Sex differences in DNA methylation in autism spectrum disorder: an epigenome-wide association study

Background Autism Spectrum Disorders (ASD) represent a heterogeneous group of neurodevelopment conditions with a significant sex disparity—males are affected 3–4 times more frequently than females. The current study aims to explore the role of DNA methylation, an epigenetic modification, in the underlying mechanisms of ASD and its sex differences. Methods Blood DNA from 95 ASD probands (48 males, 47 females) and 49 age-matched controls was analysed. Using the Infinium MethylationEPIC BeadChip, we assessed over 845,000 CpG-sites to conduct an Epigenome-Wide Association Study (EWAS). The EWAS results were further evaluated through Gene Set Enrichment Analysis to identify significantly enriched pathways. DNA methylation clocks were calculated, and differences between ASD boys and girls, as well as between ASD probands and controls, were assessed. Results Seven significant CpG-sites associated with ASD were identified, with a lower mean methylation compared with controls. The gene set enrichment analysis revealed ‘multicellular organismal signalling’ as the most significant pathway. While no sex-specific significant associations emerged, we identified global hypomethylation in boys compared with girls. The sex-specific pathway analyses revealed enriched pathways linked to cellular signalling and metabolic functions. The analysis of DNA methylation clocks (Levine’s clock and telomere length’s (TL) clock) showed sex differences in the TL clock, with ASD boys exhibiting shorter telomeres compared with ASD girls. Conclusions The findings of the study indicate that DNA methylation could be implicated in the sex-differences in ASD.