Fig. 2: Transcriptome-wide differential expression and developmental expression bias in T21 fetal brain.

Differential expression between T21 (n = 20) and controls (n = 27) was tested separately in (A) PFC and (B) hippocampus using a covariate-adjusted linear model (gestational age, sex, RIN, % mRNA reads, and estimated neuronal proportion) with empirical Bayes moderation (two-sided). p Values were adjusted using the Benjamini–Hochberg false discovery rate (FDR); effect sizes are log₂ fold-changes. Each gene was plotted by log₂ fold-change (x-axis) and −log₁₀ adjusted p-value (y-axis); points are colored to indicate upregulated, downregulated, and chromosome 21-encoded genes. Brain diagrams were constructed in the cerebroViz R package. C Venn diagrams display the overlap of all differentially expressed genes (DEGs) (left) and DEGs located on chromosome 21 (right) between PFC and hippocampus. D Genome-wide concordance of expression changes between regions is shown by plotting log₂ fold-changes in PFC (x-axis) against hippocampus (y-axis); Spearman correlation coefficient (ρ) quantifies regional concordance. E Barplots show the number of DEGs (FDR < 5%) per chromosome in PFC (top) and hippocampus (bottom), separated by direction of change. F Dot plots display expression changes for all genes on chromosome 21, arranged by genomic position (x-axis) for PFC (top) and hippocampus (bottom); colors reflect FDR significance thresholds (FDR < 5%, pink; 5% <FDR < 10%, light blue; FDR > 10%, dark blue) applied to the covariate-adjusted, empirical Bayes–moderated linear model (two-sided). G For each gene, a BrainSpan-derived t-statistic summarizing prenatal versus postnatal expression bias was computed using data from matching anatomical regions (PFC and hippocampus; “Methods”). Distributions of these developmental bias scores for T21-upregulated versus T21-downregulated genes were compared using a two-sided Mann–Whitney U test. In the PFC (top) and hippocampus (bottom), a prenatal bias was observed among under-expressed genes (p = 0.02 and p = 2.3 × 10⁻⁵) and a postnatal bias among over-expressed genes (p = 0.01 and p = 7.6 × 10⁻⁷). H Normalized expression trajectories of T21-upregulated and downregulated genes were plotted across ten developmental stages in the BrainSpan bulk RNA-seq using matching PFC (top) and hippocampus (bottom) tissues, illustrating typical temporal shifts in gene expression trajectories.