Multimodal analysis of dysregulated heme metabolism, hypoxic signaling, and stress erythropoiesis in Down syndrome

Abstract

Down syndrome (DS), the genetic condition caused by trisomy 21 (T21), is characterized by delayed neurodevelopment, accelerated aging, and increased risk of many co-occurring conditions. Hypoxemia and dysregulated hematopoiesis have been documented in DS, but the underlying mechanisms and clinical consequences remain ill defined. We report an integrative multi-omic analysis of ∼400 research participants showing that people with DS display transcriptomic signatures indicative of elevated heme metabolism and increased hypoxic signaling across the lifespan, along with chronic overproduction of erythropoietin, elevated biomarkers of tissue-specific hypoxia, and hallmarks of stress erythropoiesis. Elevated heme metabolism, transcriptional signatures of hypoxia, and stress erythropoiesis are conserved in a mouse model of DS and associated with overexpression of select triplicated genes. These alterations are independent of the hyperactive interferon signaling characteristic of DS. These results reveal lifelong dysregulation of key oxygen-related processes that could contribute to the developmental and clinical hallmarks of DS.

Keywords: CP: Developmental biology; CP: Metabolism; erythropoietin; heme; hypoxemia; hypoxia; macrocytosis; red blood cells; stress erythropoiesis.

Figure 1.. Transcriptomic signatures of dysregulated heme metabolism and hypoxic signaling in Down syndrome

(A and B) Heatmaps showing (A) normalized enrichment scores (NES) from GSEA and (B) Z scores from upstream regulator IPA for whole-blood transcriptome changes in T21 (n = 304) relative to D21 (n = 96). Asterisks represent significance (q < 0.1) after Benjamini-Hochberg adjustment for (A) and absolute Z score ≥2 for (B). (C) Volcano plots summarizing whole-blood transcriptome changes in T21 relative to D21, highlighting chromosome 21 (chr21, left), heme metabolism (middle), and HIF1A (right) genes. (D) Overlap between chr21 (n = 179), heme metabolism (n = 107), and HIF1A (n = 76) genes upregulated in T21 (fold change [FC] > 1, q < 0.1). (E) Heatmap displaying expression (log₂FC) of genes in both the heme metabolism and HIF1A signatures in T21 relative to D21. Asterisks denote significance (q < 0.1) after Benjamini-Hochberg adjustment of p values. (F) Sina plots displaying mRNA levels (log₂RPKM) for GATA1 and SLC2A1 in T21 and D21. Benjamini-Hochberg adjusted p values (q-values) are indicated. (G) Heatmap displaying expression (log₂FC) of top 10 differentially expressed genes (DEGs) in the heme metabolism and HIF1A signatures in T21 relative to D21. Asterisks denote significance (q-value < 0.1) after Benjamini-Hochberg adjustment of p values. (H and I) Sina plots displaying mRNA levels (log₂RPKM) for (H) heme metabolism and (I) HIF1A genes in T21 relative to D21. Benjamini-Hochberg adjusted p values (q-values) are indicated. (J) Sina plots showing Heme (left) and HIF1A (right) scores in T21 relative to D21. p values from linear regressions are denoted. (K) Scatterplot depicting relationship between Heme and HIF1A scores in T21. Spearman rho and p value are denoted. Points are colored by density; blue lines represent linear fit, with 95% confidence intervals in gray. (L) Sina plots showing mRNA levels (median Z scores) of genes in Heme (left) and HIF1A (right) signatures in white blood cells (WBCs) and whole blood in a subgroup of participants (n = 12), with p values denoted. (M) Scatterplot depicting expression (median Z scores) of genes from the Heme score in WBCs compared to whole blood. For sina plots, boxes represent interquartile ranges and medians, with notches approximating 95% confidence intervals.

Figure 2.. Elevated heme metabolism and HIF1A signaling are associated with multi-omic markers of stress erythropoiesis in trisomy 21

(A and B) Volcano plots depicting Spearman correlations for (A) Heme and (B) HIF1A scores against plasma proteins (n = 4,601) in T21 (n = 304, one technical replicate). Significant correlations (q < 0.1) are colored red. (C) Scatterplot comparing correlations against plasma proteins for the Heme score (x axis) and HIF1A score (y axis). Spearman rho and p value are denoted. (D and E) Sina plots (left) displaying plasma levels (relative abundance) of TFRC (D) and EPO (E) in T21 (n = 316) and euploid controls (D21, n = 103); Benjamini-Hochberg adjusted p values (q-values) are denoted. Scatterplots (right) displaying relationship with Heme and HIF1A scores in T21; Spearman rho and Benjamini-Hochberg adjusted p values (q-values) are denoted. (F) Volcano plot depicting Spearman correlations for EPO against plasma proteins. Significant correlations (q < 0.1) are colored red. (G) Scatterplots comparing relationship between plasma levels (relative abundance) of EPO (x axis) and TFRC and GDF15 (y axis) in T21, with Spearman rho and Benjamini-Hochberg adjusted p values (q-values) denoted. (H) Sina plot showing plasma levels (relative abundance) of GDF15 in T21 and D21, with Benjamini-Hochberg adjusted p values (q-values) denoted. (I) Sina plots displaying whole-blood mRNA levels of HBG1, HBG2, and BCL11A in T21 (n = 304) and D21 (n = 96), with Benjamini-Hochberg adjusted p values (q-values) denoted. (J) Heatmap comparing levels (median Z score) of complete blood count (CBC) parameters in T21 (n = 147, one technical replicate) and D21 (n = 171). Asterisks denote significance (q-value < 0.1) after Benjamini-Hochberg adjustment of p values. (K) Sina plots showing indicated CBC parameters in T21 and D21, with units and Benjamini-Hochberg adjusted p values (q-values) denoted. For scatterplots, points are colored by density; blue lines represent linear fit, with 95% confidence intervals in gray. Spearman rho and p value comparing sets of correlations are denoted. For sina plots, boxes represent interquartile ranges and medians, with notches approximating 95% confidence intervals.

Figure 3.. Hypoxic signaling associates with immune remodeling in trisomy 21

(A) Overview of major immune cell lineages with heatmaps showing (left) effect of T21 and (right) association with HIF1A score. Columns on the left depict differential abundance comparison between individuals with T21 (n = 284, one technical replicate) and euploid controls (D21, n = 90, one technical replicate), shown as log₂(fold change) from beta regression analysis. Asterisks denote significance (q-value < 0.1) after Benjamini-Hochberg adjustment of p values. Columns on the right depict association with HIF1A score, shown as log₂(fold change) associated with each unit increase in HIF1A score. Asterisks denote significance (q-value < 0.1) after Benjamini-Hochberg adjustment of p values. (B–J) Sina plots (left) displaying levels (% of indicated cell population) of specified immune cell lineages in T21 (n = 284) and D21 (n = 90), and scatterplots (right) displaying relationship with HIF1A scores in T21. Benjamini-Hochberg adjusted p values (q-values) from beta regressions are denoted. For sina plots, boxes represent interquartile ranges and medians, with notches approximating 95% confidence intervals. For scatterplots, points are colored by density; blue lines represent linear fit, with 95% confidence intervals in gray.

Figure 4.. A mouse model of Down syndrome displays dysregulated heme metabolism, hypoxia across multiple tissues, and stress erythropoiesis

(A) Heatmap displaying differential abundance (log₂ fold change) of complete blood count (CBC) parameters in Dp10 (n = 9, one technical replicate), Dp16 (n = 6, one technical replicate), and Dp17 (n = 6, one technical replicate) mice relative to wild-type (WT, equal sample sizes) mice. Asterisks denote significance (q-value < 0.1) after Benjamini-Hochberg adjustment of p values. (B) Sina plots showing transformed (log₂) CBC parameters in Dp16 and WT mice, with units and Benjamini-Hochberg adjusted p values (q-values) denoted. (C and D) GSEA of transcriptome data comparing Dp16 to WT mice, depicting normalized enrichment scores (NES) and transformed (−log₁₀) q-values for the hallmark heme metabolism (D) and hypoxia (E) signatures across tissues. Vertical dashed line indicates significance (q < 0.1) threshold. (E) Upstream regulator IPA of transcriptome data comparing Dp16 to WT mice, depicting activation Z scores. Asterisks indicate absolute Z score ≥2. (F–H) Sina plots showing mRNA levels (log₂RPKM) of select genes from the (F) hallmark heme metabolism signature in the spleen, (G) hallmark hypoxia signature in the adult heart, and (H) IPA HIF1A signature in embryonic day 18.5 (E18.5) hearts of Dp16 and WT mice, with Benjamini-Hochberg adjusted p values (q-values) denoted. (I) Sina plots showing mRNA levels (log₂RPKM) of Epo in the kidneys of Dp16 and WT mice, with Benjamini-Hochberg adjusted p values (q-values) denoted. (J) Overlap of differentially expressed genes (DEGs) between specified gene signatures in whole blood from individuals with T21 and tissues showing the highest enrichment of each signature in Dp16 mice. For sina plots, boxes represent interquartile ranges and medians, with notches approximating 95% confidence intervals.

Figure 5.. Heme metabolism and HIF1A signatures associate with expression of distinct chromosome 21 genes but not with interferon hyperactivity

(A and B) Volcano plots depicting Spearman correlations for (A) Heme and (B) HIF1A scores against whole-blood mRNA levels of chromosome 21 (chr21) genes in T21 (n = 304, one technical replicate). Significant correlations (q < 0.1) are colored red. (C) Scatterplot comparing correlations against plasma proteins for the Heme score (x axis) and HIF1A score (y axis). Spearman rho and p value are denoted. (D) Heatmaps depicting for chr21 genes: (left) differential expression in T21 (n = 304) vs. D21 (n = 96), (middle) correlations with Heme and HIF1A scores, and (right) which mouse models harbor triplicated copies. Asterisks indicate significance (q-value < 0.1) after Benjamini-Hochberg adjustment of p values. (E and F) Scatterplots displaying relationship between mRNA levels (log₂RPKM) of chr21 genes and the Heme (E) and HIF1A (F) scores in individuals with T21 (n = 304). Spearman rho and Benjamini-Hochberg adjusted p values (q-values) are denoted. (G) Sina plot displaying expression changes while a research participant was taking a JAK inhibitor (JAKi) for interferon-stimulated genes (ISGs), hallmark heme metabolism, hallmark hypoxia, and HIF1A score signatures. Red points indicate significance (q-value < 0.1) after Benjamini-Hochberg adjustment of p values. One technical replicate was used for sequencing. (H) Sina plots displaying expression changes of (left) hallmark heme metabolism genes in the spleen, (middle) hallmark hypoxia genes in the adult heart, and (right) HIF1A genes in E18.5 hearts when comparing Dp16 (n = 6) to wild-type (WT, n = 6) mice (blue) and Dp16 mice with normalized interferon receptor copy number (Dp16^2xIfnrs, n = 6) to Dp16 (n = 6) (yellow). (I and J) Sina plots displaying mRNA levels (log₂RPKM) of select genes from spleen heme metabolism signature (I) and adult heart hypoxia signature (J) in WT, Dp16, and Dp16^2xIfnrs mice, with Benjamini-Hochberg adjusted p values (q-values) denoted. (K) Scatterplot depicting mRNA levels (median Z scores) for chr21 in white blood cells (WBCs) compared to whole blood. For scatterplots, points are colored by density; blue lines represent linear fit, with 95% confidence intervals in gray. For sina plots, boxes represent interquartile ranges and medians, with notches approximating 95% confidence intervals.