A Molecular Biomarker for Prediction of Clinical Outcome in Children with ASD, Constipation, and Intestinal Inflammation

Background: In children with autism spectrum disorder (ASD) who present to the gastroenterologist with chronic constipation, on a background of enterocolonic inflammation, we have identified two distinct clinical subtypes: (1) patients who experience a sustained state of GI symptomatic remission while on maintenance anti-inflammatory therapy (fast responders) and, (2) those with recurrent right-sided fecal loading requiring regular colon cleanouts during treatment for enterocolitis (slow responders). We hypothesized that a detailed molecular analysis of mucosal biopsy tissue from the affected region of the colon would provide mechanistic insights regarding the fast versus slow response to anti-inflammatory therapy.

Objectives: The goal of this study was to compare gene expression in the right (ascending) colon from two groups of GI-symptomatic children with ASD and constipation (acute versus chronic fecal loading). We hypothesize that molecular examination of mucosal biopsy tissue from affected regions of the colon can provide mechanistic insights regarding transient versus chronic right-sided colonic hypomotility in the setting of enterocolitis in children with ASD.

Methods: Patients were identified by: (1) a confirmed ASD diagnosis, (2) enterocolitis noted upon biopsy and/or capsule endoscopy and, (3) constipation with right sided fecal loading as seen on abdominal radiographs. RNA sequencing was performed on mucosal biopsies from the ascending colon. Hierarchical cluster analysis was performed to assign samples to clusters and gene expression analysis was performed to identify differentially expressed transcripts (DETs) between samples within the clusters. A penalized regression analysis method (LASSO) was used to identify genes whose expression levels were most predictive of cluster assignment.

Results: Hierarchical clustering of colonic gene expression profiles resulted in two clusters. Gene ontology and canonical pathway analysis revealed significant differences between the two clusters with the fast responder-predominant cluster showing an up-regulation of transcripts involved in the activation of immune and inflammatory response and the slow responder-predominant cluster showing significant over-expression of genes involved in tryptophan and serotonin degradation and mitochondrial dysfunction. Regression analysis identified a single long non-coding RNA that could predict cluster assignment with a high specificity (0.88), sensitivity (0.89) and accuracy (0.89).

Conclusions: This initial comparison of gene expression profiles in the ascending colon from a subset of patients with ASD, chronic right-sided fecal loading constipation, and a slow versus fast response to therapy has identified molecular mechanisms that likely contribute to this differential response. Importantly, we have identified a transcript that, if validated, may provide a biomarker that can predict from the outset which patients will be slow responders who would benefit from an alternate therapeutic strategy in treating their constipation.