Fig. 5

Cerebellar and lymphoid gene expression changes are associated with different ADNP mutations. (A) Volcano plot of differentially expressed genes (DEGs) in the ADNP cerebellum using the NOISeq algorithm, representing the effect size M (log2 ratio) and D (difference between conditions) values. The DEG are shown in blue. (B) Gene set enrichment analysis of all DEGs in gene ontology (GO), biological processes (BP) and molecular function (MF) reveals specific Helsmoortel–Van der Aa syndrome-related pathways in the ADNP brain. (C) RT-PCR showing a significant reduction of METTL3, BECN1 and CTNNB1 mRNA levels in the ADNP cerebellum compared to the age-matched control subject. Expression values were normalized using the housekeeping genes ACTB, B2M and UBC. Data was analyzed using an unpaired student T-test. (D) Volcano plot of DEGs in the patient LCLs using the DESeq2 package, displaying the significance (-log10q) and effect size (log2FC). The DEG are shown in blue. (E) Functional gene set enrichment of GO and BP using differentially expressed genes in the ADNP LCLs as compared to age- and sex-matched controls. UMAP clustering of gene sets colored by standard deviation, variance, or mean fold-change in patient LCLs shows clear downregulation of the WNT, Hedgehog and Notch signaling pathways (marked in a red box), impairing proper neuronal development. Downregulated genes, blue; upregulated genes, red. (F) RT-PCR showing a significant increase of CBX3, CTNNAL1, SMG5 and UPF3B together with a significant decrease in WTN10A mRNA levels in patients versus control LCLs. Expression values were normalized using the housekeeping genes GAPDH, RPL13A and SDHA. Data was subsequently analyzed with a Mann–Whitney U test for unpaired measures