Table 1.
Clinical, anthropometrical and biochemical characteristics of study participants divided into three groups with increasing NAS.
Fig 1.
Overview of the concept for identifying potential, circulating biomarkers of NAFLD.
Liver secretome gene signatures were generated from DEGs between individuals with no/mild NAFLD and moderate to advanced NAFLD with subsequent filtering for candidate gene products with liver-selective expression and secretion. Abbreviations: DEGs, differentially expressed genes; NAFLD, non-alcoholic fatty liver disease; NAS, NAFLD activity score. Created with BioRender.com.
Table 2.
Frequency table of NAS, fibrosis grade and NASH of study participants divided into three groups with increasing NAS.
Fig 2.
Patients with moderate and more advanced NAFLD cluster together separating from patients with no or mild NAFLD.
(A) PCA plot of samples based on 500 most variable genes. Points indicate the relationship between liver biopsy samples (patients) across their gene expression profile. Color represent group defined by NAS score. (B) Venn diagram of DEGs compared to individuals with NAS 0–1 (false discovery rate, p <0.05). Circles that overlap share DEGs. Abbreviations: DEGs, differentially expressed genes; NAFLD; non-alcoholic fatty liver disease; NAS, NAFLD activity score; PCA, principal component analysis.
Fig 3.
Progression of NAFLD is accompanied by a change in immune system and extracellular matrix organization pathways.
(A) Overview of differentially regulated Reactome pathways (log10 of false discover rate) in individuals with NAS 2–3 and NAS 4–6 as compared to individuals with no/mild NAFLD (NAS 0–1). (B, C) Volcano plots illustrating DEGs in selected Reactome pathways, i.e., immune system and extracellular matrix organization Reactome pathways compared to individuals with no/mild NAFLD (NAS 0–1). Significantly regulated genes (p <0.05) are indicated in red (downregulation) and green (upregulation), respectively. Abbreviations: DEGs, differentially expressed genes; NAFLD; non-alcoholic fatty liver disease; NAS, NAFLD activity score.
Fig 4.
Selection of candidate genes with biomarker potential.
(A) Evaluation of gene expression by coefficient of variation across all tissues (y-axis) versus liver tissue expression levels (x-axis, TPM). A high coefficient of variation with respect to tissue selectivity in combination with high liver expression (towards the top right corner) suggests liver-selective expression. (B) Coefficient of variation versus liver tissue expression levels for all identified genes (grey dots) including all DEGs (green dots) as well as for albumin. (C) Coefficient of variation versus liver tissue expression levels for all identified genes (grey dots) including all DEGs (green dots) that were predicted as secreted. Highlighted genes (green boxes), LPA (lipoprotein A), IGFBP-1 (insulin-like growth factor-binding protein 1), SERPINF2 (serpin family F member 2) and MAT1A (methionine adenosyltransferase 1A), were selected and evaluated as gene biomarker candidates of NAFLD. Abbreviations: ALB, albumin; DEGs, differentially expressed genes; NAFLD; non-alcoholic fatty liver disease; TPM, transcript per million.
Fig 5.
Hepatic candidate gene expression levels compared to plasma concentrations of the corresponding gene product.
Left panels, gene expression levels. Right panels, plasma concentrations of the corresponding gene product. (A) Insulin-like growth factor-binding protein 1 (IGFBP-1). (B) Methionine adenosyltransferase 1A (MAT1A) and enzyme catalytic product (S-adenosylmethionine (SAM)). (C) Lipoprotein A (LPA). (D) Serpin family F member 2 (SERPINF2) and gene product alpha-2 antiplasmin (α2AP). * p <0.05, ** p <0.01 vs. NAS 0–1.