Characterisation of gene and pathway expression in stabilised blood from children with coeliac disease.

INTRODUCTION: A coeliac disease (CD) diagnosis is likely in children with levels of tissue transglutaminase autoantibodies (anti-TG2) >10 times the upper reference value, whereas children with lower anti-TG2 levels need an intestinal biopsy to confirm or rule out CD. A blood sample is easier to obtain than an intestinal biopsy sample, and stabilised blood is suitable for routine diagnostics because transcript levels are preserved at sampling. Therefore, we investigated gene expression in stabilised whole blood to explore the possibility of gene expression-based diagnostics for the diagnosis and follow-up of CD. DESIGN: We performed RNA sequencing of stabilised whole blood from active CD cases (n=10), non-CD cases (n=10), and treated CD cases on a gluten-free diet (n=10) to identify diagnostic CD biomarkers and pathways involved in CD pathogenesis. RESULTS: No single gene was differentially expressed between the sample groups. However, by using gene set enrichment analysis (GSEA), significantly differentially expressed pathways were identified in active CD, and these pathways involved the inflammatory response, negative regulation of viral replication, translation, as well as cell proliferation, differentiation, migration, and survival. The results indicate that there are differences in pathway regulation in CD, which could be used for diagnostic purposes. Comparison between GSEA results based on stabilised blood with GSEA results based on small intestinal biopsies revealed that type I interferon response, defence response to virus, and negative regulation of viral replication were identified as pathways common to both tissues. CONCLUSIONS: Stabilised whole blood is not a suitable sample for clinical diagnostics of CD based on single genes. However, diagnostics based on a pathway-focused gene expression panel may be feasible, but requires further investigation.

Celiac disease biomarkers identified by transcriptome analysis of small intestinal biopsies.

Establishing a celiac disease (CD) diagnosis can be difficult, such as when CD-specific antibody levels are just above cutoff or when small intestinal biopsies show low-grade injuries. To investigate the biological pathways involved in CD and select potential biomarkers to aid in CD diagnosis, RNA sequencing of duodenal biopsies from subjects with either confirmed Active CD (n = 20) or without any signs of CD (n = 20) was performed. Gene enrichment and pathway analysis highlighted contexts, such as immune response, microbial infection, phagocytosis, intestinal barrier function, metabolism, and transportation. Twenty-nine potential CD biomarkers were selected based on differential expression and biological context. The biomarkers were validated by real-time polymerase chain reaction of eight RNA sequencing study subjects, and further investigated using an independent study group (n = 43) consisting of subjects not affected by CD, with a clear diagnosis of CD on either a gluten-containing or a gluten-free diet, or with low-grade intestinal injury. Selected biomarkers were able to classify subjects with clear CD/non-CD status, and a subset of the biomarkers (CXCL10, GBP5, IFI27, IFNG, and UBD) showed differential expression in biopsies from subjects with no or low-grade intestinal injury that received a CD diagnosis based on biopsies taken at a later time point. A large number of pathways are involved in CD pathogenesis, and gene expression is affected in CD mucosa already in low-grade intestinal injuries. RNA sequencing of low-grade intestinal injuries might discover pathways and biomarkers involved in early stages of CD pathogenesis.

Potential blood-based markers of celiac disease.

BACKGROUND: Blood-based diagnostics has the potential to simplify the process of diagnosing celiac disease (CD). Although high levels of autoantibodies against tissue transglutaminase (anti-TG2) are strongly indicative of active CD, several other scenarios involve a need for additional blood-based CD markers. METHODS: We investigated the levels of messenger RNA (mRNA) in whole blood (n = 49) and protein in plasma (n = 22) from cases with active CD (n = 20), with confirmed CD and normalized histology (n = 15), and without a CD diagnosis (n = 14). Group differences were analyzed using Kruskal-Wallis one-way analysis of variance by ranks. We also investigated correlations between levels of potential markers, histopathology according to the modified Marsh scale, and CD risk gradient based on HLA type, using Spearman rank correlation. The relation between HLA-DQ2 gene dose effect and the expression levels of selected blood-based markers was investigated using the Mann-Whitney U test. Finally, the diagnostic performance of anti-TG2, potential blood-based CD markers, and logistic regression models of combined markers was evaluated using receiver operating characteristic (ROC) curve analysis. RESULTS: CXCL11 protein levels and TNFRSF9 and TNFSF13B mRNA levels were identified as potential CD markers. These are all affected by or involved in the regulation of the NF-κB complex. CXCL11 protein levels and IL21 and IL15 mRNA levels were correlated with histopathology according to the modified Marsh scale, as were the established CD markers. HLA genotype risk and HLA-DQ2 gene dose effect did not show any significant relations with either the potential CD markers or the established CD markers. ROC curve analysis revealed a slight, non-significant increase in the area under the curve for the combined use of anti-TG2 and different constellations of potential blood-based CD markers compared to anti-TG2 alone. CONCLUSIONS: The CD markers identified in this study further emphasize the significance of components related to NF-κB regulation in relation to CD. However, the relevance of CXCL11, TNFSF13B, TNFRSF9, and other NF-κB interacting proteins recognized by pathway analysis, needs to be further investigated in relation to diagnosis and monitoring of CD.

Analysis of single nucleotide polymorphisms in the region of CLDN2-MORC4 in relation to inflammatory bowel disease.

AIM: To investigate a possible genetic influence of claudin (CLDN)1, CLDN2 and CLDN4 in the etiology of inflammatory bowel disease. METHODS: Allelic association between genetic regions of CLDN1, CLDN2 or CLDN4 and patients with inflammatory bowel disease, Crohn's disease (CD) or ulcerative colitis were investigated using both a case-control study approach (one case randomly selected from each of 191 Swedish inflammatory bowel disease families and 333 controls) and a family-based study (463 non-Swedish European inflammatory bowel disease -families). A nonsynonymous coding single nucleotide polymorphism in MORC4, located on the same linkage block as CLDN2, was investigated for association, as were two novel CLDN2 single nucleotide polymorphism markers, identified by resequencing. RESULTS: A single nucleotide polymorphism marker (rs12014762) located in the genetic region of CLDN2 was significantly associated to CD (case-control allelic OR = 1.98, 95%CI: 1.17-3.35, P = 0.007). MORC4 was present on the same linkage block as this CD marker. Using the case-control approach, a significant association (case control allelic OR = 1.61, 95%CI: 1.08-2.41, P = 0.018) was found between CD and a nonsynonymous coding single nucleotide polymorphism (rs6622126) in MORC4. The association between the CLDN2 marker and CD was not replicated in the family-based study. Ulcerative colitis was not associated to any of the single nucleotide polymorphism markers. CONCLUSION: These findings suggest that a variant of the CLDN2-MORC4 region predisposes to CD in a Swedish population.

Gene expression profiling of duodenal biopsies discriminates celiac disease mucosa from normal mucosa.

Celiac disease (CD) is identified by histopathologic changes in the small intestine which normalize during a gluten-free diet. The histopathologic assessment of duodenal biopsies is usually routine but can be difficult. This study investigated gene expression profiling as a diagnostic tool. A total of 109 genes were selected to reflect alterations in crypt-villi architecture, inflammatory response, and intestinal permeability and were examined for differential expression in normal mucosa compared with CD mucosa in pediatric patients. Biopsies were classified using discriminant analysis of gene expression. Fifty genes were differentially expressed, of which eight (APOC3, CYP3A4, OCLN, MAD2L1, MKI67, CXCL11, IL17A, and CTLA4) discriminated normal mucosa from CD mucosa without classification errors using leave-one-out cross-validation (n = 39) and identified the degree of mucosal damage. Validation using an independent set of biopsies (n = 27) resulted in four discrepant cases. Biopsies from two of these cases showed a patchy distribution of lesions, indicating that discriminant analysis based on single biopsies failed to identify CD mucosa. In the other two cases, serology support class according to discriminant analysis and histologic specimens were judged suboptimal but assessable. Gene expression profiling shows promise as a diagnostic tool and for follow-up of CD, but further evaluation is needed.