Cell-type-resolved DNA methylation profiling in the cortex reveals female-specific X chromosome differences in autism

Sex differences are a prominent feature of many neurodevelopmental conditions, particularly autism for which approximately three males are diagnosed for every female. Although epigenetic dysregulation has been implicated in autism, the extent to which autism-associated epigenetic differences vary by sex and neural cell type remains poorly understood. Using fluorescence-activated nuclei sorting (FANS), we isolated neuron-enriched (NeuN+), oligodendrocyte-enriched (SOX10+), microglia-enriched (IRF8+) and astrocyte-enriched (NeuN-/SOX10-/IRF8-) nuclei from post-mortem prefrontal cortex tissue from 24 autistic (70.8% male) and 23 non-autistic (69.6% male) donors, and performed cell-type-resolved epigenome-wide association studies (EWASs) of DNA methylation. We identified cell-type-specific autism-associated differentially methylated positions (DMPs), with microglia harbouring the largest number of associations. Modelling the interaction between sex and diagnosis revealed a striking female-specific signature concentrated on the X chromosome in microglia. At the majority of these sex-by-autism DMPs, autistic females showed lower DNA methylation compared to control females consistent with an altered X chromosome inactivation (XCI) profile. Across the X chromosome, autism-associated DNA methylation differences were significantly larger in females than males and were greater at sites annotated to genes normally subject to XCI than at genes that escape XCI. Together, these data further support the importance of microglia in autism and suggest that altered X chromosome dosage regulation may contribute to sex-dependent molecular mechanisms in autism.