Altered Hepatic Metabolism in Down Syndrome

Abstract

Trisomy 21 (T21) gives rise to Down syndrome (DS), the most commonly occurring chromosomal abnormality in humans. T21 affects nearly every organ and tissue system in the body, predisposing individuals with DS to congenital heart defects, autoimmunity, and Alzheimer's disease, among other co-occurring conditions. Here, using multi-omic analysis of plasma from more than 400 people, we report broad metabolic changes in the population with DS typified by increased bile acid levels and protein signatures of liver dysfunction. In a mouse model of DS, we demonstrate conservation of perturbed bile acid metabolism accompanied by liver pathology. Bulk RNA-sequencing revealed widespread impacts of the Dp16 model on hepatic metabolism and inflammation, while single-cell transcriptomics highlighted cell types associated with these observations. Modulation of dietary fat profoundly impacted gene expression, bile acids, and liver pathology. Overall, these data represent evidence for altered hepatic metabolism in DS that could be modulated by diet.

Figure 1.. Altered bile acid metabolism in Down syndrome.

(A) Volcano plot summarizing results of plasma metabolomic analysis of individuals with DS (n=316) versus disomic individuals (n=103). Significantly differentially abundant (q<0.1) metabolites are colored in red. (B) Waterfall plot summarizing metabolites with significant differences in relative abundance in plasma of individuals with T21 versus D21 controls (q<0.1). Bars are color-coded by increased relative abundance in DS (blue) versus decreased in DS (red). (C) Heatmap displaying log₂(FoldChange) of plasma bile acids in those with T21 relative to D21 individuals. Color coding indicates a positive (red) or a negative (blue) log₂(FoldChange) value. Asterisks denote significance (q<0.1). (D) Sina plot displaying log₂(relative abundance) of taurolithocholic acid in the plasma of D21 individuals and those with DS. Boxes represent interquartile ranges and medians, with notches approximating 95% confidence intervals. Benjamini-Hochberg adjusted p-values (q-values) are indicated. (E) Scatterplot displaying the relationship between relative abundance of taurolithocholic acid and age in years both for D21 individuals and those with DS. Spearman rho values and p-values are shown. (F) Volcano plot summarizing results of plasma metabolomic analysis of Dp16 mice (n=13) compared to wildtype (n=9). Significantly differentially abundant (q<0.1) metabolites are colored in red. (G) Waterfall plot summarizing metabolites with significant differences in relative abundance in plasma from Dp16 mice versus wildtype. (H) Heatmap displaying log₂(FoldChange) of plasma bile acids in Dp16 mice relative to wildtype. Color coding indicates a positive (red) or a negative (blue) log₂(FoldChange) value. Asterisks denote significance (q<0.1). (I) Sina plots displaying log₂(relative abundance) of taurocholic acid and tauroursodeoxycholic acid plasma metabolites in wildtype and Dp16 mice. Benjamini-Hochberg adjusted p-values (q-values) are indicated.

Figure 2.. Evidence for widespread liver dysfunction in Down syndrome.

(A) Bar plot summarizing Ingenuity Pathway Analysis of plasma SOMAscan proteomics from individuals with DS (n=316) versus D21 individuals (n=103). Dotted line indicates the significance threshold of p < 0.05. (B) Sina plots displaying the relative abundance of various plasma proteins in disomic individuals and those with DS. Benjamini-Hochberg adjusted p-values (q-values) are indicated. (C) Enrichment plots showing enrichment scores for Gene Set Enrichment Analysis signatures of Fibrosis and Steatosis in plasma proteomics of individuals with DS. NES – Normalized Enrichment Score (D) Sina plots displaying the relative abundance of various plasma proteins in D21 individuals and those with DS. Benjamini-Hochberg adjusted p-values (q-values) are indicated. (E) Sina plots showing the concentration of various biomarkers of liver function in wildtype (n=8–11, 3–6 females) and Dp16 (n=9–11, 3–6 females) plasma. Individual data are presented with a bar at the median and p-values, as determined by Mann-Whitney U test, are shown. (F) Representative images of H&E-stained liver sections from wildtype and Dp16 mice. Arrows indicate the following features: red, enlarged periportal sinusoids; blue, bile duct; yellow, hepatic artery branches; green, periportal inflammatory cells. PV indicates portal vein and CV indicates central vein. Scale bar is 100 microns. (G) Sina plots displaying metrics of liver pathology scoring from adult wildtype (n=12, 6 females) and Dp16 (n=11, 5 females) mice. Individual data are presented with a bar at the median and p-values, as determined by Mann-Whitney U test, are shown. Boxes in Sina plots represent interquartile ranges and medians, with notches approximating 95% confidence intervals.

Figure 3.. Stellate cell dysfunction drives liver fibrosis in the Dp16 model.

(A) Volcano plot summarizing results of whole liver transcriptome analysis of WT (n=6, 3 females) versus Dp16 (n=6, 3 females) mice. Significantly differentially expressed (q<0.1) genes are colored in red. (B) Barplot summarizing the results of Gene Set Enrichment Analysis (GSEA) of gene expression changes in the liver of Dp16 mice (n=6, 3 females) versus wildtype (n=6, 3 females). (C) Heatmap showing the median z-score of the top 10 leading-edge genes from the Epithelial-Mesenchymal Transition gene set. Color coding indicates a positive (red) or negative (blue) median z-score. (D) Sina plots displaying the normalized relative expression (RPKM) of Col1a1 and Eln in both WT and Dp16 mice. Benjamini-Hochberg adjusted p-values (q-values) are indicated. Individual data are presented with a bar at the median. (E) Uniform Manifold Approximation and Projection (UMAP) plot of single cell RNA sequencing analysis of mouse liver, color coded by 16 major cell clusters, identified using Seurat. (F) UMAP plot displaying differential cellular abundance (beta regression Dp16 versus WT) of clusters from single cell RNA sequencing analysis of mouse liver. Significant (FDR < 0.1) clusters are colored according to mean fold-change. (G) Sina plots showing the Dp16 versus WT abundance of Hepatocyte Cluster 1 and Endothelial Cell Cluster 2 from single cell RNA sequencing analysis of mouse liver. Benjamini-Hochberg adjusted p-values (q-values) are indicated. Individual data are presented with a bar at the median. (H) UMAP plot displaying the Epithelial-Mesenchymal Transition gene set Normalized Enrichment Score (NES) for each cluster. (I) Bubble plot summarizing Col1a1 single-cell expression across clusters and animals, with color representing mean expression and size representing percent of cells with expression. (J) Sina plot displaying the normalized pseudobulk counts of Col1a1 expression within the Stellate Cell cluster in both WT and Dp16 animals. Benjamini-Hochberg adjusted p-values (q-values) are indicated. (K) Representative images of multiplexed stained liver sections from wildtype and Dp16 mice where DAPI is stained in blue, desmin in red, and smooth muscle actin (SMA) in yellow. Sina plot shows number of desmin+, SMA+ cells as a percent of total cells for wildtype mice (n=6, 3 females) and Dp16 mice (n=6, 3 females). Individual data are presented with a bar at the median and the p-value, as determined by a Mann-Whitney U test, is shown. Scale bars are 100 microns. (L) Representative images of picrosirius red stained (PSR) liver sections from wildtype (n=8, 4 females) and Dp16 (n=7, 4 females) mice, imaged under polarized light. Sina plot showing the percent positive PSR in wildtype and Dp16 mice. Individual data are presented with a bar at the median and the p-value, as determined by a Mann-Whitney U test, is shown. Scale bars are 100 microns.

Figure 4.. Increased copy number of the interferon receptor locus is not a major contributor to Dp16 liver dysfunction.

(A) Heatmap showing the median z-score of the leading-edge genes from the Interferon Gamma Response gene set from WT and Dp16 liver bulk transcriptome analysis. (B) Sina plot displaying relative expression (RPKM) of Cxcl9 in both wildtype and Dp16 liver bulk transcriptome analysis. Benjamini-Hochberg adjusted p-values (q-values) are indicated. (C) UMAP plot displaying the Interferon Gamma Response gene set normalized enrichment score (NES) for each cluster. (D) Bubble plot displaying the mean expression and the percent of cells per cluster expressing Cxcl9 in both wildtype and Dp16 mice. (E) Sina plot displaying the log₂ of the normalized counts of Cxcl9 expression within Endothelial Cell cluster 1 in both wildtype and Dp16 animals. Benjamini-Hochberg adjusted p-values (q-values) are indicated. Individual data are presented with a bar at the median. (F) Representative images of H&E-stained liver sections from adult wildtype, Dp16, and Dp16^2xIFNRs mice. Scale bar is 100 microns. (G) Sina plots showing the metrics of liver pathology scoring from adult wildtype (n=13, 6 females), Dp16 (n=12, 5 females), and Dp16^2xIFNRs (n=13, 7 females) mice. Individual data are presented with a bar at the median and p-values, as determined by a Mann-Whitney U test, are shown. (H) Sina plot displaying the grams/deciliter of plasma Albumin from adult wildtype (n=6, 4 females), Dp16 (n=6, 2 females), and Dp16^2xIFNRs (n=8, 1 female) mice. Individual data are presented with a bar at the median and p-values, as determined by a Mann-Whitney U test, are shown. (I) Heatmap displaying the median z-score of various plasma bile acids in adult wildtype (n=9, 4 females), Dp16 (n=13, 6 females), and Dp16^2xIFNRs (n=10, 5 females) mice.

Figure 5.. Liver dysfunction in Down syndrome is driven by hepatocyte dysfunction.

(A) Heatmaps showing the median z-score of the leading-edge genes from the Cholesterol Homeostasis, Adipogenesis, Bile Acid Metabolism, and Fatty Acid Metabolism GSEA signatures in whole liver tissue. Color coding indicates a positive (red) or negative (blue) median z-score. (B) Sina plots displaying the log₂(RPKM) of various genes. Benjamini-Hochberg adjusted p-values (q-values) are indicated. (C) UMAP plot displaying the Cholesterol Homeostasis GSEA signature enrichment in all clusters. Color coding indicates a positive (red) or negative (blue) normalized enrichment score. (D) Bubble plot displaying the mean expression and the percent of cells per cluster expressing Fasn in both wildtype and Dp16 mice. (E) Sina plot displaying the log₂ of the normalized counts of Fasn expression within hepatocyte cluster 2 in both wildtype and Dp16 animals. The q-value indicates linear regression significance after Benjamini-Hochberg correction for multiple testing. (F) UMAP plot displaying the Bile Acid Metabolism gene set normalized enrichment score (NES) in each cluster. Color coding indicates a positive (red) or negative (blue) NES. (G) Bubble plot displaying the mean expression and the percent of cells per cluster expressing Agxt in both wildtype and Dp16 mice. (H) Sina plot displaying the log₂ of the normalized counts of Agxt expression within hepatocyte cluster 2 in both wildtype and Dp16 animals. The q-value indicates linear regression significance after Benjamini-Hochberg correction for multiple testing. (I) Schematic illustrating the genes measured at various stages in the iPSC derived hepatocyte differentiation protocol, as well as microscopy images of cells in each stage. Scale bars are 50 microns. (J) Sina plots displaying the gene expression of Alb and Cyp3a4 relative to disomic iPSCs. Values from disomic cells are shown in gray and from trisomic cells are shown in blue. Individual data are presented with a bar at the median and p-values, as determined by a Mann-Whitney U test, are shown. (K) Representative images of Albumin (green) and DAPI (blue) immunofluorescent staining of disomic and trisomic iPSC derived hepatocytes. (L) Representative images of Oil Red O staining of disomic and trisomic iPSC derived hepatocytes. Scale bars are 50 microns. (M) Heatmap displaying the enrichment of various GSEA gene sets in whole mouse liver, hepatocytes, and two single cell hepatocyte clusters. Asterisks indicate significance after Benjamini-Hochberg correction for multiple testing (q-value< 0.1). (N) Heatmaps showing the median z-score of the leading-edge genes from the Bile Acid Metabolism GSEA pathway in the iHepatocytes. (O) Sina plots displaying the log₂(RPKM) of CYP27A1. Benjamini-Hochberg adjusted p-values (q-values) are indicated. (P) Heatmap displaying the log₂(fold change) of bile acids detected in the supernatant of trisomic versus disomic iPSC derived hepatocytes. Color coding indicates a positive (red) or negative (blue) log₂(fold change). (Q) Sina plot displaying the log₂(relative abundance) of Taurolithocholic acid detected in the supernatant of disomic and trisomic iPSC derived hepatocytes. Benjamini-Hochberg adjusted p-values (q-values) are indicated. P-value was determined by a Mann-Whitney U test.

Figure 6.. Liver pathology in Dp16 mice is dependent on diet.

(A) Representative images of H&E-stained liver sections from wildtype and Dp16 mice fed either a LFD or HFD. Scale bar is 100 microns. (B) Sina plots showing the metrics of liver pathology scoring from WT and Dp16 mice fed either a LFD or HFD (WT LFD n=10, 4 females, Dp16 LFD n=8, 4 females, WT HFD n=14, 4 females, Dp16 HFD n=13, 4 females). Individual data are presented with a bar at the median and p-values, as determined by a Mann-Whitney U test, are shown. (C) Volcano plot summarizing results of plasma metabolomic analysis of Dp16 LFD mice compared to wildtype LFD mice (WT LFD n=10, 4 females, Dp16 LFD n=8, 4 females). Significantly differentially expressed (q<0.1) genes are colored in red. (D) Volcano plot summarizing results of plasma metabolomic analysis of Dp16 HFD mice compared to wildtype HFD mice (WT HFD n=13, 4 females, Dp16 HFD n=13, 4 females). Significantly differentially expressed (q<0.1) genes are colored in red. Benjamini-Hochberg adjusted p-values (q-values) are indicated. (E) Scatterplot comparing fold-changes of plasma metabolites from Dp16 LFD mice compared to WT LFD mice (x axis) versus Dp16 HFD mice compared to WT HFD mice (y axis). Points are colored according to significance in both groups (red), significance in the LFD group only (green), significance in the HFD group only (yellow), or not significant in any group (gray). Significance is defined as q < 0.1 (10% FDR). (F) Sina plots displaying the relative abundance of indicated plasma bile acids. Benjamini-Hochberg adjusted p-values (q-values) are indicated.

Figure 7.. A high-fat diet contributes to global transcriptomic changes in the Dp16 liver.

(A) Volcano plots summarizing the results of transcriptome analysis of livers from Dp16 mice fed a low-fat diet (LFD) compared to wildtype mice fed a LFD and from Dp16 mice fed a high-fat diet (HFD) compared to wildtype mice fed a HFD. (wildtype LFD n=10, 4 females, Dp16 LFD n=8, 4 females, wildtype HFD n=14, 4 females, Dp16 HFD n=13, 4 females). Points in blue are in the MMU16 region triplicated in Dp16 mice, points in gray have reached a linear regression significance threshold of less than 0.1 after Benjamini-Hochberg correction for multiple testing, and points in black are not significant. (B) Venn diagrams displaying the overlap in the number of significant differentially expressed genes in the Dp16 triplicated region between HFD fed mice and LFD mice (top) or in the differentially expressed genes that are not triplicated in Dp16 mice (bottom). (C) Scatter plot comparing GSEA NES values for signatures in the plasma of Dp16 versus WT mice on LFD (x axis) and Dp16 versus WT mice on HFD diet (y axis). Points are colored according to significant in both groups (red), significant in the LFD group only (green), significant in the HFD group only (yellow), or not significant in either comparison (gray). Significance is defined as q < 0.1. (D) Scatter plots comparing the fold-changes of leading-edge genes from the cholesterol homeostasis, fatty acid metabolism, and adipogenesis Hallmark gene sets for Dp16 versus WT mice on LFD (x axis) and Dp16 versus WT on HFD (y axis). Points are colored according to significant in both groups (red), significant in the LFD group only (green), significant in the HFD group only (yellow), or not significant in any group (gray). Significance is defined as q < 0.1. (E) Sina plots displaying the log₂(RPKM) of various genes. Horizontal bars indicate median values. Benjamini-Hochberg adjusted p-values (q-values) are indicated.