Attention deficit/hyperactivity disorder (ADHD) is a highly heritable neurodevelopmental disorder, but its genetic architecture remains incompletely characterized. Rare coding variants, which can profoundly impact gene function, represent an underexplored dimension of ADHD risk. In this study, we analyzed large scale DNA sequencing datasets from ancestrally diverse cohorts and observed significant enrichment of rare protein-truncating and deleterious missense variants in highly evolutionarily constrained genes. This analysis identified 15 high-confidence ADHD risk genes, including the previously implicated KDM5B. Integrating these findings with genome-wide association study (GWAS) data revealed nine enriched pathways, with strong involvement in synapse organization, neuronal development, and chromatin regulation. Protein-protein interaction analyses identified chromatin regulators as central network hubs, and single-cell transcriptomic profiling confirmed their expression in neurons and glial cells, with distinct patterns in oligodendrocyte subtypes. These findings advance our understanding of the genetic architecture of ADHD, uncover core molecular mechanisms, and provide promising directions for future therapeutic development.

The authors have declared no competing interest.

This study was supported by a grant from Beatrice and Samuel A. Seaver Foundation (BM); and the National Institutes of Health (NIH), under grant numbers R01MH129724 (BM), 5R01MH097849 (BM), and K08MH128665 (EO). The All of Us Research Program is supported by the National Institutes of Health, Office of the Director: Regional Medical Centers: 1 OT2 OD026549; 1 OT2 OD026554; 1 OT2 OD026557; 1 OT2 OD026556; 1 OT2 OD026550; 1 OT2 OD 026552; 1 OT2 OD026553; 1 OT2 OD026548; 1 OT2 OD026551; 1 OT2 OD026555; IAA #: AOD 16037; Federally Qualified Health Centers: HHSN 263201600085U; Data and Research Center: 5 U2C OD023196; Biobank: 1 U24 OD023121; The Participant Center: U24 OD023176; Participant Technology Systems Center: 1 U24 OD023163; Communications and Engagement: 3 OT2 OD023205; 3 OT2 OD023206; and Community Partners: 1 OT2 OD025277; 3 OT2 OD025315; 1 OT2 OD025337; 1 OT2 OD025276. In addition, the All of Us Research Program would not be possible without the partnership of its participants.

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The use of de-identified data from the All of Us Research Program, accessed via the Researcher Workbench, does not constitute human subjects research and does not require IRB review according to the guidelines provided in the All of Us Responsible Conduct of Research training. Our project adheres to the ethical principles of research with human participants, as encouraged by the All of Us program, although it involves only the analysis of previously collected, de-identified data.

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

Yes

I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

Data availability: All of Us controlled tier data is available for registered institutions and cohort selection is detailed in the workbench titled "Detecting the prevalence of ultra-rare gene mutations." Due to stringent privacy protections and data sharing restrictions enforced by the All of Us Research Program, individual-level variant data cannot be shared publicly. Researchers must adhere to specific guidelines to protect participant confidentiality, which includes not reporting or disseminating any data that could allow the re-identification of participants, particularly those involving participant counts between 1 and 20 without employing approved data obscuration methods.