A serum-induced gene signature in hepatocytes is associated with pediatric nonalcoholic fatty liver disease

Abstract

Objective: Pediatric nonalcoholic fatty liver disease (NAFLD) is a growing problem, but its underlying mechanisms are poorly understood. We used transcriptomic reporter cell assays to investigate differences in transcriptional signatures induced in hepatocyte reporter cells by the sera of children with and without NAFLD.

Methods: We studied serum samples from 45 children with NAFLD and 28 children without NAFLD. The sera were used to induce gene expression in cultured HepaRG cells and RNA-sequencing was used to determine gene expression. Computational techniques were used to compare gene expression patterns.

Results: Sera from children with NAFLD induced the expression of 195 genes that were significantly differentially expressed in hepatocytes compared to controls with obesity. NAFLD was associated with increased expression of genes promoting inflammation, collagen synthesis, and extracellular matrix remodeling. Additionally, there was lower expression of genes involved in endobiotic and xenobiotic metabolism, and downregulation of peroxisome function, oxidative phosphorylation, and xenobiotic, bile acid, and fatty acid metabolism. A 13-gene signature, including upregulation of TREM1 and MMP1 and downregulation of CYP2C9, was consistently associated with all diagnostic categories of pediatric NAFLD.

Conclusion: The extracellular milieu of sera from children with NAFLD induced specific gene profiles distinguishable by a hepatocyte reporter system. Circulating factors may contribute to inflammation and extracellular matrix remodeling and impair xenobiotic and endobiotic metabolism in pediatric NAFLD.

Keywords: RNA‐sequencing; patient serum; pediatric NAFLD; transcriptomic reporter cell assay.

Figure 1.. Gene expression differences in serum-exposed hepatocyte reporter cells distinguishes children with NAFLD and control children with obesity.

Principal component analysis (PCA) plot showing the variance in gene expression of 195 differentially expressed genes (DEG) among hepatocyte reporter cells exposed to sera from children with NAFLD (n=14) and controls with obesity (n=14). The variance captured within each coordinate is shown in parenthesis. Smaller symbols represent individual samples, larger symbols represent the centroid (or average of each group), and the ellipses represent the 95^th percentile of each group.

Figure 2.. Gene signature in serum-exposed hepatocytes associated with pediatric NAFLD.

(A) Venn diagram showing the number of differentially expressed genes (DEG) identified by pairwise analyses using controls with obesity as a baseline. (B) Heatmap of the 13 genes associated with NAFLD. Data presented using log₂ fold change (L2FC) in expression compared to controls with obesity. (C) Heatmap showing hierarchical clustering of gene expression profiles induced by sera from controls with normal weight (Normal), controls with obesity (Obese), NAFLD, NASH 1a, NASH 1b, and Definite NASH using the 13 genes associated with NAFLD. Data presented using fold change in expression.

Figure 3.. Predictive modeling of disease categories versus controls with obesity using the 13-gene signature associated with pediatric NAFLD and liver function tests.

Area under the receiver operating characteristic (AUROC) curves showing the random forest predictive models using the 13-gene signature associated with NALFD, liver function tests (LFTs), and the combined model of 13-genes plus LFTs for (A) NAFL, (B) NASH 1a, (C) NASH 1b, and (D) Definite NASH.