DNA methylation profile of genes involved in iflammation and autoimmunity in inflammatory boewl disease

Objective This thesis aimed to investigate the methylation profile of 22 genes associated with thepathogenesis of IBD in patients with ulcerative colitis (UC) or Crohn’s Disease (CD) andcompare them with the methylation profile of the same genes in healthy controls. Anadditional purpose of the study was to investigate whether methylation profile of these genesbetween inflamed intestinal tissue and peripheral blood are in concordance, hoping to indicatea possible future use of methylation as biomarker.Materials & MethodsIsolated peripheral blood samples and inflamed intestinal tissue samples were collectedfrom 24 patients with IBD, 12 of them suffering from UC and 12 from CD. The control groupconsisted of 12 healthy subjects without any personal and family history of digestive diseases.The promoter methylation status of genes involved in inflammation and autoimmunity wasprofiled using the Human Inflammatory Response and Autoimmunity EpiTect Methyl IISignature PCR Array profiles. Methylation was considered to be hyper-methylated if >20%according to the instructions of the manufacturer. The microarrays were validated with Quantitative Real-time PCR.The statistical analysis was performed using non-parametricstatistical tests (non-parametric statistical tests).ResultsRegarding CD, the methylation status of IL10RA, IL13, IL13RA1 and IL17C inperipheral blood samples did not differ significantly from the methylation status of healthycontrols. Only three genes – ATF2, CXCL5 and IL12B showed higher methylation in CDcompared to controls, but they did not exceed the threshold of 20% for hypermethylation. Allother genes tested appear lower methylation than controls.Regarding UC, methylation status of CXCL6 and IL13RA1 in peripheral blood samplesdid not differ significantly from the methylation status of healthy individuals. Five genes(CXCL14, CXCL5, GATA3, IL17C and IL4R,) were found to be significantly hypermethylatedin UC patients compared to healthy individuals. Some genes show higher methylation thancontrols, but they do not exceed 20% methylation threshold for hypermethylation. All other genes show lower methylation in UC compared to controls.Active cases of both CD and UC could be promptly distinguished from healthycontrols based on the signatures provided by the methylation profiles in peripheral bloodsamples. Based on these results - despite the relatively limited number of patients - it waspossible to define distinct signatures for active CD vs. active UC. Specifically, CXCL14,CXCL5, GATA3, IL17C and IL4R genes were hyper-methylated in UC compared to CD. Inaddition, CXCL6 which did not differ significantly between UC and controls appeared hypermethylatedcompared to CD; in contrast, IL13 which did not differ significantly between CDand controls appeared hypermethylated in CD compared to UC. Moreover the real-timequantitative PCR conducted for seven genes (CCL25, IL13, IL17RA, IL17A, IL12B, CXCL5,and IL4R) confirmed the causal relationship between hyper-methylation and lower expressionof genes. Finally, it was confirmed that methylation profile in intestinal tissue and peripheralblood are in concordance. ConclusionsIn this study we have identified panels of genes that show evidence of differentialmethylation between UC and controls, as well as CD and UC. Moreover there is strongevidence that methylation level in intestinal tissue samples is well related to methylation levelin whole blood samples. Our findings suggest that these genes play an important role in IBDpathogenesis, as differential methylation status observed affects gene expression at the mRNAlevel. In conclusion, the different methylation levels between CD and UC could play a role inconfirming the diagnosis of IBD, determining the exact type of IBD (UC or CD) and possiblyestimating the activity of diseas