Integrative transcriptome network analysis of iPSC-derived neurons from schizophrenia and schizoaffective disorder patients with 22q11.2 deletion

Fig. 1. Differentially expressed genes in 22q11.2 SZ neurons and their enriched functions. a Heat map showing relative expression of 782 genes that exhibited significant change between control and SZ at p-value < 0.05 (503 increased in SZ; 279 decreased). b Bar plot presenting batch-corrected expression values of 22q11.2 genes in control and SZ samples. Three genes flanking the deleted region at either side were also included. Asterisks (*) indicate significant differential expression at the genome-wide level (FDR < 0.05). c Enriched GO terms of the DEGs as determined by the software David. d Enriched pathways for the DEGs as determined by the software Toppgene

Fig. 2. qPCR validation and NPC proliferation assay. a qPCR analysis of six DEGs using neuronal RNAs from 4–5 controls and 4–5 patients. Relative expression values were determined and pooled by phenotype. Error bars show standard error of the mean; Two-tailed t-test p-value <0.01 (**) and <0.05 (*) are indicated. b Proliferation rates of NPCs derived from control (n = 4) and 22q.11.2 (n = 4) iPSCs. An equal number of cells were plated for each sample (10,000) on day 0. Fold changes were calculated by normalizing the day 1 controls to 1.00, and the control and patient samples were pooled. Statistical significance was determined using a two-tailed Student’s t-test. The asterisk denotes statistically significant difference in proliferation at p < 0.05

Fig. 3. WGCNA modules and function enrichment. a Module preservation Z-summary statistics of 15 modules identified by WGCNA. b Barplot showing the –log10 (p-value) (y-axis) of expression differences between SZ samples and controls for 13 modules whose module structures were well preserved between SZ and control samples (Z > 10, A). c Network of top pathways in the differentially expressed pink module. Each node represents a term and an edge between two nodes indicates that the two terms share genes. d The top pathways in the pink module as determined by the software Enrichr

Fig. 4. Enrichment of DEGs in a region of chromosome 6 predicted to interact with 22q11.2. Scatterplot presenting the –log10 (p-values) (y-axis) of changes in gene expression for each gene along chromosome 6. The heat map below shows the reported Hi-C correlation between each section of chromosome 6 and the 22q11.2 region. Note that the 6p21 region, which is highlighted by a yellow box in the heat map, was enriched for differentially expressed genes, and it displayed the strongest physical interaction with 22q11.2

Fig. 5. Highest co-expression of DEGs in specific brain regions. a Interconnectedness of transcriptional coexpression networks at various developmental stages and in different brain regions based on DEGs or randomly selected genes were evaluated using RNA-seq data from the BrainSpan Atlas. A pair of genes was defined as coexpressed if their expression correlation coefficient |R| was ≥ 0.9, across all tissues from a given brain region and a given developmental stage (Table 1). Dotted lines indicate numbers of connections (i.e., edges) in networks actually observed for DEGs, while the histograms represent distributions of the numbers of edges in 10,000 simulated networks derived from randomly picked genes. b Multiple testing adjusted –log10(P-value) for the significance of difference between the numbers of observed and simulated network edges

Fig. 6. ClueGO network for top correlating DEGs and co-expressed non-DEGs in frontal cortex during the embryonic stage. The size of the nodes reflects the statistical significance of the terms. Only terms with p-values < 0.05 are shown. For grouping terms, the initial group size was set to 3 and the percentage for merging groups was set to 50%. A term can be included in more than one group. Different groups were colored differently. The group leading term (in bold) is the most significant term of the group

Fig. 7. ClueGO network for top correlating DEGs and co-expressed non-DEGs in frontal cortex during the adolescence stage. See annotation in Fig. 6