Coding and noncoding de novo variation converge on developmental trajectories of cortical layer 5–6 neurons in autism spectrum disorder

Large-scale sequencing has greatly advanced our understanding of the genetic architecture of autism spectrum disorder (ASD). Whole-exome sequencing (WES) in thousands of trios revealed a major role for de novo protein-truncating variants (PTVs) in loss-of-function (LoF) intolerant genes and identified dozens of high-confidence ASD genes, many central to neuronal development and synaptic signaling. Whole-genome sequencing (WGS) extended these findings by uncovering noncoding contributors, including rare structural and regulatory variants that disrupt gene expression during brain development. Despite these advances, the mechanisms by which diverse mutations converge on shared neurodevelopmental pathways, and the specific cell types and developmental windows most impacted, remain incompletely understood. To address this, we integrated results from large ASD WES/WGS studies with bulk and single-nucleus transcriptomic data. Stratifying LoF-intolerant ASD genes into broadly expressed versus brain-restricted subsets revealed distinct functional roles: broadly expressed genes regulate transcription, chromatin, histone modification, and splicing, whereas brain-restricted genes function mainly in synaptic processes. Clinically, the former tend to be linked to general neurodevelopmental disorders (NDDs), while the latter are more associated with ASD-predominant phenotypes. Intersecting coding and regulatory ASD variants with human prefrontal cortex (PFC) trajectories showed that both converge on the L5-6_TLE4 neuronal lineage, but at different stages: coding de novo variants disrupt postnatal programs of neuronal maturation, while regulatory promoter variants act earlier, on fetal developmental programs. These findings highlight a framework in which distinct variant classes act within different subsets of LoF-intolerant genes, shaping ASD risk through cell type– and stage-specific mechanisms.