Haplotype-resolved methylomes reveal parent-of-origin DNA methylation imbalance in autism spectrum disorder

Epigenetic dysregulation plays an essential role in autism spectrum disorder (ASD), but the parent-of-origin effects (POEs) of DNA methylation remain unknown. Here, we applied PacBio HiFi sequencing with haplotype-phased methylation profiling in 124 individuals (31 ASD quartets) to systematically dissect POE-dependent methylation. Comparative analysis of phased methylomes between probands and unaffected siblings identified 114 paternal- and 106 maternal-specific differential methylated cytosines (DMCs), 45 and 46 differential methylated regions (DMRs), and 2425 and 2693 methylation outliers (MOs), respectively. These POE methylation alterations were enriched in ASD-relevant gene categories but exhibited distinct genomic distributions and functional pathways between parental haplotypes. Furthermore, genome-wide parent-of-origin DMR analysis identified 443 allele-specific methylation (ASM) regions, from which we detected 34 differential expression ASMs and 62 ASM outliers, showing pronounced enrichment within PWS/AS locus and ASD-associated genes. Collectively, this study provides a comprehensive evidence of pervasive POE-dependent methylation imbalance and aberrant ASM at imprinting regions underlying ASD pathogenesis, offering insights into epigenetic mechanisms of complex neurodevelopmental disorders.