Abstract

Background Activity-dependent neuroprotective protein (ADNP) is a critical regulator of neurodevelopment, and most pathogenic variants reported in Helsmoortel–Van der Aa syndrome (HVDAS) are truncating variants. In contrast, the functional consequences of ADNP missense variants remain largely unclear. We integrated an ADNP variant cohort in China with variants recorded in the NCBI ClinVar database, revealing a major gap in the interpretation of ADNP missense variants. Methods We investigated a rare de novo ADNP missense variant, p.C687R, predicted to disrupt the ninth zinc finger domain. In vitro, p.C687R was overexpressed in HEK293T cells to assess subnuclear localization by immunofluorescence and chromatin binding patterns using CUT&Tag, with chromatin interactions inferred from published Hi-C datasets. CRISPR/Cas9-mediated ADNP knockout was performed for comparison. In vivo, wild-type ADNP or p.C687R was introduced into the embryonic mouse cortex at E14.5 via in utero electroporation (IUE) and neuronal development was evaluated at E18.5 and P14. Patient-derived induced pluripotent stem cells (iPSCs) from a de novo p.C687R carrier were differentiated into neural progenitor cells (NPCs) and analyzed by multi-omic profiling (RNA-seq, ChIP-seq, ATAC-seq), with lineage-specific markers examined by immunofluorescence. Results p.C687R displays altered subnuclear localization and redistributes wild-type ADNP when overexpressed in HEK293T. IUE in the mouse cortical plate revealed impaired neuronal migration and abnormal cortical arborization. Genome-wide profiling in HEK293T demonstrated a p.C687R-specific chromatin occupancy pattern, preferentially targeting histone modification-related genes. Knockout of ADNP led to upregulation of neuronal genes, including GABAergic lineage-associated genes. In patient-derived iPSCs, a distinct set of neurodevelopmental genes, including key regulators of GABAergic differentiation, showed increased bivalent histone marks (H3K4m