Supplementary Figure 7: Aberrant overrepresentation of TM1 genes causes maturational acceleration.

a, Distribution of individual signed gene significance (GS) values for TM1 genes with kME > 0.7 for different developmental stages. Center measure shows mean from n = 620 TM1 genes. GS values were defined as the Student t-test statistic (two-sided) for testing differential expression between time points and conditions. b, Number and percentage of TM1 genes that feature significantly negative correlations (two-sided Student t-test with FDR < 0.05, n = 65 time series samples) at different developmental stages. Note that control NSCs have considerably more suppressed network genes than ASD NSCs (Supplementary Table 9). c, Left: Schematic Venn diagram showing the overlap of genes in TM1 that are significantly negatively correlated with the NSC stage in ASD and control individuals. Right: Distribution of Gene Significance (GS) values of the 189 genes specifically suppressed in control NSCs between conditions. Violin plots show median (center line), IQR (box), 95% confidence interval and the kernel probability density at different values; n = 189 genes. d, Gene-gene connections for the top 30 negatively correlated TM1 genes in control NSCs. Gradual increasing point size of nodes corresponds to GS values in control NSCs (two-sided Student t-test, ASD NSCs: n = 8 samples; control NSCs: n = 5 samples; Supplementary Table 9). e, Comparative gene expression plots of quantitative real time PCR (qRT-PCR) assays and RNA-seq normalized counts for TM1 genes differentially expressed at the NSC stage; qRT-PCR: two-sided Student-t test, FBXO2: ***P = 0.0001, RASD2: ***P = 0.0009, ISLR2: *P = 0.0127, INSM2: **P = 0.0011; RNA-seq: Wald test as implemented in DESeq2, q-values shown were adjusted by the Benjamini-Hochberg procedure). Box plots show median (center line) and interquartile range (IQR), with whiskers representing the minimum and maximum; ASD NSCs: n = 8, control NSCs: n = 5. f and g, Gating strategy for flow cytometry-based quantification of FBXO2 protein levels in ASD and control NSCs (SOX2⁺ cells); Experiments were performed in all 13 subject lines with similar results; numeric values represent percentages. h, ASD NSCs show elevated FBXO2 protein levels as compared with control NSCs (*P = 0.0101, Mann-Whitney U-test); ASD NSCs: n = 8, control NSCs: n = 5. i and j, Schematic showing the experimental design for mimicking aberrant TM1 gene expression dynamics. NSCs were infected with an inducible lentiviral construct designed to control the expression levels of FBXO2 (FBXO2-GFP) alone or in combination with TM1 genes FBXO44 and CEND1 (FFC-GFP). Overexpression of GFP was used as a control (UGFP). One day later, transgene expression was induced by adding doxycycline, and neuronal differentiation of NSCs was induced the following day. k, Representative confocal images of the morphological development of control neurons (GFP) and those that started with an aberrant overrepresentation of FBXO2 (FBXO2-GFP) alone or in combination with TM1 genes FBXO44 and CEND1 (FFC-GFP). Scale bars 50 μm. Immunohistochemistry was repeated in 2 independent control lines with similar results. l, Left: Total neurite length of NSC-derived neurons at 4 dpi. ASD neurons had significantly longer neurites at 4 dpi as compared with controls (control: 114.23 ± 9.7 μm, ASD: 226.04 ± 12.22 μm, **P = 0.0016, Mann-Whitney U-test). Values represent means ± s.e.m.; ASD: n = 8 biologically independent cell lines, control: n = 5 biologically independent cell lines. Right: Neurons overexpressing FBXO2-GFP or FCC-GFP had significantly longer neurites at 4 dpi as compared with GFP expressing control neurons (GFP control: 106.74 ± 7.76 μm, n = 22 cells; FBXO2-GFP: 175.83 ± 11.58 μm, n = 31 cells; FFC-GFP: 272.9 ± 23.15 μm, n = 34 cells; one-way ANOVA with Dunnett’s multiple comparisons test, GFP vs FBXO2-GFP: *P = 0.0199; GFP vs FFC-GFP: ****P = 0.0001). Values represent means ± s.e.m.; n = 2 biologically independent control cell lines. m, Left: ASD neurons show significantly higher numbers of PSA-NCAM + cells as compared with controls at 4 dpi (**P = 0.0016, Mann-Whitney U-test). Values represent means ± s.e.m.; ASD: n = 8 biologically independent cell lines, control: n = 5 biologically independent cell lines. Right: Neurons overexpressing FBXO2-GFP or FCC-GFP had significantly higher numbers of PSA-NCAM + cells at 4 dpi as compared with GFP expressing control neurons (one-way ANOVA with Dunnett’s multiple comparisons test, GFP vs FFC-GFP_low: *P = 0.0231; GFP vs FBXO2-GFP_med: *P = 0.0348; GFP vs FFC-GFP_high: ***P = 0.0010). Values represent means ± s.e.m.; n = 5 biologically independent control cell lines for each of the three groups.