DNA methylation and transcriptional profiles of intestinal epithelium from celiac patients

Background

Celiac disease (CD) is triggered by gluten in genetically susceptible individuals and characterized by a wide clinical spectrum going from classical to potential CD. Around 50% of the heritability of CD remains unexplained. The identification of changes in DNA methylation could help discover novel genomic regions involved in the onset of CD. Since epigenetic mechanisms operate at the interface between genetic predisposition and environment, the intestinal epithelium is the disease-relevant cell type of the small-intestine to identify stable and potentially heritable epigenetic alterations involved in the pathogenesis of CD.

Hypothesis, Rationale and Aims

Epigenetics is a highly plausible mechanism that may both initiate and maintain intestinal mucosal inflammation. In CD the epigenetic and transcriptomic analysis of highly purified intestinal epithelial cells (IECs), bearing the epithelial cell adhesion molecule CD326, could reveal specific disease-associated alterations to learn more about the pathogenesis of CD in all clinical and biological manifestations.

Our aims are:

1) Enrollment of patients suspected for CD which will be diagnosed as classical or potential CD patients and a matched cohort of controls suffering from other gastrointestinal disorders
2) Profiling the genome-wide DNA methylation and gene expression of IECs derived from the enrolled patients to detect genes differently methylated and expressed in the three study groups
3) Intestinal epithelial organoids will be generated from patient’s biopsy samples to investigate if disease-associated epigenetic and transcriptomic alterations are retained in organoids
4) A functional study will be performed to demonstrate that patient-derived organoids represent a valid in-vitro disease-model.

Research Plan (Experimental design and methodologies)

To reach the aims of this exploratory cohort study:

1) Pediatric subjects suspected for CD tested positive for serum CD antibodies concentrations and patients with other gastro-intestinal (GI) disorders negative for serum CD antibodies (controls) will be consecutively enrolled and duodenal biopsies will be collected. Suspected CD patients positive for serum CD antibodies will be diagnosed as:

– classical CD –with intestinal villous atrophy

– potential CD –with normal intestinal mucosa

2) Genome-wide DNA methylation and gene expression analysis will be performed with DNA/RNA extracted from IECs separated from the other intestinal cell types to identify group-specific differentially methylated and transcripted regions

3) Intestinal epithelial organoids will be generated from randomly selected classical and potential CD patients and controls to detect epigenetic and transcriptional alterations which will be compared with the alterations observed in IECs derived from the same patients of the three study groups

4) Bidimensional and differentiated organoids will be stimulated with gliadin fragments and gluten-dependent activation will be evaluated by measuring the epithelium-derived IL-15.

Expected results and Impact

This is the first epigenetic and transcriptomic study that will be performed in highly purified IECs. The identification of changes in DNA methylation affecting gene expression could help discover novel genomic regions involved in CD onset. Specific epigenetic and transcriptional profiles of IECs able to distinguish the different manifestations of CD could be transferred in clinical practice offering a new diagnostic tool for the diagnosis of CD in all clinical manifestations. The generation of gut epithelial organoids could give a valid experimental model to explore the gluten-dependent activation pathways potentially involved in CD pathogenesis.