Modest alcohol consumption attenuates expression of fibrosis-associated genes in patients with non-alcoholic fatty liver Disease (NAFLD)

Abstract

BACKGROUND: NAFLD, the liver manifestation of the metabolic syndrome, increases the risk for cardiovascular disease. Modest alcohol consumption is advocated for cardiovascular health, but whether it benefits or worsens NAFLD is unclear. We used gene expression analysis to determine if modest alcohol consumption potentiated or antagonized the changes in liver gene expression that accompany fibrosis progression in NAFLD. METHODS: 85 patients with biopsy-proven NAFLD who had completed alcohol questionnaires were selected from our repository on the basis of fibrosis stage (early:F0-1 vs. advanced:F3-4) and alcohol exposure (unexposed: none within 2 years vs. exposed: <2 drinks/day) to generate 4 study groups: early fibrosis/unexposed, early fibrosis/exposed, advanced fibrosis/unexposed, advanced fibrosis/exposed. Total RNA was extracted from frozen liver biopsies. Adequate RNA was obtained from 72 samples, hybridized to Affymetrix HG U133A 2.0 arrays, and resultant data analyzed via an empirical Bayes method after normalization. In unexposed patients, linear modeling was done using the R package limma to identify differentially expressed genes in livers with advanced fibrosis (n=19) versus early fibrosis (n=20). Significance was set at a False Discovery Rate of 5% after Benjamini-Hochberg correction. The Wilcoxon rank-sum test with multiple testing threshold q<.05 was then used to compare expression in exposed and unexposed patients with advanced fibrosis in order to assess antagonism or potentiation by alcohol exposure. Permutation analysis was also performed to confirm the findings. RESULTS: In the 39 non-drinking (unexposed) patients, 585 genes were found to be differentially expressed in patients with advanced fibrosis vs. early fibrosis, identifying a group of fibrosis-associated genes. In the 13 alcohol-exposed patients with advanced fibrosis, expression of only one of these genes (COL16A1) was potentiated, whereas the fibrosis-associated pattern of 338 other genes (including genes associated with extracellular matrix (COL1A2, FBN1); PDGF signaling (STAT1); oxidoreductases (ALDH1A3, KDM5A); integrin cell surface interactions (LAMA2); and vascular development (CXCL12,VEGFC, TGFB2) was antagonized. Histology demonstrated that the alcohol-exposed patients were more likely to have early fibrosis than unexposed patients (62.5% vs. 51.2%; p=.002). They were also younger (47±2 vs. 53±2 years; p=0.015) and more likely to be male (51.5% vs. 20.5%; p=.002). Race, BMI, diabetes mellitus, hypertension, and triglyceride levels did not differ significantly by exposure. CONCLUSIONS: In this cohort of NAFLD patients, alcohol exposure was associated with less advanced fibrosis and generally attenuated the changes in liver gene expression that accompanied fibrosis progression in non-drinkers.