ADNP missense variant p.C687R disrupts chromatin regulation and GABAergic differentiation in Helsmoortel-Van der Aa syndrome.

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 (H3K4me3/H3K27me3). Although their promoters remained in an open chromatin state, these genes were transcriptionally silent in pluripotent cells but became more activated upon GABAergic differentiation. LIMITATIONS: This study is based on a single patient-derived line in combination with complementary experimental models. In the heterozygous endogenous context, distinguishing increased functional activity from dosage-related effects requires further investigation. Replication in additional patient-derived or engineered lines is required to determine the generalizability. CONCLUSIONS: Our results suggest that p.C687R may exert gain-of-function-like effects in experimental systems and underscore chromatin-mediated regulation of GABAergic lineage genes in HVDAS. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13229-026-00713-4.