Fig. 4: Differential expression analysis in affected hNPCs.

A Venn diagram of gene overlap of THOC6 ^W100*/W100* and THOC6^E188K/E188K affected genes and all syndromic intellectual disability genes included in the SysID database. Overlap significance tested by one-sided Fisher’s exact test. DEG, differentially expressed genes; ASG, alternatively spliced genes. B Violin plot of coding sequence (CDS) length of combined DEGs in affected cells compared to non-significant genes. NotSig, non-significant genes (dark grey); UP, upregulated (coral); DOWN, downregulated (dark blue). Significance, two-tailed unpaired t test, p = <0.0001 (****). C Violin plot of isoform number of combined DEGs in affected cells compared to non-significant genes. NotSig, non-significant genes (dark grey); UP, upregulated (coral); DOWN, downregulated (dark blue). Significance, two-tailed unpaired t test, p = 0.0373. D Percentage of gene type by condition for combined DEGs in affected hNPCs. NotSig, non-significant genes; UP, upregulated. DOWN, downregulated; lncRNA, long non-coding RNA (lavender); PP, processed pseudogene (turquoise); TUP, transcribed unprocessed pseudogene (grey); PCG, protein-coding gene (blue). E DAVID biological pathway enrichment analysis of combined upregulated genes (top, red) and downregulated genes (bottom, blue) in THOC6 affected hNPCs. F qPCR relative abundance quantifications (2^-ΔΔCt) for MEG3, ESRG, MEG8, and NEAT1 in hNPCs. n = 3 experimental replicates of 3 biological replicates per genotype. Genotypes: THOC6^+/+ (black), THOC6^E188K/+ (dark purple), THOC6^E188K/E188K (purple), THOC6^W100*/+ (dark green), THOC6^W100*/W100* (green). Data shown as mean ± SEM. Significance, two-tailed unpaired t test. This experiment was repeated twice independently and showed similar results. G RNA FISH probing for MEG3 and MALAT1 in affected and unaffected hNPCs. Cell inset showing MEG3 expression and localization differences with yellow arrows in merged image. Scale bar 50 μm. UNAFF, unaffected (THOC6^W100*/+); AFF, affected (THOC6^W100*/W100*) H Representative Western blot images (left) and protein abundance quantification (normalized to B-actin control) for validation of top downregulated (blue) and upregulated (coral) genes across genotypes. Triangles specify genotypes with E188K variant, and circles specify the W100* variant. N = 3 technical replicates per genotype (n = 6 total replicates per condition). This experiment was performed once. Source data are provided as a Source Data file.