Investigating the role of DNA methylation in coeliac disease

Coeliac disease (CD) is a chronic autoimmune disease characterised by mucosal inflammation in the small intestine in response to dietary gluten in genetically susceptible individuals. Current screening and diagnostic tests for CD are suboptimal as accurate serology and histology requires active consumption of gluten. Additionally the gastroscopy procedure is invasive and carries risks associated with anaesthesia and oesophageal perforation. Furthermore, genetic testing for CD susceptibility is only useful for its negative predictive value, as up to 56% of the Australian general population carry the disease susceptibility alleles. Multiple environmental factors have also been associated with CD development, but the underlying mechanisms are unknown. Gene-environment interactions are mediated by epigenetic modifications of the genome, and changes to epigenetic profiles can occur in response to changes in the environment. DNA methylation is a potential mechanism to partially explain how interactions between genetic and environmental factors combine to trigger disease development. Altered DNA methylation profiles have been observed in other gastrointestinal inflammatory disorders and have identified new pathways integral to the disease process. Altered DNA methylation profiles are also potential disease biomarkers with utility in disease screening if associated with disease state and identified in an easily accessible tissue. In the context of CD, DNA methylation profiles unique to CD irrespective of whether individuals are consuming gluten could be a valuable screening tool, particularly if the DNA methylation markers were present in an easily accessible tissue such as saliva. This thesis investigated the role of DNA methylation in CD and the feasibility of using saliva as the tissue source for these studies. To achieve this, the feasibility of saliva and the oral mucosa as an alternative tissue source for DNA methylation studies in intestinal disorders was first examined (Chapter 2). The DNA methylation profiles of saliva and intestinal mucosa, obtained via upper duodenal endoscopy, were compared within individuals with and without a CD. We found that DNA methylation profiles generated from saliva were of high quality and suitable for downstream analysis. DNA methylation profiles were positively correlated between saliva and intestinal mucosal tissues within an individual (intra-individual), and the differentially methylated regions that were identified mapped to genes with tissue specific functions. Interestingly, DNA methylation profiles in saliva exhibited greater variability between individuals, compared with intestinal mucosal profiles. This was proposed to either be a result of differences in disease status, CD vs non-CD, or that the CD individuals adhered to a gluten-free diet (GFD), while the other individuals did not. To investigate the impact of diet on DNA methylation profiles, we examined the effect of a GFD on saliva DNA methylation profiles in healthy individuals (Chapter 3). The relationship between microbiome composition and health has been well established, and studies have shown how the microbiome can influence DNA methylation. Therefore, in addition to DNA methylation profiles, the oral microbiome in healthy individuals was also characterised. This portion of the study utilised healthy non-CD individuals in order to eliminate any effect that CD may have on DNA methylation and the microbiome. No variations in microbial diversity or alterations in microbial species composition or abundance were observed following a 6 week GFD. Similarly, no difference in saliva DNA methylation profiles were observed, which was not surprising given the absence of change in saliva microbiome profiles before and after the uptake of a GFD. This suggests that the variability observed in saliva samples in Chapter 2 could be due to disease state. Finally, DNA methylation profiles in saliva were compared between CD and non-CD individuals to identify whether epigenetic mechanisms are involved in the development of CD (Chapter 4). DNA methylation profiles of CD individuals who had been diagnosed at least 2 years previously; were on a GFD; and who were currently asymptomatic, were compared to the DNA methylation profiles in age and sex-matched non-CD healthy controls. CpG sites within a region near the SLC17A3 gene were identified as being differentially methylated between the two groups, however the results could not be validated within a larger cohort, with methylation differences in the opposite direction being observed. DNA methylation has emerged as a potential mechanism for which interactions between genetic and environmental factors trigger development of disease. Identification of altered levels of methylation associated with disease states, whether preceding or following disease, offers the potential for discovering new pathways integral to the disease process. This thesis shows that saliva is a feasible DNA source alternative for epigenetic studies in intestinal disorders, whilst also showing a short-term GFD does not impact DNA methylation profile or the oral microbiome in healthy individuals. DNA methylation profiles in saliva from asymptomatic GFD-treated CD individuals did not differ significantly from non-CD healthy controls. Further research is required to confirm the direction of the methylation differences identified in the SLC17A3 region. Expanding the CD cohort to include newly diagnosed CD individuals with active disease may also produce different DNA methylation profiles that may be an important step in understanding the mechanisms that lead to the development of disease and identify potential biomarkers for future screening tools