 november 2019 om 12.45 uur

door

Suzanne van Sommeren

geboren op 17 december 1984

te Luyksgestel

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Promotores

Prof. dr. R.K. Weersma

Prof. dr. C. Wijmenga

Beoordelingscommissie

Prof. dr .G. Bouma

Prof. dr. A. Zhernakova

Prof. dr. H.J. Verkade

Paranimfen

M.T.J. (Thecla) Wortelboer

A.M.L. (Marjolein) IJbema-Blom

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CHAPTER

1

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The inflammatory bowel diseases (IBD) are characterised by chronic, relapsing immune mediated inflammation of the gastrointestinal tract. Although IBD only has two main enti-ties, Crohn’s disease (CD) and ulcerative colitis (UC), it encompasses a heterogeneous clinical disease spectrum with a complex underlying pathogenesis.

Clinical aspects

Patients presenting with idiopathic inflamma-tion in the gastro-intestinal tract are strived to be classified as UC or CD patients mainly accord-ing to clinical and endoscopic features.

Inflammation in UC is restricted to the co-lon, in which the rectum is practically always affected and can extend proximally in a contin-uous fashion. The main classification is based on disease location: at diagnosis the disease is confined to the rectum (proctitis) in 30–60% of patients, extended inflammation till the flexura lienalis (left sided disease) is seen in 16–45% of patients and extended inflammation beyond the flexura lienalis resulting in pancolitis is seen in

15–35% of patients.1,2_{Symptoms of ulcerative}

colitis can be related directly to the inflamma-tion of the colon or colon with (bloody) diar-rhoea as a key symptom. Accompanying abdom-inal pain occurs more often in colitis, urgency occurs more often in proctitis. Because the in-flammation is restricted to the mucosa, UC be-haves in a non-stricturing and non- penetrating fashion; however, the severity of inflammation and thereby the severity of symptoms can vary greatly. In severe cases systemic inflammatory reactions (fever, tachycardia) are seen. The most severe form of UC, acute severe ulcerative colitis, is associated with complications such as gross bleeding, toxic megacolon, in which the colon dilatates, and (micro)perforation, and has a

mortality of 1%.3 _{Furthermore, longstanding}

inflammation of the colon increases the risk off colorectal cancer (CRC), which necessitates

surveillance colonoscopies.4_{Next to CRC, the}

main indications for surgery are medication refractory disease and refractory acute severe

ulcerative colitis, in which case most often a (procto)colectomy is performed. Despite a range of available medical therapies up to 15.6% of patients undergo surgery within 10 years of the

diagnosis.5

A typical aspect of CD is the widespread and discontinuous disease location (mouth to anus), mainly affecting the terminal ileum (47%), the colon (28%) or both (21%). In only 3% of the pa-tients the upper gastrointestinal tract is affected

(e.g. above the terminal ileum).6_Moreover

dis-ease behaviour in CD is more varied compared to UC. A distinction is made between non-stric-turing and non-penetrating (or inflammatory) disease, stricturing disease and penetrating disease. The main classification is based on lo-cation and disease behaviour and is made with

the Montreal classification.7_{The variance in}

disease location and behaviour explains the variety of symptoms. Inflammatory disease affecting the colon resembles UC. Inflamma-tion in the terminal ileum can result in vague abdominal pain and loss of weight. Stricturing inflammation results in stenosis with accompa-nying symptoms of nausea, abdominal pain and weight loss. Penetrating disease results through (micro)perforation in abscesses or fistula with skin or other organs. CD often presents with the inflammatory form, the stricturing and pen-etrating forms are relatively rare at diagnosis (only 17% for stricturing disease and 13% for

penetrating disease respectively);6_however,

through prolonged inflammation strictures, or penetrating complications occur frequently in the course of the disease and they are frequent indications for surgery, with around 50% of pa-tients needing surgery within 10 years of the di-agnosis. Unfortunately, due to the susceptibility of the entire gastrointestinal tract to developing inflammation, CD tends to reoccur, as a result of which one third of the patients require multiple

surgeries.8_{After multiple resections and with}

extensive disease, the remaining small intes-tines might not be sufficient to sustain

nutri-tional uptake, resulting in intestinal failure.9

Although the characteristics of UC and CD de-scribed seem to be clearly distinctive, it is not always possible to distinguish between the two

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entities. If a clear distinction cannot be made, the disease is called undetermined IBD (IBD-U). Sometimes a definite diagnosis can be made through the course of the disease; however, in some patients this remains uncertain. Further-more, rectal sparing or the presence of backwash ileitis in UC might be misinterpreted as CD. On the other hand, 5–10% of the patients diagnosed

with UC eventually turn out to have CD.10

The relapsing nature is shared between CD and UC. Although clinical risk factors have been identified to predict a severe disease course or complications (for example young age at diag-nosis, smoking and extensive small intestine disease for CD and pancolitis and deep

ulcer-ations in UC )11,12_{no individual prognosis can}

be made.

Adding to the heterogeneity of the disease spectrum are the extraintestinal manifestations (EIM) of IBD, which can affect multiple organ systems and can impact quality of life more than IBD itself. 63.4% of IBD patients suffer from at least one EIM during their lifetime. Some EIMs like erythema nodosum, peripheral arthritis and oral aphthous ulcers occur with active in-testinal disease whereas other EIMs like axial spondylarthropathy/ankylosing spondylitis, uveitis and primary sclerosing cholangitis

fol-low their own clinical course.13_{It is believed}

that there is a shared pathogenic mechanism between IBD and its EIMs; however, this link is not yet clarified. One theory suggests activation of the immune response by translocated bacte-ria through the inflamed and leaky intestinal

barrier.14_{Therefore, a crucial component of the}

treatment of EIMs is successful treatment of in-testinal inflammation. Furthermore, some con-ditions are frequently seen in IBD patients as a consequence of metabolic abnormalities due to intestinal inflammation. Examples are osteopa-thy, nephrolithiasis or thromboembolic events. A third category of diseases occurs more often in patients with IBD, but is not IBD- specific like the EIM. These associated diseases like insuline- dependent diabetes mellitus, thyroid disease and vitiligo probably share immune mediated pathways with IBD, possibly due to a shared

ge-netic background.15

Epidemiology

IBD is a ‘disease of the young’, with the peak age of onset in the second to fourth decade of

life.16_{The incidence has been rising in recent}

years; in the Netherlands in 2010 the annual incidence for IBD was 40.36/100,000, for CD 17.49/100,000 and for UC 21.47/100,000. The prevalence of IBD in the Netherlands is high, with 830/100,000 inhabitants (CD: 331/100,000; UC: 475/100,000), and is

com-parable to the prevalence in Europe.16,17_IBD

has always been considered a disease of indus-trialised countries, with the highest prevalence in Europe and North America. Recently, and simultaneously with the industrialization of developing countries the incidence of IBD is

rising in, for example, Asia.16_{Since IBD occurs}

more often in urban areas compared to rural

areas18,19_{and the fact that children of migrants}

from low-incidence to high-incidence countries show an increase in incidence this suggests

en-vironmental risk factors are driving factors.20,21

Treatment

Because IBD is an immune mediated disease treatment is focused on suppression of the immune system. The first goal is inducing re-mission and because IBD tends to flare up, the next goal is to maintain remission in order to prevent long-term damage and complications. This means that patients might have to take life-long medication. There are multiple definitions of remission and treatment strategies and ther-apy regimes to achieve this. It falls beyond the scope of this introduction to go into great detail, but a key feature of the medication used is that they are not specific in suppressing the immune system, resulting in significant infectious and oncological risks. In order to expand the thera-peutic arsenal and make medication more spe-cific great effort is put into development of new medication. Examples of new biologicals are Vedolizumab, which is a monoclonal antibody

that blocks the gut specific a4b7 integrin,22_and

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interleukin 23.23_{Based on new insights in}

dis-ease pathogenesis, new therapy targets and new drugs are still being developed.

One of the biggest clinical challenges in im-proving the therapeutic strategy is to select the right treatment for individual patients (person-alised treatment). Till this day it remains impos-sible to predict the effect of a drug on the disease, the duration of this effect and occurrence of side effects in individual patients. This challenge is further complicated by the unpredictable de-velopment of anti-drug-antibodies that greatly diminish the clinical effect of monoclonal anti-bodies like Infliximab. Furthermore, because the disease course is unpredictable patients of-ten develop a significant flare of disease activ-ity before treatment escalation has taken place. Ideally, clinicians would like to offer individual patients a treatment strategy with a predicted good effectiveness and minimal side-effects at the correct time during the disease course.

Impact

Considering that IBD is a chronic disease, with the impact of symptoms described above, the tense treatment and the erratic prognosis, its in-fluence on daily life can be tremendous, dimin-ishing patients’ quality of life, their ability to

participate in society, sexuality and fertility.24

The financial burden of IBD is significant, which is not surprising because IBD often affects young people and their ability to work, and taking into

account the considerable costs of medication.25

The costs per patient even exceeds the costs for other chronic diseases like COPD, hypertension

and diabetes.26

Pathogenesis

Similar to the complexity of the disease spec-trum is the complexity of the disease patho-genesis. It is believed that IBD is caused by an inappropriate reaction of the immune system in response to commensal gut bacteria in a ge-netic susceptible host. Besides gege-netic factors,

disease susceptibility is thought to be influenced by the gut microbiome and several environmen-tal factors. Furthermore it is believed that there are complex interactions between the different factors.

Environmental factors and the

microbiome

The influence of environmental factors (the

ex-posome) has been well established for years,27,28

but has been highlighted by the recent increase in incidence in developing countries that adopt the Western lifestyle. Smoking increases the

risk of developing CD29_{and is associated with a}

more severe disease course.30_{On the other hand}

UC occurs more often in former smokers.29_Diet

can influence the risk of IBD through dietary

macronutrients (fibres,31 _{saturated fat}32_),

mi-cronutrients (vitamin D,33_zinc34_{) or by altering}

the microbiome.35_{It is possible that factors like}

(childhood) antibiotic use36_{and hygienic}

fac-tors during childhood37_{also mainly influence}

disease risk through adjusting the microbiome. The role of the microbiome has only been rec-ognised since a few years, driving a tremendous effort in research in this field. The key feature is a reduction of the diversity of the microbiome in IBD, which is more pronounced in CD than in UC.38–40_{As mentioned above, the microbiome}

is sensitive to influences of numerous external and internal factors, amongst which is the ge-netic background.

Genetic background

Locus identification

The heritability of IBD was demonstrated by seg-regation in families and a higher disease concor-dance in monozygotic twins than in dizygotic twins. Concordance is higher in CD compared to UC, suggesting that the heritable component

is higher for CD.41–43_{Moreover, it was clear}

that the heritability of IBD doesn’t behave in a Mendelian fashion and, as is typical for a com-plex disease, multiple genetic factors underlie

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disease pathogenesis. Hereafter, tremendous efforts were undertaken to unravel the genetic background of IBD.

Linkage and candidate gene studies

The first IBD gene to be identified in 1996 was

NOD244_{, after a locus (called IBD1) was found to}

be associated with Crohn’s disease. It was iden-tified by a linkage study, in which genetic mark-ers are tested for cosegregation (linkage) with disease status in affected families. In 2001 three causal mutations (R702W, G908R and L1007fs)

in NOD2 were found.45,46_{The NOD2 locus still}

remains the locus with the greatest risk in caus-ing disease (or effect size): heterozygotes have an odds ratio (OR) of 2–4, homozygotes have an OR

of 20–40.45,46_{By means of linkage studies}

mul-tiple loci were identified; however many of them failed replication. Besides NOD2 IBD5/5q31 (harboring SLC22A4, SLC22A5), IBD3/6p21

(HLA region) showed consistent replication.47

Results from candidate gene studies also suffered from replication issues. In candidate gene studies markers in genes with suspected in-volvement in disease pathogenesis are selected. In case of the IBD studies candidate genes have roles in the innate or adaptive immune system or the intestinal mucosal barrier. Only a few associations, like CARD9 and IL18RAP, were

consistently replicated,48_{and some tested loci}

that would later turn out to be associated, like

the IL10 locus, were missed.49

Genome-wide association studies

With the development of genotyping arrays, the research of IBD genetics took a new turn. In ge-notyping arrays genetic markers (by means of single nucleotide polymorphisms – SNPs) are strategically selected based on linkage disequi-librium (LD) patterns, enabling the coverage of the majority of the genome by a relatively small selection of SNPs. In a genome-wide association study (GWAS) these SNPs are tested in a large selection of cases and controls to find SNPs that are significantly associated. This study design enables hypothesis-free testing and testing ge-netic variants with modest to small effect sizes, which wasn’t possible with the linkage and

candidate gene studies. In order to correct for the multiple statistical tests done in a GWAS stringent criteria have to be met to claim an association.

Interestingly, the first GWAS in IBD was

per-formed in a Japanese CD population.50_Later

studies were primarily focused on European (derived) populations. Therefore, there has re-mained a relative gap of knowledge in the ge-netic background of IBD in non-European pop-ulations. At the start of the research presented in this thesis in 2010 17 GWAS were published,

nine in CD,50–58_{six in UC}59–64_{and two in early-}

onset IBD.65,66_{As could be expected, many of}

the candidate genes in associated loci play roles in the innate (CARD9, MST1) or adaptive im-mune system (PTPN22, ICOSLG) or the intes-tinal mucosal barrier (CDH1, HNF4α, LAMB1, MUC19). Interestingly, candidate genes in path-ways that were not implicated in the pathogen-esis of IBD before turned out to be associated, like IRGM and ATG16L1 in CD, which together with NOD2 play crucial roles in autophagy. Au-tophagy is the process of degradation of dam-aged cytoplasmic components by the lysosome and has been studied in the context of CD after

genetic association became apparent.67,68_The

importance of certain pathways is stressed by the large enrichment of candidate genes found to be associated. The Th17/IL23 pathway is such an example with many key genes residing in IBD associated loci (IL23R, IL12B, JAK2, STAT3, IL17REL, CCR6).

Because the first studies included relatively small sample sizes, they first picked up the ‘low-hanging fruit’: the loci with relatively large effect sizes like TNFSF15 in the Japanese

pop-ulation (OR 2.17)50_{and IL23R in a combined}

Jewish and non-Jewish European collection (OR

2.2–3.8).51_{As it became clear that most genetic}

variants had much smaller effect sizes, with ORs below 1.5, it became more important to increase statistical power by increasing sample sizes. Therefore, the next effort was combining GWAS data in meta-analyses. Shortly after starting this research, two large meta-analyses in CD and UC were published, increasing the total number of

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many loci associated with CD or UC were shared, implicating large overlap in disease pathogene-sis. Also, many genetic loci are shared with other immune mediated diseases. This led with the combined effort of multiple research groups to the development of a customised genotyping platform that primarily targets genetic loci in-volved in 11 immune mediated diseases, the Immunochip. In 2012, a large meta-analysis of previously reported GWAS data, combined with an extra cohort of individuals genotyped

on the Immunochip was published.71_{In total,}

75,000 individuals were analysed, which gave enough statistical power to increase the num-ber of IBD associated loci with 71 (to a total of 163 loci). Two-third of these loci are shared be-tween CD and UC, 30 seem to be CD specific and 23 seem to be UC specific.

Uncovering functional implications

Although the GWASs and meta-analyses where very successful in identifying disease risk loci, many challenges remained. GWAS platforms were designed to cover large stretches of the ge-nome, the SNPs that act as genetic markers are (mostly) not causal variants but tag causal ants through LD. For some risk loci causal vari-ants have been found, for example the NOD2

and IRGM loci;45,46,72,73_{however, for most of}

the loci the causal variants remain unknown. The dense coverage of loci on the Immunochip aids in solving this problem. Another possible solution is sequencing the locus.

The next challenge is to determine the effect of causal variants and which genes are influenced by genetic variation. The most obvious expla-nation through which genetic risk variants can influence disease pathogenesis is by influencing gene expression. Many associated SNPs have a strong correlation with gene expression, the

so-called eQTL effect.74_{And although this}

par-ticular SNP is not likely to cause a disruption in gene expression itself, the eQTL effect can point out the candidate gene for a locus and we can learn more about a genetic risk factor if we un-derstand how gene expression is influenced by that risk factor. Other approaches use network analyses (based on for example gene ontology or

protein-protein interaction) to find enrichment of certain pathways in the entire set of genes

residing within associated loci.75,76_These

meth-ods were used mostly to select candidate genes in the published GWAS.

Finally a correlation will have to be made be-tween genetic risk factor, the gene(s) influenced by genetic variation, it’s role in a signalling path-way and the influence of this pathpath-way on disease pathogenesis.

Aim and outline of the thesis

The complexity of the clinical disease spectrum and the response to treatment are probably a reflection of (subtle) differences in disease pathogenesis. In order to gain insight into the disease development and disease course and improve quality of life by realising personalised treatment, we need to gain full insight in the pathogenesis of IBD.

In this thesis we focus on expanding knowledge about the genetic basis of disease pathogenesis.

Part I starts by describing our research fo-cussed on identification of genetic risk loci and candidate genes and explains how the effect of genetic variation on gene expression plays a role.

Chapter 2 is a replication study of three

previ-ously reported associated loci resulting from a genome-wide association study (GWAS) per-formed in ulcerative colitis (UC). The candidate genes residing in the loci (CDH1, HNF4A and LAMB1) play roles in the intestinal mucosal barrier integrity. We analysed these loci in an independent Dutch UC cohort. Chapter 3 de-scribes the largest meta-analysis published at the time, containing nearly 100,000 individuals including a substantial proportion of individu-als from East-Asian, Indian and Iranian descent. This study plays two roles: identifying new IBD risk loci and investigating the overlap in genetic background of IBD across populations.

Next we focus on the role of gene expression. In the study presented in chapter 4 we selected SNPs with known influence on gene expres-sion from GWAS results that did not reach the

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genome wide significant threshold before. We hypothesised that these genetic loci were more likely to be truly associated to IBD because they have functional consequences. We performed a replication study in an independent Crohn’s disease (CD) cohort. In the research presented in

chapter 5, we investigated the effect of genetic

risk on gene expression of genes in the Th17/ IL23 pathway. This pathway is very import-ant in IBD from both a biological and genetic perspective.

In part II, we focus on shared genetics with other diseases. The clinical spectrum of IBD is broad and includes several extraintestinal manifestations. Chapter 6 presents the over-lap in genetic factors and sharing of biological pathways between IBD and its extraintestinal manifestations. In chapter 7 we focus on a dis-ease occurring after allogeneic hematopoietic cell transplantation and clinically resembling Crohn’s disease: gastro-intestinal graft-vs.-host disease. We identify risk loci and examine wether there is overlap with IBD.

Chapter 8 gives an overview of the results of

the work presented and provides a general dis-cussion and future perspectives.

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inflammatory bowel disease: differences between Jews and non-Jews. Gut 1993;34:517–524. 44. Hugot JP, Laurent-Puig P, Gower-Rousseau

C, et al. Mapping of a susceptibility locus for Crohn’s disease on chromosome 16. Nature. 1996;379:821–823.

45. Hugot JP, Chamaillard M, Zouali H, et al. As-sociation of NOD2 leucine-rich repeat variants with susceptibility to Crohn’s disease. Nature. 2001;411:599–603.

46. Ogura Y, Bonen DK, Inohara N, et al. A frameshift mutation in NOD2 associated with susceptibility to Crohn’s disease. Nature. 2001;411:606–606. 47. Mathew CG, Lewis CM. Genetics of inflammatory

bowel disease: progress and prospects. Hum Mol Genet. 2004;13:R161-R168.

48. Zhernakova A, Festen EM, Franke L, et al. Genetic analysis of innate immunity in Crohn’s disease and ulcerative colitis identifies two susceptibil-ity loci harbouring CARD9 and IL18RAP. Am J Gastroenterol. 2008;82:1202–1210.

49. Castro-Santos P, Suarez An, Lopez-Rivas L, et al. TNFa and IL-10 gene polymorphisms in inflam-matory bowel disease. Association of -1082 AA low producer IL-10 genotype with steroid depen-dency. Am J Gastroenterol. 2006;101:1039–1047. 50. Yamazaki K, McGovern D, Ragousssis J, et al. Sin-gle nucleotide polymorphisms in TNFSF15 confer susceptibility to Crohn’s disease. Hum Mol Genet. 2005;14:3499–3506.

51. Duerr RH, Taylor KD, Brant SR, et al. A genome- wide association study identifies IL23R as an inflammatory bowel disease gene. Science 2006.314:1461–1463.

52. Franke A, Hampe J, Rosenstiel P, et al. Systematic association mapping identifies NELL1 as a novel IBD disease gene. PLoS ONE. 2007;2:e691. 53. Hampe J, Franke A, Rosenstiel P, et al. A genome-

wide association scan on nonsynonymous SNPs identifies a susceptibility variant for Crohn dis-ease in ATG16L1. Nat Genet. 2007;39:207–211 54. Libioulle C, Louis E, Hansoul S, et al. Novel Crohn disease locus identified by genome-wide associa-tion maps to a gene desert on 5p13.1 and modulates expression of PTGER4. PLoS Genet. 2007;3:e58. 55. Parkes M, Barrett JC, Prescott NJ, et al. Se-quence variants in the autophagy gene IRGM and multiple other replicating loci contribute to Crohn’s disease susceptibility. Nat Genet. 2007; 39:830–832.

56. Raelson JV, Little RD, Ruether A, et al. Ge-nome-wide association study for Crohn’s

disease in the Quebec Founder population identifies multiple validated disease loci. PNAS. 2007;104:14747–14752.

57. Rioux JD, Xavier RJ, Taylor KD, et al. Ge-nome-wide association study identifies new sus-ceptibility loci for Crohn disease and implicates autophagy in disease pathogenesis. Nat Genet. 2007;39:596–604.

58. The Wellcome Trust Case Control Consortium. Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared con-trols. Nature. 2007;447:661–678.

59. Fisher SA, Tremelling M, Anderson CA, et al. Ge-netic determinants of ulcerative colitis include the ECM1 locus and five loci implicated in Crohns’ disease. Nat Genet. 2008;40:710–712. 60. Franke A, Balschun T, Karlsen TH, et al. Sequence

variants in IL10, ARPC2 and multiple other loci contribute to ulcerative colitis susceptibility. Nat Genet. 2008;40:1319–1323.

61. Silverberg MS, Cho JH, Rioux JD, et al. Ulcerative colitis-risk loci on chromosomes 1p36 and 12q15 found by genome-wide association study. Nat Genet. 2009;41:216–220

62. UK IBD Genetics Consortium, Barrett JC, Lee JC, et al. Genome-wide association study of ulcer-ative colitis identifies three new susceptibility loci, including the HNF4A region. Nat Genet. 2009;41:1330–1334.

63. Franke A, Balschun T, Sina C, et al. Genome-wide association study for ulcerative colitis identifies risk loci at 7q22 and 22q13 (IL17REL). Nat Genet. 2010;42:292–294.

64. McGovern DPB, Gardet A, Torkvist A, et al. Genome-wide association identifies multiple ulcerative colitis susceptibility loci. Nat Genet. 2010;42:332–340.

65. Kugathasan S, Baldassano RN, Bradfield JP, et al. Loci on 20q13 and 21q22 are associated with pe-diatric-onset inflammatory bowel disease. Nat Genet. 2008;40:1211–1215.

66. Imielinski M, Baldassano RN, Griffiths A, et al. Common variants at five new loci associated with early-onset inflammatory bowel disease. Nat Genet. 2009;41:1335–1342.

67. Plantinga TS, Crisan TO, Oosting M, et al. Crohn’s disease-associated ATG16L1 polymor-phism modulated pro-inflammatory cytokine responses selectively upon activation of NOD2. Gut. 2011;60:1229–1235.

68. Kuballa P, Huett A, Rioux JD, et al. Impaired au-tophagy of an intracellular pathogen induced by

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69. Franke A, McGovern DPB, Barrett JC, et al. Ge-nome-wide meta-analysis increases to 71 the number of confirmed Crohn’s disease suscepti-bility loci. Nat Genet. 2010;42:1118–1126. 70. Anderson CA, Boucher G, Lees CW, et al.

Me-ta-analysis identifies 29 additional ulcerative coli-tis risk loci, increasing the number of confirmed associations to 47. Nat Genet. 2011;43:246–255. 71. Jostins L, Ripke S, Weersma RK, et al. Host-

microbe interactions have shaped the genetic architecture of inflammatory bowel disease. Nature. 2012;491:119–124.

72. McCarroll S, Huett A, Kuballa P, et al. Deletion polymorphism upstream of IRGM associated with altered IRGM expression and Crohn’s disease. Nat Genet. 2008;40:1107–1112.

73. Prescott NJ, Dominy KM, Kubo M, et al. Indepen-dent and population-specific association of risk variants at the IRGM locus with Crohn’s disease. Hum Mol Genet. 2010;19:1828–1839. 74. Gilad Y, Rifkin SA, Pritchard JK. Revealing the

architecture of gene regulation: the promis of eQTL studies. Trends Genet. 2008;24:408–415. 75. Raychaudhuri S, Plenge RM, Rossin EJ, et al.

Identifying relationships among genomic dis-ease regions: predicting genes at pathogenic SNP associations and rare deletions. PLOS Genetics. 2009;5:e1000534.

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Part

1

Identification of genetic risk factors for

Inflammatory Bowel Disease and the

influence on gene expression

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chapter

2

HNF4α and CDH1 are associated

with ulcerative colitis in a Dutch cohort

Suzanne van Sommeren, Marijn C. Visschedijk, Eleonora A.M. Festen, Dirk J de Jong, Cyriel Y Ponsioen, Cisca Wijmenga, Rinse K. Weersma, Inflammatory Bowel Diseases. 2011;17(8)1714–8.

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ABSTRACT

Background and Aims: Inflammatory Bowel Disease (IBD) consist-ing of ulcerative colitis (UC) and Crohn’s disease (CD) are complex disorders with multiple genes contributing to disease pathogene-sis. A recent genome wide association scan identified three novel susceptibility loci for UC comprising HNF4α, CDH1 and LAMB1. We performed an analysis of these three loci in an independent cohort.

Materials and methods: 821 UC patients and 1,260 healthy controls of central European Caucasian descent were genotyped for SNPs: rs6017342 (HNF4α), rs1728785 (CDH1) and rs6949033 (LAMB1). Differences in allele and genotype distribution in cases and controls were tested for significance by the χ²-test.

Results: Allelic association analysis showed that SNP rs6017342 in the HNF4α locus was strongly associated with

UC (p-value = 1.04 × 10–11_{, OR = 1.56, CI = 1.37–1.77) and}

SNP rs1728785 (CDH1) was associated with a P-value of 0.01 (OR = 1.23, CI = 1.05–1.44). SNP rs6949033 in LAMB1 was not associated in our cohort (p-value = 0.12, OR = 1.11, CI = 0.97–1.26). We found an association for SNP rs6949033 (LAMB1) for disease limited to the rectum limited disease (P-value = 0.02). However this association was lost after correcting for multiple testing. No further specific subphenotype associations were identified. Conclusion: This is the first independent study to replicate the HNF4α and CDH1 loci as susceptibility loci for UC. The main candidate genes in these risk loci play important roles in the main-tenance of the integrity of the epithelial barrier, highlighting the importance of the mucosal barrier function for UC pathogenesis.

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23 INTRODUCTION

Inflammatory Bowel Disease (IBD) are common, chronic gastrointestinal inflammatory disorders with a prevalence of 100–200/100.000 in the developed countries. They comprise two ma-jor forms; Crohn’s disease (CD) and ulcerative

colitis (UC).1,2_{The aetiology of CD and UC is}

complex and consists of an aberrant immune response to the commensal bacterial flora in a genetically susceptible host. It is thought that this aberrant response is due to a combination of factors including environmental and genetic factors, causing defects in both innate and

adap-tive immunity and epithelial barrier function.2

The genetic basis of IBD had long been preciated through family studies with a ap-proximately 30,0% concordance of IBD among

monozygotic twins.3_{IBD are complex genetic}

disorders with multiple genes contributing to the disease pathogenesis. Until recently the focus in genetic research in IBD was mainly on CD, however, in recent years the attention also turned to UC, with six Genome Wide As-sociation Studies (GWAS) identifying 18 UC associated loci. These studies highlighted both disease- specific loci and other loci that are

shared between UC and CD.4–10

As part of the Wellcome Trust Case Control Consortium phase 2 (WTCCC2), the UK IBD Genetics Consortium identified three novel susceptibility loci for UC comprising HNF4α, CDH1 and LAMB1 which all play a role in

epi-thelial barrier function.4_{Given the central role}

of the epithelium in regulating inflammatory responses, the importance of the intestinal bar-rier in limiting access of toxins and microbes to underlying tissues and the antimicrobial nature of the immune responses in UC, intestinal bar-rier dysfunction is an important factor in UC

pathogenesis.11

Replication studies are important to support the differences between true positive

associa-tions and false ones.12_{Essential in a replication}

study is the presence of independent popula-tions using large sample sizes with matched controls and disease phenotypes mostly com-parable with those used in the initial studies.

We undertook a study in a Dutch cohort of UC patients and tested these three new associated loci (HNF4α, CDH1, LAMB1) in 821 UC patients and 1,260 controls.

MATERIALS AND METHODS

Patients and controls

Cases consisted of 838 Dutch UC patients. Cases were collected at the Academic Medical Cen-ter, Amsterdam (n = 409) the University Med-ical Center St. Radboud, Nijmegen (n = 206) and the University Medical Center Groningen, Groningen (n = 223) the Netherlands. Healthy controls consisted of 1,260 Dutch blood bank donors collected at the University Medical Center Groningen, Groningen. All cases and controls were of European Caucasian descent. Patients were diagnosed according to accepted clinical, endoscopic, radiological, and

histo-logical findings.2_{For UC patients, phenotypes}

were described according to age of onset, max-imum extent of disease (proctitis, left-sided, or extensive), necessity of colectomy, and the occurrence of malignancy and extra intestinal manifestations. Clinical data of the study pop-ulation are presented in table 1.

In all cases, informed consent was obtained using protocols approved by the local tional review board in all participating institu-tions. All patients and controls gave informed consent and DNA samples were handled anonymously.

DNA samples and SNP genotyping

All individuals in the study populations pro-vided blood samples, DNA was extracted

ac-cording to standard protocols.13_{Samples that}

displayed undetermined genotypes for all tested SNPs were excluded from analysis (n = 17), as-suming insufficient DNA quality. All excluded samples were cases, so the final number of cases was 821.

For this study the three SNPs showing ge-nome-wide significant association in the

WTCCC2 GWA study4_{were selected: SNP}

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and SNP rs886774 (LAMB1). Probably due to an unknown polymorphism in the primer, SNP rs886774 (LAMB1) gave technical problems: genotypes of a group of individuals could not reliably be called. This SNP was replaced by a

proxy SNP rs6949033 (D’ and r2_{= 1).}

Genotyp-ing was performed at the Department of Ge-netics, University Medical Center Groningen, using Taqman technology and SNP genotyping assays for PCR obtained from Applied Biosys-tems (Nieuwerkerk a/d IJssel, the Netherlands) between December 2009 and March 2010. Ge-notyping of the SNPs was successful with a call rate > 95%.

Statistical analysis

All genotypes obtained were tested for Hardy- Weinberg equilibrium by χ²-testing. Deviation from Hardy-Weinberg equilibrium was defined when observed genotypes differed significantly from expected genotypes. Differences in allele

Table 1. Clinical characteristics of ulcerative colitis patients and healthy controls.

cases controls Total number 821 1,260 Sexa Male 393 50.4% 765 61.6% Female 386 49.6% 476 38.4% Ageb Mean (years) 44.2 50.8 Median (years) 42.9 52.4 Age at diagnosisc Mean (years) 31.3 Median (years) 29.0

Early onset (<18 years) 71 11.0%

Adult onset (≥18 years) 575 89.0%

Disease extentd Proctitis 70 11.8% Left-sided 174 29.4% Extensive 339 57.4% Extra-intestinal manifestationse ₇₁ _15.9% Colectomyf ₁₆₇ _27.5% Colorectal cancerg ₂ _0.5%

a,b_{Data available for >95% of samples,}c,d,f_{data available for >70% of samples,}e,g_{data available for >50% of cases.}

and genotype distribution in cases and controls were tested for significance by the χ²-test, and odds ratios (OR) and confidence intervals (CI) were calculated. Association between SNPs and sub-phenotypes were calculated using a with-in-cases analysis. The sub-phenotype analyses consisted of limited disease (proctitis) against leftsided and extensive disease; and extensive disease against proctitis and left-sided. The sub-phenotype analyses were also tested for sta-tistical significance using the χ²-test. P-values from the sub-phenotype analysis were corrected for multiple testing with Bonferroni’s correction for six analyses. To test for interaction between two loci an epistasis analysis was performed. Combined allele frequencies for paired SNPs were compared between cases and controls and tested for significance by χ²-testing. All analyses were performed using Plink association analysis

toolset.14_{All significant thresholds were set at}

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25 RESULTS

Genotyping success rate and Hardy-Weinberg equilibrium

After excluding cases with insufficient DNA quality, 97.7% of the cases and 98.0% of the controls were successfully genotyped. Controls showed no deviation from Hardy-Weinberg equilibrium for all tested SNPs.

Replication of susceptibility loci from the UC-GWA study

Allelic association analysis results are shown in table 2. SNP rs6017342 in the HNF4α locus was strongly associated

with UC (p-value = 1.04 × 10−11_{, OR = 1.56,}

CI = 1.37–1.77), which reached genome-wide significant association. Bases on genotype

fre-quency, the P-value is 6.65 × 10−11_{(OR = 1.56,}

CI = 1.37–1.79). Results from the sub-phe-notype analyses are shown in table 3. The association could not be specified for limited or severe UC sub-phenotypes in the sub-phe-notype analysis. SNP rs1728785 (CDH1) was associated to UC with a P-value of 0.01 (OR of 1.23, CI = 1.05–1.44). The P-value for the gen-otype association analysis was 0.03 (OR = 1.23, CI = 1.05–1.45). This locus was not associated with a distinct sub-phenotype. We did not find an association of rs6949033 (LAMB1) with UC. We found an association for SNP rs6949033 (LAMB1) for disease limited to the rectum lim-ited disease (P-value = 0.02). However this as-sociation was lost after correcting for multiple testing.

No statistically significant epistasis was seen for the three SNPs. The strongest interaction was seen between rs1728785 (CDH1) and rs6017342 (HNF4α) with a p-value of 0.05.

DISCUSSION

We confirmed the association between UC and two susceptibility loci previously identified by the WTCCC2 and UK IBD Genetics Consortium: HNF4α and CDH1. The HNF4α locus was also strongely associated with UC in the WTCCC2

Table 2. Allelic association anal

ysis for 821 UC patients and 1,260 health

y contr ols. SNP Chr Position a Gene Risk allele RAF contr ols RAF cases P v alue OR 95% CI rs6017342 20q13.12 42.498.442 HNF4 α C 0.49 0.60 1.04 × 10 –11 1.56 1.37–1.77 rs1728785 16q22.1 67.148.731 CDH1 C 0.78 0.81 0.01 1.23 1.05–1.44 rs6949033 b 7q31.1 107.282.453 LAMB1 A 0.42 0.44 0.12 1.11 0.97–1.26 aPosition NCBI B uild 36.1 c oor dina tes bPr ox y f or rs886774 (D’ and r 2=1). St atis tic all y signi fic an t associa tions in bold. SNP, single n uc le otid e pol ymorphism; Chr, c hr

omosome; RAF, risk allele fr

eq uenc y; OR, od ds r atio; CI, c on fid enc e in ter val.

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26

Table 3. S ub-phenotype anal ysis. SNP Gene

Sub phenotype anal

ysis Minor allele P-v alue a OR 95% CI rs6949033 LAMB1 limit ed disease A 0.02 0.64 0.44–0.93 rs6949033 LAMB1 ext ensi ve disease A 0.69 0.95 0.75–1.21 rs1728785 CDH1 limit ed disease A 0.20 1.33 0.86–2.07 rs1728785 CDH1 ext ensi ve disease A 0.08 0.76 0.56–1.04 rs6017342 HNF4 α limit ed disease A 0.96 0.99 0.69–1.43 rs6017342 HNF4 α ext ensi ve disease A 0.34 1.12 0.88–1.43 aUnc orr ect ed f or m ul tiple t es ting. St atis tic all y signi fic an t associa tions in bold. SNP, single n uc le otid e pol ymorphism; OR, Od d’ s r atio; CI, C on fid enc e in ter val

GWA study (P-value = 8.5 × 10–17_{). The CDH1}

lo-cus was associated with a P-value of 2.8 × 10–8

in the WTCCC2 GWA study, comparable to the LAMB1 locus (P-value = 3.0 × 10–8_{) which could}

not be confirmed in the current study. The direc-tion of the associadirec-tion for the associadirec-tions is the same as in the WTCCC2 GWA study. However, the magnitude of association for both HNF4α and CDH1 is even larger.

The HNF4α locus showed the strongest asso-ciation with UC. This locus was also the most associated locus in the WTCCC2 and UK IBD Consortium GWA study. It is extraordinary to discover a locus this strongly associated with UC after so many performed GWA studies. This can be explained by the simple fact that this SNP

was not tested on previously used platforms.6,7

The HNF4α, or hepatocyte nuclear factor 4 alpha gene is the most likely candidate gene in this locus at 20q13. HNF4α is a nuclear transcrip-tion factor, controlling expression of multiple

genes.15_{From a functional perspective, HNF4α}

is important for epithelium homeostasis, cell function and cell architecture in the liver and

the gastrointestinal tract.16–19_{In the}

gastro-intestinal tract, the protein controls the ex-pression of several components of the cell-cell junction in the intestinal epithelium. In mice, loss of HNF4α leads to increased paracellular permeability through impairment of cell-cell junctions, indicating that HNF4α is crucial for

the barrier function of the intestinal mucosa.18

Another important function of HNF4α is its role as a transcriptional regulator of ion transport: loss of HNF4α in mice initiates loss of mucosal homeostasis through a decline in mucosal ion transport. This loss of mucosal homeostasis triggers a chronic inflammatory response in the colon of the mice and worsens tissue damage in

experimental colitis.20,21_{Both the impairment}

of the integrity of the intestinal epithelium and loss of mucosal homeostasis have been shown to be primary events in UC pathogenesis, which makes HNF4α a very attractive candidate gene for the disease. Other genes surrounding HNF4α are TTPAL, ADA, SERINC3 and PKIG. ADA (ad-enosine deaminase) has been associated with severe combined immunodeficiency disease

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(SCID), causing a dysfunction of both B and T lymfocytes with impaired cellular immunity and decreased production of

immunoglobu-lins.22_{However, nor ADA or the other genes}

are functionally as interesting as HNF4α. The second UC associated locus we confirmed is the CDH1 locus. The CDH1 locus at 16q22 comprises three genes of which CDH1 is the most plausible candidate gene. CDH1 encodes E-cadherin, a protein that plays an important

role in cell-cell adhesions.23_{Several studies}

sug-gest that loss or mislocalization of E- cadherin causes IBD through disruption of cell-cell

contacts and increased permeability.24, 25

In-terestingly, different bacteria are able to medi-ate adhesion to epithelial cells and disrupt the cell-cell adhesions by means of down- regulating

E- cadherin.26_{In this way E-cadherin is}

import-ant for invasion and adhesion of pathogens, which may contribute to IBD pathogenesis. Furthermore, CDH1 mutations are associated with multiple epithelial tumours, like gastric cancer, esophageal cancer and colorectal can-cer.27–29_{In these tumors, loss of E-cadherin}

causes increased proliferation, invasion, and

metastasis.30,31_{Especially the association}

be-tween CDH1 mutations and colorectal cancer (CRC) is interesting, because patients with UC

are more prone to develop CRC.32_The

associa-tion between CDH1 and both traits gives us an indication that there might be a shared genetic background between the two diseases.

CDH3, encoding P-cadherin, is the second gene in the 16q22 locus. P-cadherin, like E- cadherin is a member of the cadherin super-family. In contrast to E-cadherin, P-cadherin has not been related with IBD in functional stud-ies, therefore CDH1 was selected as the most probably candidate gene in the original GWAS

study.4_{The third gene in this locus is a zinc}

finger protein, ZFP90, no specific function for this gene is known. Finemapping of this region and additional functional studies are needed to clarify which gene is actually the causative gene within the locus.

Interestingly, E-cadherin and HNF4α interact in the Wnt/β-catenin signaling pathway. Loss of HNF4α induces mislocalization of E-cadherin,

which results in destabilized cell-cell junctions

and increased intestinal permeability.18_These

findings suggest that having defects in both genes would destabilize cell-cell junction fur-ther, increasing the risk of IBD. We did not see epistasis between the HNF4α and CDH1 risk variants in our dataset, but this could be ex-plained by lack of statistical power.

The fact that we could not replicate the asso-ciation between the LAMB1 locus and UC could be due to the lack of statistical power. Post hoc power analysis revealed that for LAMB1 the power to detect an association with an OR of 1.11 was only 32%. Other possible causes are genetic heterogeneity, or a difference in disease phenotypes between the British discovery co-hort and our Dutch coco-hort. Cases in this study more often have extensive disease than the population in the WTCCC2 GWA study. Also, cases described in our study have a lower age at diagnosis, and undergo colectomy more often than the British cases. These differences are statistical significant (P<0.0001). Differences might have arisen because the Dutch cases were all selected at tertiary referral centers. Moreover, in the subphenotype analysis we found a trend towards association for less extensive disease. These observations suggest that our negative findings were due to more severe disease phe-notypes in our cohort and that LAMB1 mostly influences risk for mild UC.

In conclusion, this is the first independent study to replicate the HNF4α and CDH1 loci as susceptibility loci for UC. The main candidate genes in these risk loci play important roles in the maintenance of the integrity of the epithe-lial barrier, highlighting the importance of the mucosal barrier function for UC pathogenesis.

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CHAPTER

3

Association analyses identify

38 susceptibility loci for inflammatory

bowel disease and highlight shared

genetic risk across populations

Jimmy Z Liu*, Suzanne van Sommeren*, Hailiang Huang, Siew C Ng, Rudi Alberts, Atsushi Takahashi, Stephan Ripke, James C Lee, Luke Jostins, Tejas Shah, Shifteh Abedian, Jae Hee Cheon, Judy Cho, Naser E Dayani, Lude Franke, Yuta Fuyuno, Ailsa Hart, Ramesh C Juyal, Garima Juyal, Won Ho Kim, Andrew P Morris, Hossein Poustchi, William G Newman, Vandana Midha, Timothy R Orchard, Homayon Vahedi, Ajit Sood, Joseph Y Sung, Reza Malekzadeh, Harm-Jan Westra, Keiko Yamazaki, Suk-Kyun Yang, The International Multiple Sclerosis Genetics Consortium, The International IBD Genetics Consortium, Jeffrey C Barrett, Behrooz Z Alizadeh, Miles Parkes, Thelma BK, Mark J Daly, Michiaki Kubo, Carl A Anderson, and Rinse K Weersma

Nature Genetics 2015;47(9):979–86 *These authors contributed equally

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ABSTRACT

Ulcerative colitis and Crohn’s disease are the two main forms of inflammatory bowel disease (IBD). Here, we report the first trans-ethnic association study of IBD, with genome-wide or Immunochip genotype data from an extended cohort of 86,640 European individuals and Immunochip data from 9,846 individuals of East-Asian, Indian or Iranian descent. We implicate 38 loci in IBD risk for the first time. For the majority of IBD risk loci, the direction and magnitude of effect is consistent in European and non-European cohorts. Nevertheless, we observe genetic heterogeneity between divergent populations at several established risk loci driven by a combination of differences in allele frequencies (NOD2), effect sizes (TNFSF15, ATG16L1) or a combination of both (IL23R, IRGM). Our results provide biolog-ical insights into the pathogenesis of IBD, and demonstrate the utility of trans-ethnic association studies for mapping complex disease loci and understanding genetic architecture across diverse populations.

University of Groningen Genetic susceptibility for inflammatory bowel disease across ethnicities and diseases van Sommeren, Suzanne