SUMMARY

Coeliac disease is a common food intolerance in Western populations, in which it has a prevalence of about 1%. In early infancy, when the transition is made to a gluten-containing diet (particularly wheat-derived products), the disease may manifest with fatty diarrhea, belly cramps, and abdominal distention. In nearly all cases these clinical symptoms disappear when patients adhere to a (life-long) gluten-free diet. However, in modern societies with a high standard of pediatric care, the proportion of patients that present with coeliac disease as an adolescent or adult is progressively growing. In these age groups coeliac disease patients show less specific clinical features like fatigue, anemia, osteoporosis, short stature, dermatitis herpetiformis (coeliac disease of the skin), and possibly reproductive failure. Whatever the clinical presentation of coeliac disease may be, it is always the result of an intestinal inflammation provoked by dietary gluten. This inflammation leads to typical changes in the intestinal mucosa: the infiltration of lymphocytes into the epithelial layer and the deeper tissue layer (lamina propria); enlargement of the crypts between the villi (crypt hyperplasia); and disappearance of the villi (villous atrophy). Thus, the intestine gets the appearance of a flat mucosa. The inflammation also changes the permeability of the intestinal epithelial barrier for salt and sugar molecules (referred to as a leaky gut ). The loss of the villi results in a dramatic reduction of the absorptive surface of the intestine, leading to microand macro-nutrient deficiency that results in the various chronic health problems of untreated coeliac disease patients. Coeliac disease is a multifactorial disorder, which means that it is caused by the interaction of environmental factors (e.g. gluten; breast feeding; infections) and heritable factors (susceptibility genes). The genetic component of coeliac disease is apparent from the enhanced risk of siblings when one sib is affected, and from comparisons between monozygous and dizygous twins. Patients with autoimmune diseases also have an increased risk of developing coeliac disease, suggesting that these diseases share common risk genes and causative mechanisms. The main genetic risk factor for developing coeliac disease is the presence of major histocompatibility complex (MHC) genes on chromosome 6p21.3 that code for the heterodimer HLA-DQ2 or HLA-DQ8. However, the presence of the MHC genes coding for these HLA molecules contribute just 40% of the genetic risk. The presence of these gene variants is necessary, but not sufficient, to develop the disease. Coeliac disease is a complex genetic disorder, meaning that multiple genes are involved in the pathogenesis, each making a minor contribution to the susceptibility. The identity of these genes, their total number, and the combinations in which they occur is, for the most part, an enigma. In this thesis we describe a combination of genetics and gene expression profiling used to identify genes and molecular pathways that contribute to the cause and pathology of coeliac disease. 191

Chapters 1 and 2 provide additional background information on coeliac disease in general and an overview of the various genetic approaches that have been employed. In addition, gene expression profiling is discussed as a method to gain insight into the pathology that may aid the genetic studies through the identification of molecular pathways affected by the disease. The integration of both genetics and genomics is emphasized to facilitate the identification of candidate genes and gene networks involved in the pathogenesis. In section II the application of high-throughput technologies is presented for the study of gene expression in the duodenal mucosa of coeliac disease patients. In chapter 3 we examined the effect that tissue heterogeneity may have on the assessment of gene expression measurements. We observed that mucosal inflammation and differentiation (represented by IFNG and TM4SF4 expression, respectively) were inversely correlated, and dependent on the extent of the mucosal restructuring. Interestingly, variability in gene expression between separately sampled biopsies derived from one individual was more or less independent of the variation in the histology of the same samples. This variation in gene expression between samples was present, and even comparable in size, in control samples. In other words, gene expression variation is present regardless of the pathological stage. This led us to propose a model where gene expression mosaicism in the duodenum under conditions of changing inflammation may give rise to tissue heterogeneity ( patchiness ). Chapters 4 and 5 describe the use of microarrays to study genome-wide gene expression in the duodenums of coeliac disease patients. These two studies differed in the microarray platform (cdna versus oligonucleotides) and the type of patient samples used (case-control versus remission sequence). The two sets of differentially expressed genes thus obtained had hardly any overlap, although the biological processes they represent were comparable. The alterations induced by the pathology of coeliac disease point to an enhanced proliferation in the mucosa and an arrest in the terminal differentiation of enterocytes. This impaired differentiation particularly affected the uptake and processing of lipids, sterols, sugars, peptides, and iron. These deficiencies might contribute to the diverse clinical features seen in coeliac disease. We also observed an impaired detoxification system that was previously also described for inflammatory bowel disease (IBD). This may suggest that, like coeliac disease, IBD is also characterized by a differentiation defect. In section III we describe how molecular pathway analysis was applied to conduct genetic (chapter 6) and gene expression studies (chapter 7). Based on the altered intestinal permeability in coeliac disease patients, and the previously identified susceptibility gene MYO9B, we focused on the tight junction gene network. The expression pattern of genes of the claudin family appeared to have 192

been evolutionary conserved for over 75 million years. Three pairs of claudins showed tight co-regulation. Patients showed variable patterns of claudin gene expression marked by extreme outliers. Particularly genes involved in signal transduction and regulation of the cytoskeleton showed trends of changed gene expression. We also observed a remarkable induction of the growth factor EGF in patients. The genetic association study of the same gene network was based on the use of single nucleotide polymorphisms (SNPs) and information on linkage disequilibrium from the International HapMap Consortium. We identified two tight junction genes that were associated with coeliac disease, and to a lesser extent with IBD. This suggests that both gastrointestinal disorders, coeliac disease and IBD share a genetic barrier defect. The adaptive immune response provoked by gluten in coeliac disease patients is characterized by a T helper cell type 1 reaction in which interferon-gamma plays a pivotal role. In section IV in which we examine candidate genes related to the immune defense, we describe the genetics and gene expression of the IFNG gene. In chapter 8 we demonstrate that the extent of the mucosal remodeling is correlated with the level of IFNG gene expression. However, the genetic study performed with the CA-repeat from the first intron of IFNG, a commonly used marker for this gene, showed no genetic association in our population of Dutch patients. In chapter 9 we re-examine the IFNG gene, but now by using SNP markers and could demonstrate that this gene is weakly associated with coeliac disease in the Dutch population. Finally, we performed a genetic and gene expression study on four members of the SPINK (serine protease inhibitors of the Kazal type) gene family. SPINK genes are involved in tissue preservation and bacterial containment, and could therefore be considered as part of the innate immune system. Three of these SPINK genes mapped to two candidate regions for coeliac disease (chromosomes 5q32 and 9p21-13), which made them attractive positional and functional candidate genes. None of these SPINK genes, however, showed genetic involvement to coeliac disease in the Dutch population. Finally, in the General Discussion, we evaluate our approach and our interpretations of the data, which are than incorporated in possible models of the pathogenesis of coeliac disease. 193