Fig. 1.

ANKS1B microdeletions reduce AIDA-1 expression in patient-derived neurons. a Monogenic microdeletions (red bars) in patients span multiple exons in the ANKS1B gene. Black bars represent patients with additional CNVs in other genes. Green arrows represent putative transcription start sites. b ANKS1B encodes AIDA-1B, AIDA-1D, and AIDA-1C protein isoforms. c (Left) Western blots show that all three major AIDA-1 isoforms are expressed in total lysate (TOT) and enriched in synaptic (SYN) and postsynaptic density (PSD) fractions in postmortem human and mouse brain (5 μg lysate). (Right) Lentivirus-mediated knockdown of AIDA-1 in primary rat neurons (DIV 14-21) using two different shRNAs (sh1 and sh2 = AIDA-1 specific; shNT = scrambled control shRNA) confirms antibody specificity (20 μg lysate). d Pedigree of EIN-1 and EIN-2 families. e (Top) IPSCs generated from probands (EIN-1-1 and EIN-2-1) and unaffected mothers (EIN-1-M and EIN-2-M) express Oct-4 and Sox-2 pluripotency markers. (Bottom) Induced neurons (iNs) generated from forced NGN2 expression (GFP) in iPSCs show typical neuronal morphology and express the mature neuronal marker MAP2. Nuclei from non-neuronal cells are co-cultured rat astrocytes added to improve neuronal viability and maturation. Scale bars = 10 μm. f Western blots of AIDA-1 and PSD95 show that AIDA-1 isoforms are significantly reduced in probands EIN-1-1 and EIN-2-1 compared to unaffected mothers EIN-1-M and EIN-2-M (10 μg lysate). g Quantitation of AIDA-1 expression normalized to neuronal marker PSD95 from Family EIN-2. N = 5–7 biological replicates. Bar graphs show mean ± SEM, two-sided Student’s t-test, *p < 0.05 ***p < 0.001. h RT-qPCR of exons 4, 13, and 20 spanning the ANKS1B gene and normalized to neuronal marker MAP2 are reduced in probands from both families. N = 4–5 biological replicates for each family. Bar graphs show mean ± SEM, one-sided Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001, ^#p = 0.052