A novel Adnp frameshift variant disrupts Wnt signaling leading to chromatocytoskeletal defects and autism-related behavior in mice
Background: Heterozygous de novo variants in the transcription factor Activity-Dependent Neuroprotective Protein (ADNP) cause a severe neurodevelopmental disorder, termed the Helsmoortel-Van der Aa syndrome (HVDAS), that is characterized by autism, intellectual delay, and malfunctions in several other organ systems. The ADNP gene is essential for embryonic development and interacts with components of several chromatin remodeling complexes, though the exact pathophysiological mechanisms remain hidden.  Methods: We used CRISPR/Cas9 genome editing to create a 14-base pair deletion in the murine Adnp gene to study the effects of the variant in a heterozygous disease-relevant mouse model.  Results: Heterozygous mice carrying the c.2463_2476del (p.Leu822Hisfs*6) deletion are viable and fertile. Introduction of the 14-base pair deletion reduced cellular Adnp levels in the brain as well as its associations to chromatin, paralleled by a genome-wide increase in chromatin accessibility. Morphology analysis showed mild neuroanatomical changes in brain regions related to cognition, memory and learning, and motor skills. Behavioral testing in heterozygous Adnp mice confirmed cognitive defects in the Morris water maze test, increased anxiety in the elevated plus maze test, repetitive behavior after marble burying, and impaired social interactions. Transcriptome sequencing of the frontal cortex, an essential region involved in executive functions, cognition, and motor control, showed predominant downregulation of the Wnt signaling pathway. Cytoskeletal abnormalities were further coupled to synaptic plasticity deficits with dysregulated transcription factors implicated in lineage specification, as well as alterations in neuronal cell numbers. Adnp can also directly regulated mechanisms of synaptic plasticity by interacting with Camk2a and Dbn1. The frameshift variant interfered with these complexes and caused aberrant Camk2a phosphorylation at synapses. Mass spectrometry of the frontal cortex of heterozygous Adnp mice identified changes of multiple chromatin interacting proteins and cytoskeletal components, confirming chromatocytoskeletal dysregulations detected in the bulk transcriptome. Ultimately, Hi-C analysis detected fine-scale, locus-specific differences in 3D genome architecture in the heterozygous Adnp 14-base pair deletion mice, which are associated with transcriptional changes.  Conclusions: We generated a disease-relevant heterozygous Adnp mouse model that parallels many of the molecular and behavioral aspects observed in Helsmoortel-Van der Aa patients. We demonstrate that Adnp has a primary nuclear role involving Wnt signaling coupled with cytoskeletal deficits. Our novel mouse model is thus a valuable model to study the human disorder and may be used for preclinical studies. Overall design: RNAseq of the frontal cortex in adolescent WT and HET littermates. Differential gene expression seven WT and seven HET mice was analyzed 13,383 expressed genes.