Background & Aims Gluten challenge studies are instrumental in understanding the pathophysiology of celiac disease. Our aims in this study were to reveal early gluten-induced transcriptomic changes in duodenal biopsies and to find tools for clinics. Methods Duodenal biopsies were collected from 15 celiac disease patients on a strict long-term gluten-free diet (GFD) prior to and post a gluten challenge (PGC) and from 6 healthy control individuals (DC). Biopsy RNA was subjected to genome-wide 3’ RNA-Seq. Sequencing data was used to determine the differences between the three groups and was compared to sequencing data from the public repositories. The biopsies underwent morphometric analyses. Results In DC vs. GFD group comparisons, 167 differentially expressed genes were identified with 117 genes downregulated and 50 genes upregulated. In PGC vs. GFD group comparisons, 417 differentially expressed genes were identified with 195 genes downregulated and 222 genes upregulated. Celiac disease patients on a GFD were not “healthy”. In particular, genes encoding proteins for transporting small molecules were expressed less. In addition to the activation of immune response genes, a gluten challenge induced hyperactive intestinal wnt-signaling and consequent immature crypt gene expression resulting in less differentiated epithelium. Biopsy gene expression in response to a gluten challenge correlated with the extent of the histological damage. Regression models using only four gene transcripts described 97.2% of the mucosal morphology and 98.0% of the inflammatory changes observed. Conclusions Our gluten challenge trial design provided an opportunity to study the transition from health to disease. The results show that even on a strict GFD, despite being deemed healthy, patients reveal patterns of ongoing disease. Here, a transcriptomic regression model estimating the extent of gluten-induced duodenal mucosal injury is presented.
BackgroundThere is an unmet need for novel treatments, such as drugs or vaccines, adjunctive to or replacing a burdensome life-long gluten-free diet for coeliac disease. The gold standard for successful treatment is a healed small intestinal mucosa, and therefore, the outcome measures in proof-of-concept studies should be based on evaluation of small intestine biopsies. We here evaluated morphometric, immunohistochemical and messenger RNA (mRNA) expression changes in coeliac disease patients challenged with gluten using PAXgene fixed paraffin-embedded biopsies.MethodsFifteen coeliac disease patients were challenged with 4 g of gluten per day for 10 weeks and 24 non-coeliac patients served as disease controls. A wide array of histological and immunohistochemical staining and mRNA-based gene expression tests (RT-qPCR and RNAseq) were carried out.ResultsDigital quantitative villous height: crypt depth ratio (VH: CrD) measurements revealed significant duodenal mucosal deterioration in all coeliac disease patients on gluten challenge. In contrast, the Marsh-Oberhuber class worsened in only 80% of coeliac patients. Measuring the intraepithelial CD3+ T-lymphocyte and lamina propria CD138+ plasma cell densities simultaneously proved to be a meaningful new measure of inflammation. Stainings for γδ T cells and IgA deposits, where previously frozen samples have been needed, were successful in PAXgene fixed paraffin-embedded samples. Messenger RNA extraction from the same paraffin-embedded biopsy block was successful and allowed large-scale qRT-PCR and RNAseq analyses for gene expression. Molecular morphometry, using the mRNA expression ratio of villous epithelium-specific gene APOA4 to crypt proliferation gene Ki67, showed a similar significant distinction between paired baseline and post-gluten challenge biopsies as quantitative histomorphometry.ConclusionRigorous digitally measured histologic and molecular markers suitable for gluten challenge studies can be obtained from a single paraffin-embedded biopsy specimen. Molecular morphometry seems to be a promising new tool that can be used in situations where assessing duodenal mucosal health is of paramount importance. In addition, the diagnostically valuable IgA deposits were now stained in paraffin-embedded specimens making them more accessible in routine clinics.
Canonical Wnt/β-catenin signaling regulates the homeostasis of intestinal epithelium by controlling the balance between intestinal stem cell self-renewal and differentiation but epigenetic mechanisms enacting the process are not known. We hypothesized that epigenetic regulator, Polycomb Repressive Complex-2 (PRC2), is involved in Wnt-mediated epithelial homeostasis on the crypt-villus axis and aberrancies therein are implicated both in celiac disease and in intestinal malignancies. We found that PRC2 establishes repressive crypt and villus specific H3K27me3 signature on genes responsible for e.g. nutrient transport and cell killing in crypts and e.g. proliferation and differentiation in mature villi, suggesting that PRC2 facilitates the Wnt-governed intestinal homeostasis. When celiac patients are on gluten-containing diet PRC2 is out-of-bounds active and consequently its target genes were found affected in intestinal epithelium. Significant set of effective intestinal PRC2 targets are also differentially expressed in colorectal adenoma and carcinomas. Our results suggest that PRC2 gives rise and maintains polar crypt and villus specific H3K27me3 signatures. As H3K27me3 is a mark enriched in developmentally important genes, identified intestinal PRC2 targets are possibly imperative drivers for enterocyte differentiation and intestinal stem cell maintenance downstream to Wntsignaling. Our work also elucidate the mechanism sustaining the crypt hyperplasia in celiac disease and suggest that PRC2-dependent fostering of epithelial stemness is a common attribute in intestinal diseases in which epithelial hyperplasia or neoplasia prevails. Finally, this work demonstrates that in intestine PRC2 represses genes having both pro-stemness and pro-differentiation functions, fact need to be considered when designing epigenetic therapies including PRC2 as a drug target.
Approximately 20%-25% of childhood acute lymphoblastic leukemias carry the ETV6-RUNX1 (E/R) fusion gene, a fusion of two central hematopoietic transcription factors, ETV6 (TEL) and RUNX1 (AML1). Despite its prevalence, the exact genomic targets of E/R have remained elusive. We evaluated gene loci and enhancers targeted by E/R genome-wide in precursor B acute leukemia cells using global run-on sequencing (GRO-seq). We show that expression of the E/R fusion leads to widespread repression of RUNX1 motif-containing enhancers at its target gene loci. Moreover, multiple super-enhancers from the CD19 + /CD20 + -lineage were repressed, implicating a role in impediment of lineage commitment. In effect, the expression of several genes involved in B cell signaling and adhesion was down-regulated, and the repression depended on the wild-type DNA-binding Runt domain of RUNX1. We also identified a number of E/R-regulated annotated and de novo noncoding genes. The results provide a comprehensive genome-wide mapping between E/R-regulated key regulatory elements and genes in precursor B cell leukemia that disrupt normal B lymphopoiesis.
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