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

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Discussion and future perspectives

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DISCUSSION AND FUTURE PERSPECTIVES

The field of human genetics has seen an enormous increase in knowledge over the last few years due to larger cohorts, the development of new high-throughout genotyping and sequencing techniques, and new methods for analysis and data integration. A host of new trait-associated genomic regions have been uncovered, and current work is focused on their biological interpretation and the first steps towards their translation to clinical practice (see chapter 1).

In this final chapter I will focus on several aspects of genetics in inflammatory bowel disease (IBD) and position my work in a broader perspective. I will discuss the identification of novel and causal risk variants in IBD, the exploration of the role of rare variants in IBD and primary sclerosing cholangitis (PSC) and discuss the progress of the translation of genetic variants into clinical practice. Finally, I will summarize my concluding remarks on the perspectives for the future of the IBD genetics field.

SECTION I PLACING THE WORK DESCRIBED IN THIS

THESIS IN A BROADER PERSPECTIVE

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

There are several approaches for identifying new genetic risk variants in IBD. The first genetic studies in IBD were linkage and candidate gene association studies. Linkage studies map genetic risk loci by testing a series of marker alleles for co-segregation (linkage) with disease status through different generations in a family.

A total of 10 linkage and association studies for IBD were performed in the period from 1996 to 2004, including the studies that identified the risk variants in NOD2. Nowadays

NOD2 variants remain the strongest genetic risk variants for Crohn’s disease (CD), with

odds ratios (ORs) varying from 2 to 41_.

In the early 2000s new techniques provided the opportunity to perform genome wide association studies (GWAS). GWAS typically focus on associations between complex traits and 100,000–500,000 single nucleotide polymorphisms (SNPs), selected to tag a maximum of genetic variation over the whole genome. Unlike linkage studies, GWAS are not restricted to sibling pairs and families, and thus have more statistical power to detect

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genetic risk loci of small to moderate effect sizes, ORs (1.0-1.5). Due to possibility of false positives because of the large number of tests, there is need to correct for multiple testing and a strict protocol for replication. In Chapter 2 of this thesis, we demonstrate that we can lower the number of tests and detect new risk variants by selecting SNPs that show a correlation to gene expression [cis-expression quantitative trait loci (eQTLs)] for association analysis. We identified, among others, the loci containing UBE2L3 and BCL3 as likely novel risk genes for CD. The association for UBE2L3 has later been confirmed in a large cohort (~50,000) of GWAS meta-analysis2_{. BCL3 is not detected as a CD gene}

in large meta-analysis, which suggests this is a false positive finding.

The genome-wide statistical significance for true positive associations is commonly set to a P-value <5 × 10−8_{. Replication studies are important to differentiate between}

true-positive associations and false-positive associations. In Chapter 3 we confirm the association between ulcerative colitis (UC) and two (out of three) susceptibility loci previously identified by the Wellcome Trust Case Control Consortium 2 (WTCCC2) and UK IBD Genetics Consortium in an independent cohort of 821 UC patients and 1260 unrelated healthy controls: HNF4-α and CDH13_.

Besides the importance of replication, gain of power by increasing initial sample size is crucial for identifying new associations with lower odds ratios. Most IBD risk loci overlap with those of other immune-mediated diseases. This knowledge was applied in the design of the Immunochip: a chip composed of all genetic variants correlated to immune-mediated diseases. Jostins et al. increased the number of IBD risk loci to 163 by performing a meta-analysis of all GWAS and Immunochip data from a total of 75,000 individuals4_{. Next, Liu et al. performed a combined GWAS and Immunochip data}

meta-analysis with genetic data from 96,486 individuals including 9,846 individuals of non-European ancestry (East-Asian, Indian and Irian descent) and allowed for the identification of 38 additional risk loci for IBD, increasing the total number of IBD risk loci to 2005_{. 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 IBD risk loci to 2426_.

Although this last study contained IBD cases not previously used in genetic studies, and although this study identified additional associated loci, at this moment it would, in my opinion, not be cost-effective to further invest in enlarging the cohort size to identify novel associations. In 2010, it has already been demonstrated that increasing the number of associated loci, does not significantly increase the explained disease

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variation anymore7_{. I think, the search for part of the ‘hidden heritability’ has to shift}

towards variants that affect protein function and are thus more likely to have greater penetrance. It could be that such variants are low frequency (minor allele frequency (MAF) between 1% and 5%) and rare (MAF < 1%) variants of higher effect size (i.e., high odds ratios > 2.0). The exploration of the role of rare variants in complex diseases is described in the next part (Part II).

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

Because natural selection acts against variants that confer large effects on disease susceptibility (such as variants leading to Mendelian diseases), variants that affect protein function are usually low frequent variants. Sequencing a large case-vs-control cohort is the method of choice to comprehensively ascertain low frequency and rare variants that have effect sizes higher than those detected by GWAS.

Prior to the publication of Chapter 4, four re-sequencing studies have been performed that investigated IBD in European populations8–11_{. In Chapter 4, we aimed to investigate}

the contribution of rare variants to UC susceptibility. We performed a deep-pooled targeted resequencing study of 122 known UC risk genes. We confirmed known rare variants in the IL23R (rs41313262, rs76418789, rs11209026), CARD9 (rs141992399, rs200735402) and JAK2 (rs41316003) genes, most with similar AFs to those reported in other studies. We additionally identified an association with variants in the MUC2 gene on UC susceptibility in the Dutch population (gene-based analyses p-value of 9.2 x 10-5_),

which suggests a population-specific contribution of rare variants to UC susceptibility. It has been demonstrated that rare variants are even more population-specific compared to common variants12_{. More recently, another population specific mutation was}

identified by using whole exome sequencing (WES) in an Ashkenazi Jewish population which identified a frameshift mutation in CSF2RB13_.

Other studies identified the R179X variant in RNF186 as protective against UC14_{, and}

a missense variant in SLC39A8 associated with Crohn’s disease, which influences the gut microbiome15_{. In 2016, a large whole-genome sequencing (WGS) was performed in}

16,432 IBD cases and 18,843 controls. In this study, a 0.6% frequency missense variant in ADCY7 that doubles the risk of UC was discovered, known variance were confirmed

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and they detected a burden of very rare, damaging missense variants in known Crohn’s disease risk genes. Despite the robust statistical power, no other new low-frequency risk variants were identified16_.

WES and WGS are still very expensive and large cohorts are needed to have enough power to detect exome variants that contribute to disease. The Illumina Exome Array is an alternative for WES and WGS. The array is based on coding variants seen several times in existing re-sequencing datasets. In Chapter 5 we used the Illumina Exome array to study low and rare frequency variants in PSC. Until now, 24 genetic loci that are associated with the susceptibility of PSC have been identified ( Ji et al, 2017). 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). We identified 5 genome-wide low-frequency variants associated with PSC. The replication phase is currently on going, we will study 87 variants, based on our findings in the discovery phase, and in four independent cohorts of 2401 PSC cases and 5088 populations matched healthy controls. In the last few years, rare, causal genetic variants in IBD have been identified through a combination of a specific Exome-chip, imputation, sequencing and fine-mapping. This has brought us new low-frequency variants with high odds ratios. However, the amount of identified variants is much lower than expected, and these variants probably explain less of the hidden heritability than hoped for17_{. This trend does not}

only apply to IBD alone, also the other complex diseases (like Diabetes Mellitus type II, hypercholesterolemia, Alzheimer) identified rare variants with various success. One of the positive stories is the identification of PCSK9, which led to the development of new therapies to treat hypercholesterolemia18_.

The latest WGS in IBD detected a burden of very rare, damaging missense variants in known CD risk genes, suggesting that more comprehensive sequencing studies will continue to improve understanding of the biology of complex diseases16_{. For the future,}

I think that rare variant association studies good be as successful as those for common variants. There is a need for development of computationally efficient methods, accepted standards for combining sequence data sets and genetics burden tests/polygenetics risk scores for precision risk prediction. This has to be combined with a careful selections of genes based on pathway or candidate genes analyses, and to re-sequence these genes in population-specific case-control cohorts. Additionally, specific phenotypic populations likely to be enriched for rare variants, such as early onset IBD19_{or families with high}

disease prevalence 20_{, can be used.}

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Part III- The translation of genetic variants into clinical practice

Over the last years, the research field of genetics in IBD and PSC has seen a tremendous gain of knowledge. Since the start of my PhD in 2010, the number of associated susceptibility loci has grown from 99 to 242. Moreover, a lot of causal genes have been identified. However, understanding the functional and clinical consequences of the associated loci has not followed at the same pace. I will describe the steps that have been undertaken in translational research from different angles and explain what these steps mean for the future of IBD genetics.

Genotype-phenotype studies

In order to properly study genotype-phenotype correlations across different cohorts, it is important to use a uniform classification system. For IBD, the Montreal criteria are used in clinical practice. In Chapter 6 we aimed to validate the Montreal classification as a phenotype to use in genotype-phenotype correlation studies, by letting 30 observers with different professions (gastroenterologist specialist in IBD, gastroenterologist in training and IBD-nurses) and experience levels with IBD patient care score 20 de-identified medical records with the appropriate representation of the IBD sub-phenotype. We found a good to excellent inter-observer agreement for all Montreal items except for disease severity in UC (poor). This implies that we can use the Montreal classification for genotype-phenotype studies, but we have to be careful with the interpretation of UC disease severity.

A large international collaboration used the Montreal classification to study the genotype-phenotype interaction in 34,819 patients (19,713 with CD, 14,683 with UC), genotyped on the Immunochip array21_{. Three loci (NOD2, MHC, and MST1 3p21) were}

associated with subphenotypes of IBD, mainly with disease location. However, little or no genetic association with disease behavior was detected. Cleynen et al. showed that it was possible to distinguish colonic from ileal Crohn’s disease using prediction models, based on the genetic risk score. This supports a continuum of disorders within IBD, much better explained by three groups (ileal Crohn’s disease, colonic Crohn’s disease, and ulcerative colitis) than by the two groups, Crohn’s disease and ulcerative colitis, that are currently used21_{. I think that in the near future this will have consequences for}

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Crohn’s disease could be treated more similar as ulcerative colitis, this could have be impact on the treatment with Vedolizumab for example. Future research is necessary to confirm this hypothesis.

VEO-IBD

Of my special interest is a group of IBD patients who develop IBD at a very early age, so called Very Early Onset IBD (VEO-IBD). The VEO-IBD group experiences a more severe disease course and VEO-IBD patients more frequently have a positive family histories for IBD, both of which shows that VEO-IBD has a higher genetic penetrance than adult-onset IBD. Although, I treat only adult patients, we can learn a lot form the genetically background of VEO. VEO cases are more likely to display Mendelian-like forms of IBD, characterized by highly penetrant variants of higher effect size, as already shown by mutations identified in the IL10R gene22_{. In clinical practice, WES or targeted}

panels for genotyping are used as the golden standard in children with VEO-IBD to identify the pathogenic variants and to guide decisions on treatment modalities23_{. Many}

VEO-IBD patients are found to carry a genetic defect in IL-10/IL-10R, which often leads to resistance to standard immunosuppressive therapy. In this specific patient group Hematopioetic Stem Cell Transplantation (HSCT) should be considered early as a potentially curative therapeutic option24_.

The use of HSCT in adult IBD has been investigated and did not result in a statistically significant improvement of sustained disease remission at 1 year, while it was associated with significant toxicity25_{. In my opinion it would be interesting to test patients with}

adult-onset IBD and a refractory disease course on the VEO-IBD gene panel. If we can identify patients with deficiencies in IL-10/IL-10R, HSCT could result in an effective treatment for adults as well. It would be great if we can add also adult-specific rare variants to this panel, given the time-consuming, difficult research in rare variants in adult IBD, I think this will happen in the more later in the future.

Pharmacogenetics

Another clinical use of genetic knowledge is pharmacogenetics. Pharmacogenetics can play several different roles in a complex disease such as IBD. Firstly, each genetic risk locus can be looked upon as being a potential drug target for therapy. In our review, Chapter 1, we describe the difficulties in drug development. Examples of medication targeting proteins with a genetic association with IBD are Ustekinumab (

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Cilag), a human antibody against IL-12 and IL-2326_{, and Tofacitinib (Pfizer)}27_{a selective}

JAK-inhibitor28_{. A recently performed GWAS discovered new IBD susceptibility loci,}

including the genes ITGA4, ITGAV and ITGB8, which encode the proteins for the IBD drug Vedolizumab6_.

Secondly, we are always looking for drugs based on biological pathways. In silico studying of direct protein-protein interactions of IBD risk genes linked to medication databases can identify a second circle of potential drug targets, beyond just the known IBD risk genes. A preliminary study identified 113 drugs that could potentially be used in IBD treatment. Fourteen are known IBD drugs, 48 drugs have been, or are being investigated in IBD, 19 are being used or being investigated in other inflammatory disorders treatment, and 32 are investigational new drugs that have not yet been registered for clinical use29_{. This kind of research is still in the beginning phase, more}

known candidate genes and extensive public drug databases are needed for to improve this drug identification method.

Thirdly, genetic variants can be used to predict drug toxicity. IBD treatment is complicated by drug toxicity, such as for example thiopurine-induced myelosuppression. This can be predicted and prevented by testing for thiopurine methyltransferase (TPMT) variation before the start of thiopurine treatment30_{. A large Dutch study investigated}

the screening for variants in TPMT with the conclusion that screening for TMPT did not reduce the proportion of patients with hematologic adverse drug reactions during thiopurine treatment for IBD30_{. Currently, the standard of care is routine measurements}

of leukocyte levels and liver test values in the first weeks after starting thiopurine treatment, followed by routine measurements of thiopurine metabolites.

Over the past few years, many additional genetic variants influencing thiopurine related site effects have been identified. A common missense variant NUDT15 in a Korean population was associated with thiopurine-induced myelosuppression31_{. This finding}

was replicated in a Japanese population, where the NUDT15 R139 variant was also found to be associated with early severe hair loss 32_{. In the European population, a recent study}

in 329 cases and 635 controls, showed that carriage of any NUDT15 coding variant conferred a 22-fold increase in the odds of thiopurine induced myelosuppression (P = 2.0 x 10-8_{), independent of TPMT genotype and thiopurine dose. Pre-treatment}

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An additional novel 6bp in-frame deletion in the NUDT15 was also detected33_{. The HLA}

class II allele HLA-DQA1-HLA-DRB1 was shown to be associated with a higher risk of developing pancreatitis after thiopurine administration34_.

If we are able to identify more genetic related adverse drug events or genetic variants with drug levels (ea biologicals like anti-TNF therapy) in future studies it could be cost-effective to develop a pharmacogenetics gene panel. With the development of quicker and cheaper genetic testing I think it will be a feasible and cost-effective option for the distant future.

Fourthly, a hot topic in IBD is the microbiome. Multi-omics studies (studies focusing on integrating different levels of that contribute to the disease, microbiome, transcriptome methylation, epigenetics). have so far revealed important interplays between the host’s genetics, clinical disease behavior and the composition of the microbiome35,36_{. From}

oncological immunotherapy studies we have learned that the gut microbiome can affect the efficacy of these immunotherapies37,38_{.These findings stress the importance}

of integrating multi-omics data from large cohorts in order to find therapies targeting patients with specific risk profiles. The ongoing identification of causal genetic variants and multi-omics studies will greatly improve our understanding of IBD, and subsequently the treatment of IBD patients, over the coming years.

Disease-modifying genes

In order to identify genetic variants influencing disease behavior, so called disease-modifying genes, we performed a within-case analysis comparing “extreme phenotypes” (in chapter 7). We have identified a variant in WWOX and in lncRNA RP11-679B19.1, as a disease-modifying genetic variant associated with recurrent fibrostenotic CD (P = 6.09 × 10-11_{) by a genome-wide approach including 322 recurrent fibrostenotic CD cases}

and 619 purely inflammatory CD cases. WWOX is a known tumour suppressor gene and evidence in literature supports the involvement of WWOX in fibrosis formation40,41_.

Our expression analyses confirmed the idea of WWOX as tumour suppressor gene, by enhanced expression of Transforming Growth Factor-beta (TGF-β) in WWOX risk-allele carriers. CD patients carrying the WWOX risk allele appear to have a profibrotic profile. To avoid fibrotic complications, it might be advisable to refrain from prescribing anti-inflammatory medication that enhances TGF-β signalling in intestinal fibroblasts in these patients.

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Other disease modifying genes with disease prognosis have been identified in FOXO3,

XACT, a region upstream of IGFBP1, and the major histocompatibility (MHC) region42_.

Like the WWOX gene, these associations did not contribute to disease susceptibility, but are purely disease modifying. If we know which patient is at risk of developing a severe disease course42_{; we have to think of a top-down strategy and start with biological}

therapy in the early phase to avoid complications.

In the next section I will summarize concluding remarks on the perspectives for the future of the IBD genetics field.

Section II- Concluding Remarks

The focus of genetics of complex diseases in general has shifted from identifying genetic regions towards the identification of causal variants and the effects on gene expression and function. An extra challenge in adult IBD is the high number of risk alleles in the noncoding part of the genome, which demands the use of WGS for screening. Research will therefore be focused more and more on computationally challenging WES, WGS and fine-mapping studies. This has to be combined with careful selections of genes based on pathway or candidate genes analyses and specific phenotypic populations likely to be enriched for rare variants, such as early onset IBD19_{or families with high}

disease prevalence 20_{, can be used. Because the difficulties these techniques are facing, I}

think is a slowly progressing field.

Although precision medicine is not yet feasible, the developments in the genetics of IBD are coming closer to directly improving patient care. With the possibility of quicker and cheaper technologies, in my opinion, it will be possible (and in future hopefully cost-effective) to design an IBD panel with known SNPs related to VEO-IBD, adverse drug responses and disease-modifiers.

The developments of new high-throughout genotyping and sequencing techniques have also given us the opportunity to study the microbiome within IBD. This was not the focus of my thesis, but it is an important topic in the IBD field. The progressive unravelling of the gut flora will help us understanding the complex aetiology of IBD. Importantly, it can hopefully be used as drug targets or predictors of efficacy of therapies like their role in immunotherapy38_{. Studying IBD with a multi-omics approach will be,}

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