Attention-deficit hyperactivity disorder (ADHD) is a prevalent childhood-onset neuropsychiatric condition marked by persistent inattention, impulsivity, and hyperactivity, often extending into adulthood. Higher-level executive functions, mediated by frontal-striatal-parietal and frontal-cerebellar networks, are significantly impaired in individuals with ADHD. These functions include motor and interference inhibition, working memory, sustained attention, and temporal information processing. While the etiology remains unclear, ADHD exhibits substantial heritability, suggesting a significant genetic component. Research increasingly points to the involvement of rare monogenic variants in its pathogenesis. This study investigates a family with severe ADHD to identify a potential genetic cause and elucidate the underlying mechanisms.

Existing literature highlights the substantial heritability of ADHD, indicating a strong genetic basis. Studies suggest that rare monogenic variants play a crucial role in ADHD's pathogenesis. Previous research has explored the involvement of various genes and pathways, but a comprehensive understanding remains elusive. The role of cell adhesion molecules (CAMs), particularly cadherins, in brain development and function has been established, with some evidence linking cadherin gene abnormalities to psychiatric disorders such as autism and schizophrenia. However, prior to this study, cadherins had not been directly implicated in the pathophysiology of ADHD. This research aimed to fill this gap in knowledge by focusing on a family exhibiting severe ADHD, conducting genetic analysis to identify a disease-associated gene, and investigating the physiological consequences of identified mutations.

This study employed a multi-faceted approach. First, a consanguineous Bedouin family with three siblings presenting severe ADHD was clinically characterized using semi-structured interviews, questionnaires (CELF-5, Conner's Parent Rating Scales-Revised, WISC-III), and observations. Genetic analysis involved linkage analysis to identify a shared homozygous locus, followed by whole-exome sequencing (WES) of an affected individual to pinpoint the causal mutation. The identified mutation in *CDH2* was validated using Sanger sequencing and compared to a control group. Computational protein modeling and biochemical peptide cleavage assays using furin protease were performed to analyze the impact of the mutation on N-cadherin protein processing and maturation. To investigate the in vivo effects, CRISPR/Cas9-mediated knock-in mice harboring the human mutation in the mouse *Cdh2* ortholog were generated. These mice underwent a comprehensive behavioral and cognitive assessment using tests such as the open-field test, elevated plus-maze, Y-maze, resident-intruder test, three-chamber sociability test, acoustic startle response (ASR), pre-pulse inhibition, and rotarod. The effect of acute methylphenidate (MPH) administration was also examined. Ex vivo studies on hippocampal neurons from the mutant mice included immunofluorescence (IF) analysis of synaptic vesicle markers (synaptobrevin2, vGlut1), FM1-43 dye loading and unloading assays to assess the readily releasable pool (RRP), and synaptopHluorin (sypHy) imaging to monitor synaptic vesicle recycling. Electrophysiological recordings in acute brain slices were used to measure spontaneous miniature excitatory postsynaptic currents (mEPSCs) and short-term synaptic plasticity (STP). Quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA) were used to analyze tyrosine hydroxylase (TH) expression and dopamine levels in the ventral midbrain (vMB) and prefrontal cortex (PFC). Finally, whole-transcriptome RNA-seq analysis was performed on micro-dissected vMB and PFC tissues to identify differentially expressed genes (DEGs) and perform gene ontology (GO) analysis.

The study identified a homozygous missense mutation (c.355C>T; p.H150Y) in *CDH2* in three siblings with severe ADHD. This mutation resides in a region crucial for furin protease cleavage and N-cadherin maturation. Biochemical assays demonstrated significantly reduced cleavage efficacy of the mutated peptide compared to the wild type. Knock-in mice carrying the human mutation exhibited core behavioral features of hyperactivity in open-field tests, increased startle amplitude, and potential working memory deficits. These hyperactivity symptoms were exacerbated by acute methylphenidate administration. Ex vivo analysis revealed that *Cdh2*-mutant neurons had smaller presynaptic vesicle clusters, a reduced readily releasable pool (RRP) size, and attenuated evoked transmitter release. Spontaneous miniature excitatory postsynaptic current (mEPSC) frequency was also significantly lower in these neurons, indicating impaired synaptic transmission. Short-term synaptic plasticity, specifically frequency facilitation, was also reduced. Furthermore, qPCR and ELISA analyses revealed significantly reduced tyrosine hydroxylase (TH) expression and dopamine levels in the prefrontal cortex of mutant mice, suggesting impaired dopaminergic neurotransmission. Whole-transcriptome analysis identified numerous differentially expressed genes (DEGs) in the ventral midbrain and prefrontal cortex, including genes related to cell adhesion, synapse organization, and neuronal function. Many of these DEGs were previously linked to ADHD.

This study presents compelling evidence for a causal link between a novel *CDH2* mutation and familial ADHD. The findings highlight the crucial role of N-cadherin in synaptic function and dopaminergic neurotransmission, emphasizing the importance of proper N-cadherin maturation for normal brain development and function. Impaired N-cadherin function, due to the identified mutation, leads to alterations in synaptic properties, affecting both the size and release probability of synaptic vesicles. The observed reduction in dopamine levels in the prefrontal cortex is consistent with the “hypo-dopaminergic” hypothesis of ADHD. The aggravated hyperactivity response to methylphenidate in mutant mice suggests a potential mechanistic explanation for the variable response to stimulant medication observed in ADHD patients. The identified differentially expressed genes provide further insights into the complex molecular mechanisms underlying ADHD.

This study provides the first evidence of a monogenic, non-syndromic form of familial ADHD caused by a mutation in *CDH2*. The findings reveal a critical role for N-cadherin in the pathophysiology of ADHD through its impact on synaptic transmission and dopaminergic function. The results provide potential avenues for future research into novel therapeutic strategies for ADHD, particularly for individuals unresponsive to stimulant medications. Further research could explore the specific molecular pathways downstream of N-cadherin that contribute to ADHD symptomatology and evaluate the potential of targeting these pathways for therapeutic intervention.

The study focused on a single family, limiting the generalizability of findings to other populations. The mouse model, while successfully recapitulating key features of ADHD, may not fully capture the complexity of the human condition. The ex vivo studies were limited to hippocampal neurons, and future research should investigate other brain regions. Finally, the precise mechanisms through which the *CDH2* mutation affects dopamine levels require further investigation.