University of Groningen Inflammatory Bowel Disease Visschedijk, Marijn

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University of Groningen

Inflammatory Bowel Disease

Visschedijk, Marijn

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Visschedijk, M. (2018). Inflammatory Bowel Disease: 'New genes, rare variants & moving towards clinical

practice'. Rijksuniversiteit Groningen.

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Inflammatory Bowel Disease

“New genes, rare variants &

moving towards clinical practice”

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Marijn Visschedijk

Inflammatory bowel disease: new genes, rare variants & moving towards clinical practice. PhD thesis – Department of genetics and Department of gastroenterology

University of Groningen, University Medical Centre Groningen

Cover: Gillian van de Boer-Visschedijk, “de kroon op mijn werk/ziekte van Crohn/ schaakkoningin”

Lay-out and design: David de Groot, persoonlijkproefschrift.nl Printed: Ipskamp Printing, proefschriften.net

The studies presented in this thesis were supported by the ZonMw, AGIKO stipendium grant (92.003.577).

Printing of this thesis was financially supported by University Medical Center of Groningen, Graduate School of Medical Sciences, the University of Groningen, Groningen, the Netherlands. Takeda Nederland, Ferring B.V., Dr. Falk Pharma Benelux B.V., Mylan Healthcare B.V., Teva Netherlands B.V., Norgine.

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Inflammatory Bowel Disease

“New genes, rare variants &

moving towards clinical practice”

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 van Promoties

De openbare verdediging zal plaatsvinden op woensdag 5 september 2018 om 12.45 uur

door

Marijn Caroline Visschedijk

geboren op 7 september 1982

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4 Promotores Prof. dr. R. K. Weersma Prof. dr. C. Wijmenga Copromotores Dr. C.C. van Diemen Dr. E.A.M. Festen Beoordelingscommissie Prof. dr. H.M. Boezen Prof. dr. G. Bouma Prof. dr. P. Olinga

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5 Paranimfen

Lieke M. Spekhorst

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7 Part III The translation of genetic variants into the clinical practice

Chapter 6 Performance of the Montreal classification for inflammatory bowel diseases.

World J Gastroenterol. 2014 Nov 7;20(41):15374-81.

113

Chapter 7 Genomic and expression analyses identify a disease-modifying variant for fibrotic Crohn’s disease.

J Crohns Colitis. 2018 Jan 19. doi: 10.1093/ecco-jcc/jjy001

129

Chapter 8 General discussion and future perspectives. 145

Addendum Nederlandse Samenvatting List of publications

Curriculum Vitae Dankwoord

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PREFACE AND OUTLINE OF THE THESIS

Inflammatory bowel disease (IBD) comprises the chronic relapsing inflammatory disorders Crohn’s disease (CD) and ulcerative colitis (UC). The prevalence of IBD in the Western World is approximately 1 in 1000 individuals and there is an increasing trend in incidence and prevalence in developing countries. Family history is a risk factor for developing IBD, with a peak incidence in early adult life, although individuals of any age can be affected. UC is characterized by inflammation that is limited to the colon: it begins in the rectum, spreads proximally in a continuous fashion. By contrast, CD involves any part of the gastrointestinal tract in a non-continuous fashion and, unlike UC, is associated with complications such as strictures, abscesses and fistulas.

In addition to inflammation of the gut, 25% of the IBD patients have extra-intestinal manifestations (EIM). Frequently occurring EIM are immune-mediated and involve multiple organ systems like the skin (erythema nodosum, pyoderma gangrenosum), the eyes (uveïtis, episcleritis), spine (ankylosing spondylitis, AS), or the liver (primary sclerosing cholangitis, PSC). PSC is an extraintestinal manifestation of IBD, although PSC is also seen without IBD. Patients with PSC suffer from a highly increased frequency (62–83%) of IBD (called PSC with concomitant IBD, or PSC-IBD).

IBD is a complex disease, meaning that its aetiology is multifactorial: genetic, epigenetic, microbiome and environmental factors interact and give rise to the disease. Environmental factors like smoking, medication, appendectomy and diet have been implicated to play an role in the pathogenesis of IBD.

Over the last few years, the field of human genetics has seen an enormous gain of knowledge due to the development of high-throughput techniques, growing cohorts and new methods of analysis. IBD is one of the complex traits that have been extensively studied. At the start of this thesis project in 2010, 99 genetic risk loci for IBD had been identified, making it the most successfully Genome Wide Association (GWA) studied immune-mediated disease. GWAS typically ascertain at least 300.000 common single nucleotide polymorphisms (SNPs) throughout the genome, and for each of these variants the association is tested for the disease versus healthy controls. Because of the large number of genetic markers and statistical tests performed, strict statistical thresholds (P < 5 x 10-8_{) are required for proof of genome-wide significance. As well,} replication studies are important to confirm true-positive signals.

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11 Preface and outline of the thesis

To perform additional large-scale studies a new genotyping chip was designed which targeted 186 regions on the genome that are known to play a role in immune mediated diseases: the Immunochip. During the course of this thesis a large transancestry association study in IBD with genome-wide and immunochip genotyped data of 86,640 European and immunochip data 9,846 individuals of East Asian, Indian or Iranian descent increased the number of known risk loci to 200. While increasing insight in disease pathogenesis has been obtained through the identification of disease pathways the identified common loci in UC only explained ~8.2% of the disease variance and ~13.1% in CD. Recently in 2017, a new GWAS of ~12,000 IBD cases, not previously used in other studies, and meta-analysis of in total 59,957 IBD cases was performed, which increased the total of identified risk loci to 242. Almost all previously identified SNPs (228 out of 232) showed a signal in the same direction, although not all (44 SNPs not) showed nominal evidence of replication (P < 0.05).

A number of PSC GWAS and large-scale genotyping studies have been performed with the latest immunochip-based study of nearly ~3500 subjects, extending the number of identified risk loci to 24.

Because of the highly correlated nature (so-called linkage disequilibrium) of human genetic polymorphisms, association signals often span broad genomic regions including multiple genes and transcribed sequences. So far, it has often been difficult to assign the likely “causal gene” driving the association signal. GWAS have typically been focussed on common SNPs with a high prevalence in the general population (SNPs with minor allele frequency (MAF) above 5%). However low frequency (minor allele frequency (MAF) between 1% and 5%) and rare (MAF < 1%) variants of higher effects (ie, high odds ratios) could also explain a substantial fraction of common complex diseases. In order to ascertain these low frequency and rare variants, sequencing of known genetic risk loci, whole exomes or genomes, or by targeted genotyping using the Illumina exome chip array are the methods of choice. So far, multiple studies in IBD have been undertaken with mixed success. Up to this time, for PSC there have been no sequencing or exome chip array studies performed.

Of particular relevance to IBD and PSC genetics and complex disease genetics in general is the translation of genetic knowledge into clinical practice. So far, a few studies for genetic associations studies related to clinical subphenotypes have been performed.

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In this thesis, my aim is to elaborate on the partially uncovered genetic background of IBD and PSC and investigate in the translation of our genetic findings into the clinical practice.

Outline of the thesis

This thesis addresses three topics in IBD genetics

I. Identifying additional common risk variants for inflammatory bowel disease II. Identification and exploring the role of rare variants in complex diseases III. Translation of genetic variants into clinical practice

In Chapter 1, we give an overview of the clinical picture of IBD. We describe how the IBD genetic risk loci were identified by high-throughput genotyping technologies and the overlap of the risk loci with underlying biological pathways. Finally, we will discuss how these pathways can be used for therapeutic targeting and how this can help pave the way towards ‘precision medicine’.

Part I- Identifying additional common risk variants for inflammatory bowel disease

In Chapter 2 we aimed to identify additional CD risk genes. We hypothesized that part of the genetic risk loci is probably partly hidden among signals discarded by the multiple testing correction needed in the analysis of GWA studies. We prioritized 10 SNPs that are influencing gene-expression of nearby genes (cis-eQTL SNPs) and tested the SNPs for association with CD in two independent cohorts of Dutch CD patients (1539) and healthy controls (2648).

In Chapter 3 we followed up the identification of three genes that were associated to UC in a GWAS study performed in the UK IBD Genetics Consortium. Here we analyse rs6017342 (HNF4-α), rs1728785 (CDH1), and rs6949033 (LAMB1) in a independent Dutch cohort of 821 UC patients and 1260 healthy controls.

Part II- Identification and exploring the role of rare variants in complex diseases

Chapter 4 aims to investigate the contribution of rare, large effect genetic variants to UC

susceptibility. We performed a deep pooled targeted resequencing study of 122 UC genes in 790 Dutch UC patients. The Genome of the Netherlands project provided sequence data of 500 healthy controls. After an extensive quality control, follow-up genotyping of

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13 Preface and outline of the thesis

177 variants was performed in an independent cohort of 1021 Dutch UC cases and 1166 Dutch controls. Next, nineteen selected SNVs were genotyped for replication in 1064 German UC cases and 3576 general-population-based German controls.

In chapter 5 we investigated the contribution of rare variants in PSC. We genotyped a large international cohort of 1,243 PSC cases and 10,038 population-matched controls using the Illumina Exome array (Illumina, Inc., San Diego, CA). Association analyses revealed 90 rare genetic loci associated with PSC. Exome array results were validated using the same samples as used in the discovery stage. The replication phase is yet on going, we will replicate 87 variants in four independent cohorts of 2401 PSC cases and 5088 population matched healthy controls.

Part III- The translation of genetic variants into clinical practice

In Chapter 6 we aim to validate the Montreal classification system for CD and UC. We selected 20 de-identified medical records with an appropriate representation of the IBD sub phenotypes. 30 observers scored the records; the observers had different professions (gastroenterologist specialist in IBD, gastroenterologist in training and IBD-nurses) and experience level with IBD patient care.

Chapter 7 aims to identify disease-modifying genes associated with recurrent

fibrostenotic CD. Multiple genetic variants are associated with susceptibility for CD, but little is known about genes influencing CD behaviour. We performed a within-cases analysis comparing “extreme phenotypes” using a genome-wide approach (166,251 SNPs) in two independent case-control cohorts totalling 322 fibrostenotic and 619 purely inflammatory (non-penetrating/non-fistulising) CD cases.

After the identification of a disease-modifying gene, we performed expression analysis of genes in the Transforming Growth Factor-beta (Tgf-β) pathway in human resected tissue of CD patients.

Chapter 8 provides an overview of the results of the studies and future perspectives

University of Groningen Inflammatory Bowel Disease Visschedijk, Marijn