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.

Keywords: CP: Metabolism; CP: Stem cell research; Down syndrome; bile acids; liver; metabolism.

Figure 1.. Altered bile acid metabolism in Down syndrome

(A) Volcano plot summarizing results of plasma metabolomic analysis of individuals with T21 (n = 316) versus controls (n = 103). Significantly differentially abundant (q < 0.1) metabolites are red. (B) Waterfall plot summarizing plasma metabolites with significant differences in individuals with T21 versus controls (q < 0.1). (C) Heatmap displaying log₂(foldchange) of plasma bile acids in those with T21 relative to controls. Asterisks denote significance (q < 0.1). (D) Sina plot displaying log₂(relative abundance) of taurolithocholic acid in the plasma of D21 individuals versus T21. Boxes represent interquartile ranges and medians, with notches approximating 95% confidence intervals. (E) Scatterplot displaying the relationship between relative abundance of taurolithocholic acid and age in years in D21 and T21. Spearman rho values and p values are shown. (F) Volcano plot summarizing results of plasma metabolomic analysis of Dp16 mice (n = 13) versus wild-type (n = 9). Significantly differentially abundant (q < 0.1) metabolites are red. (G) Waterfall plot summarizing plasma metabolites with significant differences between Dp16 and wild-type mice. (H) Heatmap displaying log₂(foldchange) of plasma bile acids in Dp16 mice relative to wild-type. Asterisks denote significance (q < 0.1). (I) Sina plots displaying log₂(relative abundance) of indicated plasma metabolites in wild-type and Dp16 mice. For (D and I), the 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 controls (n = 103). Dashed line indicates the significance threshold of p < 0.05. (B) Sina plots displaying the relative abundance of various plasma proteins in D21 versus T21. (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 plasma proteins in D21 and T21. (E) Sina plots showing the concentration of biomarkers of liver function in wild-type (n = 8–11, 3–6 females) and Dp16 (n = 9–11, 3–6 females) plasma. (F) Representative images of H&E-stained liver sections from wild-type 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 bars, 100 μm. (G) Sina plots displaying metrics of liver pathology scoring from adult wild-type (n = 12, 6 females) and Dp16 (n = 11, 5 females) mice. For (B and D), boxes represent interquartile ranges and medians, with notches approximating 95% confidence intervals. Benjamini-Hochberg adjusted p values (q values) are indicated. For (E and G), individual data are presented with a bar at the median, and p values, as determined by Mann-Whitney U test, are shown.

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

(A) Volcano plot summarizing results of whole liver transcriptome analysis of wild-type (n = 6, 3 females) versus Dp16 (n = 6, 3 females) mice. Significantly differentially expressed (q < 0.1) genes are colored in red. (B) Bar plot summarizing the results of gene set enrichment analysis of gene expression changes data in (A). (C) Heatmap showing the median Z score of the top 10 leading-edge genes from the epithelial-mesenchymal transition gene set. (D) Sina plots displaying the normalized relative expression (RPKM) of Col1a1 and Eln in wild-type and Dp16 mice. (E) Uniform manifold approximation and projection (UMAP) plot of single-cell RNA sequencing (scRNA-seq) analysis of mouse liver, color coded by cell clusters identified using Seurat. (F) UMAP plot displaying differential cellular abundance of clusters from scRNA-seq analysis of mouse liver. Significant (false discovery rate [FDR] <0.1) clusters are colored by mean fold-change. (G) Sina plots showing the Dp16 versus wild-type abundance of hepatocyte cluster 1 and endothelial cell cluster 2 from scRNA-seq analysis of mouse liver. (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 wild-type and Dp16 animals. (K) Representative images of multiplexed stained liver sections from wild-type and Dp16 mice where DAPI is stained in blue, desmin in red, and smooth muscle actin (SMA) in yellow. Sina plot showing number of desmin+ and SMA+ cells as a percentage of total cells in wild-type mice (n = 6, 3 females) and Dp16 mice (n = 6, 3 females). (L) Representative images of picrosirius-red-stained (PSR) liver sections from wild-type (n = 8, 4 females) and Dp16 (n = 7, 4 females) mice, imaged under polarized light. Sina plot shows the total percent positive PSR staining in wild-type and Dp16 liver. For (K and L), 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, 100 μm.

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 IFN gamma response gene set from wild-type and Dp16 liver bulk transcriptome analysis. (B) Sina plot displaying relative expression (RPKM) of Cxcl9 in wild-type and Dp16 liver bulk transcriptome analysis. (C) UMAP plot displaying the IFN 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 wild-type and Dp16 mice. (E) Sina plot displaying the log₂ of the normalized counts of Cxcl9 expression within endothelial cell cluster 1 in wild-type and Dp16 animals. (F) Representative images of H&E-stained liver sections from adult wild-type, Dp16, and Dp16^2xIFNRs mice. Scale bars, 100 μm. (G) Sina plots showing liver pathology scoring from adult wild-type (n = 13, 6 females), Dp16 (n = 12, 5 females), and Dp16^2xIFNRs (n = 13, 7 females) mice. p values, determined by a Mann-Whitney U test, are shown. (H) Sina plot displaying the grams/deciliter of plasma albumin from adult wild-type (n = 6, 4 females), Dp16 (n = 6, 2 females), and Dp16^2xIFNRs (n = 8, 1 female) mice. p values, as determined by a Mann-Whitney U test, are shown. (I) Heatmap displaying the median Z score of plasma bile acids in adult wild-type (n = 9, 4 females), Dp16 (n = 13, 6 females), and Dp16^2xIFNRs (n = 10, 5 females) mice. For (B and E), Benjamini-Hochberg adjusted p value (q value) are indicated. For (B, E, and G), individual data are presented with a bar at the median.

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. (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 gene set NES in each cluster. (D) Bubble plot displaying the mean expression and the percent of cells per cluster expressing Fasn in both wild-type and Dp16 mice. (E) Sina plot displaying the log₂ of the normalized counts of Fasn expression within hepatocyte cluster 2 in wild-type and Dp16 animals. (F) UMAP plot displaying the bile acid metabolism gene set NES in each cluster. (G) Bubble plot displaying the mean expression and the percent of cells per cluster expressing Agxt in both wild-type and Dp16 mice. (H) Sina plot displaying the log₂ of the normalized counts of Agxt expression within hepatocyte cluster 2 in both wild-type and Dp16 animals. (I) Schematic illustrating iPSC-derived hepatocyte differentiation protocol, with microscopy images of cells in each stage. Scale bars, 50 μm. (J) Sina plots displaying the gene expression of Alb and Cyp3a4 relative to disomic iPSCs. Values from control cells are shown in gray and from T21 cells are shown in blue. p values, as determined by a Mann-Whitney U test, are shown. (K) Representative images of albumin (green) and DAPI (blue) immunofluorescent staining of D21 and T21 iPSC-derived hepatocytes. Sina plot displaying the concentration of albumin in the supernatant of iPSC-derived hepatocytes. (L) Sina plot displaying the concentration of albumin in the supernatant of iPSC-derived hepatocytes. (M) Sina plot displaying the concentration of urea in the supernatant of iPSC-derived hepatocytes. (N) 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). (O) Heatmap showing the median Z score of the leading-edge genes from the bile acid metabolism GSEA pathway in the iHepatocytes. (P) Sina plot displaying the log₂(RPKM) of CYP27A1. (Q) 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(foldchange). (R) Sina plot displaying the log₂(relative abundance) of taurolithocholic acid detected in the supernatant of disomic and trisomic iPSC-derived hepatocytes. Nominal and Benjamini-Hochberg adjusted p values (q values) are indicated. For (E, H, and P), q values determined by DEseq2 are shown. For (J–M), p values determined by a Mann-Whitney U test are shown. For (J–R), four separate cell lines were used per genotype, with 2–4 replicates per line.

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

(A) Representative images of H&E-stained liver sections from wild-type and Dp16 mice fed either an LFD or an HFD. Scale bars, 100 μm. (B) Sina plots showing the metrics of liver pathology scoring from wild-type and Dp16 mice fed either an LFD or an HFD (wild-type LFD n = 10, 4 females, Dp16 LFD n = 8, 4 females, wild-type 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 versus wild-type LFD mice (wild-type LFD n = 10, 4 females, Dp16 LFD n = 8, 4 females). (D) Volcano plot summarizing results of plasma metabolomic analysis of Dp16 HFD mice versus wild-type HFD mice (wild-type HFD n = 13, 4 females, Dp16 HFD n = 13, 4 females). Significantly differentially expressed (q < 0.1) genes are red. (E) Scatterplot comparing fold changes of plasma metabolites from Dp16 LFD mice versus wild-type LFD mice (x axis) versus Dp16 HFD mice versus wild-type HFD mice (y axis). Points are colored according to significance in both groups (red), LFD group only (green), HFD group only (yellow), or not significant (gray). (F) Sina plots displaying the relative abundance of indicated plasma bile acids. Individual data are presented with a bar at the median. For (C–F), Benjamini-Hochberg adjusted p values (q values) are indicated, with q > 0.1 significant.

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 versus wild-type mice fed a low-fat diet (LFD) and from Dp16 mice versus wild-type fed a high-fat diet (HFD) (wild-type LFD n = 10, 4 females, Dp16 LFD n = 8, 4 females, wild-type HFD n = 14, 4 females, Dp16 HFD n = 13, 4 females). Points in blue are in the MMU16 region triplicated in Dp16 mice. (B) Venn diagrams displaying the overlap in the 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) Scatterplot comparing GSEA NES values for signatures in the livers of Dp16 versus wild-type mice on LFD (x axis) and Dp16 versus wild-type mice on HFD diet (y axis). (D) Scatterplots comparing the fold changes of leading-edge genes from the cholesterol homeostasis, fatty acid metabolism, and adipogenesis hallmark gene sets for Dp16 versus wild-type mice on LFD (x axis) and Dp16 versus wild-type on HFD (y axis). (E) Sina plots displaying the log₂(RPKM) of various genes. Horizontal bars indicate median values. Benjamini-Hochberg adjusted p-values (q values) are indicated. For (C–E), 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 using GSEA or DESeq2.