Extended Data Fig. 7: Perturbed haematopoiesis in Down syndrome.

7a) UMAP of Down syndrome FBM scRNA-seq (n=4, k=16,743, 12-13 PCW) (Supplementary Table 20). Abbreviations as in Fig. 1a. 7b) Top 30 PySCENIC-inferred differentially active TFs in Down syndrome vs. non-Down syndrome FBM scRNA-seq HSC/MPPs, MEMPs and MKs (Supplementary Tables 7, 20). TFs (red) described in text. 7c) Proportions of erythroid lineage cell states in Down syndrome (n=4) and age-matched non-Down syndrome FBM scRNA-seq data (n=2, where n=biologically independent samples). **** p < 10⁻¹⁵, 2-sided, by chi-squared test. Abbreviations: eryth = erythroid. 7d) Heat map showing cell-cycle gene enrichment in Down syndrome and age-matched non-Down syndrome FBM erythroid lineage cell states. Colour indicates relative enrichment. 7e) Representative images from single-cell HSC/MPP methylcellulose cultures, showing relative erythrocyte colony size/structure in Down syndrome (top; n=2 biological independent samples; PCW=17, 19; k=246) and non-Down syndrome (bottom; n=3 biologically independent samples; PCW=17, 19, 21; k=365) FBM; scale bar; 400µm. 7f) Heat map of erythropoiesis-implicated genes across Monocle 3-inferred erythroid development pseudotime in Down syndrome and non-Down syndrome FBM scRNA-seq datasets (all genes shown are DEGs across both pseudotime trajectories; one-sided Moran’s I statistical test; Supplementary Tables 32, 41, 42). Log-transformed, normalized and scaled GEX (upper limit of 3). 7g) Dotplot showing chromosome 21 TFs differentially expressed in Down syndrome and non-Down syndrome FBM scRNA-seq datasets (two-sided Wilcoxon rank-sum statistical test with Benjamini-Hochberg procedure for multiple testing correction; adjusted-p-value=<0.05; Supplementary Table 21). Dot-size = average log2 fold-change in Down syndrome expression. Colour = log(log(adjusted-p-value)). 7h) Dotplot showing TFs for differentially active regulons in Down syndrome vs. non-Down syndrome FBM scRNA-seq (see panel b). 7i) Top: Heat map showing TNF expression in Down syndrome and non-Down syndrome FBM scRNA-seq cell states. Bottom: TNFα-signalling pathway enrichment (see Methods, Supplementary Table 43). Dot-size = normalized enrichment score (NES) for TNFα-signalling pathway. Line = ±log₁₀(0.25). 7j) Sankey-plot of putative TNF superfamily interactions in Down syndrome FBM scRNA-seq (see Methods; Supplementary Table 44). Fold-change expression in Down syndrome relative to non-Down syndrome (red scale). Combined expression in Down syndrome/non-Down syndrome (blue scale).

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