A Single-Cell Atlas of DNA Methylation in Autism Spectrum Disorder Reveals Distinct Regulatory and Aging Signatures

Autism spectrum disorder (ASD) is a common, genetically and clinically heterogeneous neurodevelopmental condition. Despite this diversity, studies of postmortem brain tissue have revealed convergent molecular changes across the cortex, including reduced synaptic function in subsets of excitatory and inhibitory neurons and increased glial reactivity. Whether these features are reflected in cell type–specific epigenetic signatures remains unknown. Here, we present the first single-cell analysis of DNA methylation (DNAm) coupled with transcriptomics in ASD. Using snmCT- seq, we profiled DNAm and gene expression from over 60,000 nuclei across 49 donors. We identified thousands of differentially methylated regions (DMRs) in ASD, enriched in promoters and regulatory elements active during both prenatal development and in adult cortex. ASD-related methylation changes were spatially localized but uncorrelated with gene expression, and were small in magnitude compared to robust age-associated effects. Age-DMRs were concentrated in excitatory neurons, enriched in known cognitive aging pathways, and revealed distinct roles for CG and non- CG methylation in the aging brain. Finally, we explored age-by-diagnosis interactions, identifying a reduction in inhibitory neuron abundance with age in ASD relative to controls, highlighting this area as a promising direction for future research.