Hepatic gene expression profiles differentiate presymptomatic patients with mild versus severe nonalcoholic fatty liver disease

Abstract

Clinicians rely upon the severity of liver fibrosis to segregate patients with well-compensated nonalcoholic fatty liver disease (NAFLD) into subpopulations at high- versus low-risk for eventual liver-related morbidity and mortality. We compared hepatic gene expression profiles in high- and low-risk NAFLD patients to identify processes that distinguish the two groups and hence might be novel biomarkers or treatment targets. Microarray analysis was used to characterize gene expression in percutaneous liver biopsies from low-risk, "mild" NAFLD patients (fibrosis stage 0-1; n = 40) and high-risk, "severe" NAFLD patients (fibrosis stage 3-4; n = 32). Findings were validated in a second, independent cohort and confirmed by real-time polymerase chain reaction and immunohistochemistry (IHC). As a group, patients at risk for bad NAFLD outcomes had significantly worse liver injury and more advanced fibrosis (severe NAFLD) than clinically indistinguishable NAFLD patients with a good prognosis (mild NAFLD). A 64-gene profile reproducibly differentiated severe NAFLD from mild NAFLD, and a 20-gene subset within this profile correlated with NAFLD severity, independent of other factors known to influence NAFLD progression. Multiple genes involved with tissue repair/regeneration and certain metabolism-related genes were induced in severe NAFLD. Ingenuity Pathway Analysis and IHC confirmed deregulation of metabolic and regenerative pathways in severe NAFLD and revealed overlap among the gene expression patterns of severe NAFLD, cardiovascular disease, and cancer.

Conclusion: By demonstrating specific metabolic and repair pathways that are differentially activated in livers with severe NAFLD, gene profiling identified novel targets that can be exploited to improve diagnosis and treatment of patients who are at greatest risk for NAFLD-related morbidity and mortality.

Figure 1. Hierarchical clustering analysis

Hierarchical clustering of the top 100 differentially expressed probes from the 72 NAFLD patients separated the samples into two main groups: mild NAFLD and severe NAFLD. Data are presented in heat map format in which patient samples are shown in rows and genes (probes) in columns. Red color corresponds to genes that are up-regulated in severe NAFLD as compared to the mean, and green color corresponds to genes that are down-regulated in severe NAFLD as compared to the mean.

Figure 2. Quantitative RT-PCR of human NAFLD RNA samples

Bar graphs represent the fold difference in gene expression for mild NAFLD as compared to severe NAFLD. Dark bars represent the average gene expression in mild NAFLD from 7 unique patients while the light bars represent the average gene expression in severe NAFLD from 7 unique patients. RPL35 was used as the control gene for all analyses. Results are means ± SE.

Figure 3

Hepatic accumulation of markers of tissue repair and regeneration and liver progenitor cells is greater in severe NAFLD compared to mild NAFLD. Photomicrographs of SHH, GLI2, K7, SOX9 and α-SMA IHC in patients with mild and advanced NAFLD are shown (400x magnification). Liver sections stained for SHH (brown, arrowheads) show greater numbers of positive cells in severe NAFLD (B) compared to mild NAFLD (A). Liver sections double stained for K7 (brown) and GLI2 (red, nuclear, arrow head) reveals significantly higher grade of co-staining (arrows) in livers with severe NAFLD (D) than in mild NAFLD (C). Photomicrographs illustrate parallel accumulation of liver progenitor cells co-expressing GLI2 (red, nuclear) and SOX9 (blue, nuclear), along with α-SMA-positive myofibroblastic cells (brown) in severe NAFLD (F) compared to mild NAFLD (E). Many bile ductular cells in severe NAFLD (1000x magnification) express SOX9 (blue, nuclear, arrows); such cells generally co-express GLI2 (red, nuclear, arrow heads)(G). Figure and table summarizing the semi-quantitative immunohistochemistry results are shown in the Supplementary Materials and Methods.