Genomics of Coeliac Disease - Molecular Signatures of the Pathogenesis

Coeliac disease, the intolerance for dietary gluten common in Western populations, is a multifactorial disorder, meaning that it is caused by the interaction of environmental factors and multiple susceptibility genes. The aim of this thesis was to gain insight into the pathogenesis by means of a genomics approach. We carried out integrated genetic analysis and gene expression studies to identify, and gain better understanding of genes and mechanisms behind the pathology and etiology of coeliac disease. In the introducing chapter we review genetic studies on coeliac disease and introduce new methods for gene expression profiling. First, we examined whether tissue heterogeneity (‘patchiness’) may have an effect on gene expression measurements performed on duodenal biopsies. We observed that mucosal inflammation and differentiation (represented by IFNG and TM4SF4 gene expression, respectively) were inversely correlated, depending on the extent of mucosal restructuring. However, the extent of variance in gene expression measurements was similar in cases and controls, and thus independent of lesion severity. Consequently, we proposed a model where gene expression mosaicism in the duodenum might result into tissue heterogeneity (‘patchiness’). Second, we applied microarrays to study genome-wide gene expression in the duodenums of coeliac disease patients. Alterations induced by the pathology pointed to enhanced cell proliferation in the mucosa and arrested terminal differentiation of enterocytes. This impaired differentiation affected the uptake and processing of lipids, sterols, sugars, peptides, and iron. These deficiencies contribute to the wide variety of clinical features in coeliac disease. We also observed an impaired detoxification system, previously also described for inflammatory bowel disease (IBD). This suggests that, like coeliac disease, IBD is also characterized by a differentiation defect. Third, we analyzed the tight junction gene network using genetics and gene expression profiling. The expression pattern of genes of the claudin family appeared to have 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. Genetic association analysis of this 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 suggested that both gastrointestinal disorders, coeliac disease and IBD share a genetic barrier defect. Fourth, we examined candidate genes and gene families in the inflammatory and immune pathways. Both the IFNG gene (pivotal in the Th1 adaptive response), and SPINK4 (expressed by goblet cells) showed differential expression that correlated well with the extent of mucosal remodeling. Weak genetic association was only observed for INFG in the Dutch population, but not for the positional candidate genes SPINK1, -2, -4, and –5. 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

Neospora Caninum-like oocysts observed in feces of free-ranging red foxes (Vulpes vulpes) and coyotes (Canis latrans)

The aim of this study was to examine the feces of free-ranging foxes and coyotes for the presence of Neospora caninum oocysts. Feces were collected from 271 foxes and 185 coyotes in the Canadian province of Prince Edward Island, processed by sucrose flotation, and examined by light microscopy for the presence of coccidian oocysts. In 2 fox and 2 coyote samples, oocysts morphologically and morphometrically similar to oocysts of N. caninum were observed. DNA was extracted from these samples and subjected to nested polymerase chain reaction (PCR) using primers to the N. caninum-specific Nc5 genomic sequence. Through DNA sequencing, alignment of the sequences of at least 3 clones from each isolate to sequences deposited in GenBank revealed 95-99% similarity to the Nc5 sequence of N. caninum. PCR using primers specific for Hammondia heydorni failed to yield an amplification product from these DNA samples

The interferon gamma gene in celiac disease: augmented expression correlates with tissue damage but no evidence for genetic susceptibility.

Contains fulltext : 59364.pdf (publisher's version ) (Closed access)Celiac disease (CD) is a complex genetic disorder characterized by gluten intolerance. The Th1 immune response, with a key position for interferon gamma (IFN-gamma), is an important determinant of intestinal remodeling in CD. We aimed at further ascertaining the role of IFN-gamma, either as a genetic factor in the etiology, or as a facilitator of disease initiation/progression. Duodenal biopsies were sampled across distinct histopathological stages of the disease, including refractory CD (RCD), and used to determine IFN-gamma gene (IFNG) expression by real-time RT-PCR. INFG expression correlated with the extent of tissue restructuring, reaching a 240-fold higher expression in total villous atrophy compared to healthy tissue. CD and RCD patients with similar lesions had comparable expression levels. Interestingly, patients in complete remission still had 7.6-fold residual over-expression. An INFG marker was tested in three cohorts of Dutch patients for both genetic linkage and association. Linkage analysis yielded no significant scores for IFNG or its flanking markers. In addition, IFNG allele frequencies were not differently distributed between cases and controls. Likewise, all alleles were randomly transmitted to affected children in parents-case trios. There is no evidence for IFNG as a predisposing gene in CD, despite its enhanced expression in patients in complete remission

Comparison of serological methods for the diagnosis of Neospora caninum infection in cattle

The aims of this study were to evaluate the performance and agreement of various commercial and in-house Neospora caninum antibody assays used in dairy cattle in North America, and to investigate reproducibility of two assays performed in different laboratories. From 1998 to 2005, three enzyme linked immunosorbent assays (ELISAs, a competitive ELISA-VMRD Inc., an indirect ELISA-Biovet Inc., and another indirect ELISA-Herdchek IDEXX Corp.), two indirect fluorescent antibody tests (IFATs, VMRD Inc., and in-house USDA) and one N. caninum agglutination test (NAT, in-house USDA) were utilized to test 397 randomly selected dairy cattle serum samples from 34 herds in eastern Canada for antibodies to N. caninum. The manufacturers' recommended cut-off values were used to evaluate test performance and agreement between tests. One IFAT (VMRD Inc.) performed well (sensitivity and specificity: 0.97 and 0.97, respectively) using reference sera (n = 452), therefore, results from this IFAT on the 397 samples could subsequently be used as the reference standard to calculate test characteristics for the other assays. Only 11% of the 397 sera were found to be N. caninum-positive with the IFAT. Prevalence-adjusted bias-adjusted kappa (PABAK) ranged from 0.06 to 0.99. Positive agreement was moderate to very good (Ppos = 0.25-0.96). Negative agreement was very good for all assays (Pneg > 0.94) except NAT (Pneg = 0.66). Sensitivity was ≥0.89 for all assays except the NAT, which had a significantly lower sensitivity (0.66). Specificity was high (>0.94) for all assays except for one indirect ELISA (specificity = 0.52). This indirect ELISA did not perform satisfactorily when used in 1998, but an improved version of the ELISA performed as one of the best assays in 2004. Reproducibility of the competitive ELISA was excellent, but the reproducibility of the indirect ELISA that was improved was low (concordance correlation coefficient = 0.90 and 0.36, respectively). The performance characteristics observed for most assays in this study make them useful for screening antibodies to N. caninum in cattle