MULTIOMIC CHARACTERIZATION OF GASTRIC MUSCULARIS PROPRIA IN IDIOPATHIC GASTROPARESIS

Background: Idiopathic gastroparesis (IG) is an increasingly prevalent subtype of gastroparesis and causes significant morbidity and decreased quality of life. Prior studies have shown loss of Interstitial Cells of Cajal (ICC), key regulators of gastrointestinal motility, as well as anti-inflammatory macrophages in gastroparesis. Our aim was to determine druggable molecules and signaling for human IG using a deep multiomics approach through untargeted transcriptomics and proteomics on gastric muscle tissue.

Methods: We enrolled 13 IG patients (43±2 yrs, 10 F) and 18 non-diabetic controls (NDC; 46±10 yrs, 14 F) patients from Gastroparesis Clinical Research Consortium sites and Mayo Clinic, respectively. Bulk RNA-seq and Gel-based Liquid Chromatography/Mass-Spectrometry (LC-MS) were performed on mucosa-free gastric muscle. Differentially expressed genes and proteins were determined. Gene Set Enrichment Analysis and Search Tool for Retrieval of Interacting Genes and Proteins (STRING) were used to generate signalling pathways. Next, transcriptomic, and proteomic datasets were integrated. EnrichmentMap (EM) analysis in Cytoscape was used to determine networks of integrated STRING pathways. Finally, CiberSort was used to determine distribution of immune cell markers.

Results: RNA-seq detected 16755 genes, of which, 2569 (1338 up, 1231 down in IG compared to NDC) were significantly different (FDR<0.05, FC≥|1.5|). Mitochondrial genes involved in oxidative phosphorylation (OXPHOS; COX6C, SDHA, SDHB) and apoptosis (AIFM1, GHITM, HTRA2), as well as OXPHOS pathways (KEGG, Hallmark, FDR<0.001) were increased in IG. Using CiberSort, a significant increase in memory B-cell, and CD8+ T-cell, and a decrease in M2-associated (anti-inflammatory) macrophage markers were found in IG. Proteomics analysis detected 6444 named proteins, of which, ~50% were significantly different (FDR<0.05, FC≥|1.5|; 1485 up, 1541 down in IG). Top pathways (Hallmark: “Oxidative Phosphorylation”; Reactome: “Electron Transport Chain”, FDR<0.001), and proteins (TRAP1, WASL, SDHA) were noted. Overlap of the 2 datasets revealed 1241 significantly different gene-protein pairs (574 up, 667 down in IG). EM analysis showed that network joining “Signaling by SCF-Kit”, “Kit receptor Signaling”, and “PDGFR-beta signaling” pathways as the largest downregulated network, whereas “OXPHOS system in mitochondria” and “Metabolism” was the primary upregulated network in IG (Figure 1).

Conclusions: Our deep multiomics profiling of gastric muscularis propria in IG has uncovered oxidative phosphorylation and mitochondrial dysfunction, as well as confirmed Kit-signaling to be important in pathophysiology of IG. Loss of genes associated with anti-inflammatory macrophages, and upregulation of memory B-cell, and cytotoxic CD8+ T-cells further makes a case for determining immunobiology of idiopathic gastroparesis.